Workshop 4° Curso

Scientific and
Doctorado en Ciencias Technological

de recursos Naturales Bioresource Nucleus
1er Workshop y 4to Curso
Internacional
Los Desafíos en el Nuevo Escenario Mundial
AVANCES EN CIENCIA Y TECNOLOGÍA

DE RECURSOS NATURALES
LIBRO DE RESUMENES
23, 24 y 25 de Noviembre
Sede Pucón, Universidad
de La Frontera
El contenido de los trabajos expuestos en el presente
Libro de Resúmenes es de exclusiva responsabilidad de
los respectivos autores y coautores.
COMITÉ ORGANIZADOR
Presidente
MARIA DE LA LUZ MORA
Director Ejecutivo
PABLO CORNEJO RIVAS
Secretario General
ALEX SEGUEL FUENTEALBA
Coordinadores
PAULA AGUILERA ÑONQUEPAN
SEBASTIAN MEIER ROMERO
Asesoría Logística
CARMEN HERNÁNDEZ CIFUENTES
AGRADECIMIENTOS
A los estudiantes del Doctorado en Ciencias de Recursos Naturales de la Universidad de La Frontera

por su valiosa contribución en la organización del Simposio.
A la Secretaria del Programa de Doctorado en Ciencias de Recursos Naturales de la Universidad de

La Frontera Sra. Ángela Mardones por su apoyo y cooperación.
COMITÉS DE EVALUACIÓN, 4º CURSO
INTERNACIONAL: AVANCES EN CIENCIA Y
TECNOLOGÍA DE RECURSOS NATURALES
SIMPIOSIOS 1, 2 Y 3
Dra. Carolina Shene, Univ. Frontera

Dr. Andrés Quiroz, Univ. Frontera
Dr. Christian Figueroa, Univ. Austral
Dr. Claudio Ramírez, Univ. Talca
Mg. Fernando Perich, Univ. Frontera
SIMPOSIOS 4, 5 Y 6
Dra. Liliana Gianfreda, U. Federico II, Italy

Dra. Rosario Azcón, Est. Exp. Zaidín, CSIC, España
Dr. David Crowley, Univ. California, USA
Dr. Fernando Borie, Univ. Frontera
Dr. Nanthi Bolan, UNISA, Australia
INDICE
Nº pág.
PROGRAMA GENERAL i
SIMPOSIO 1. AVANCES EN BIOTECNOLOGÍA AGROALIMENTARIA 1

PRESENTACIONES ORALES
PRODUCCIÓN MICROBIANA DE LÍPIDOS 3

C. Shene, A. Leyton, B. Quilodrán, I. Hinzpeter, M. Rubilar
ACTIVIDAD BIOLÓGICA DE COMPUESTOS POLIFENÓLICOS Y SUS APLICACIONES EN LA 6

INDUSTRIA DE ALIMENTOS
M. Rubilar , C. Gutiérrez, C. Jara, Villarroel, M., C. Shene
ADVANCED TECHNOLOGY IN FOOD COLOR: DESCRIPTION OF THE KINETIC 9

ENZYMATIC BROWNING IN FRUIT SLICES USING NON-UNIFORM COLOR INFORMATION
FROM DIGITAL IMAGES
Roberto Quevedo
TECHNOLOGICAL INNOVATION IN BIOPROCESS 18

Rodrigo Bórquez Yáñez
PRESENTACIONES PANELES 23
INMOVILIZACIÓN DE PAPAÍNA EN ALUMINOSILICATOS NANOTUBULARES SINTÉTICOS 25

Arancibia N, Manquián K, Contreras R and G Zúñiga
SOLVENT EXTRACTION OLIGOSACCHARIDES OF LUPINE FLOUR 26

Medina H and Shene C
INCORPORACIÓN DE FLAVONOIDES AGLICONA Y GLICOSILADOS EN UNA MATRIZ 27

BIOPOLIMÉRICA DE CARBOXIMETILCELULOSA SÓDICA: ESTRUCTURA QUÍMICA Y
PERMEABILIDAD AL VAPOR DE AGUA
Silva A, Ihl M and Bifani V
MECHANICAL PROPERTIES, CHEMICAL INTERACTIONS AND STRUCTURE OF 28

STARCH/CMC/MONTMORILLONITE NANOCOMPOSITE FILMS
Silva A, Garrido E, Mora M.L, Bifani V and Theng B
ZEBRAFISH (Danio rerio) LARVAL REARING: PROGRESS IN ESTABLISHING A MODEL 29

ORGANISM FOR STUDIES OF NUTRITION AND GROWTH APPLIED TO CULTURED FISHES
Ulloa P, Lizama C., Araneda C, Neira R and Iturra P
OPTIMIZATION OF AN AQUEOUS EXTRACTION OF MUCILAGE FROM FLAXSEED CAKE 30

M. Verdugo and M. Rubilar
SIMPOSIO 2. ECOLOGÍA QUÍMICA COMO HERRAMIENTA BIOTECNOLÓGICA 31

CHEMICAL ECOLOGY: AN INTERDISCIPLINARY DISCIPLINE 33

Andrés Quiroz
APPLICATION OF INSECT CHEMICAL ECOLOGY TO BLUEBERRY AND GRAPE 34
PRODUCTION IN THE GREAT LAKES REGION OF THE UNITED STATES
Rufus Isaacs, Luis Teixeira, Keith Mason, and Steve Van Timmeren
BIOTECHNOLOGY OF CROP PROTECTION 35

Claudio C. Ramirez
ECOLOGICAL AND EVOLUTIONARY GENOMICS OF HERBIVORE INSECTS 36

Christian Figueroa
INSECTICIDE RESISTANCE IN PEST OF FRUITS IN CHILE 37

Eduardo Fuentes-Contreras
PHARMACOLOGICAL AND PHYSIOLOGICAL PROPERTIES OF EXTRACTS AND PURE 38

COMPOUNDS USED IN POPULAR MEDICINE
Alejandro Urzúa
MÉTODOS PARA DETERMINACIÓN DE CAPACIDAD ANTIOXIDANTE EN PLANTAS 45

NATIVAS CHILENAS
Alfaro S, Scheuermann E and Quiroz A
LETHAL AND NEUROTOXIC EFFECTS OF EXTRACTS FROM THE RED MARINE ALGA 46
Ceramium rubrum (HUDSON) (RHODOPHYTA, FLORIDEOPHYCEAE) DETERMINED BY THE
Artemia franciscana (KELLOG, 1906) BIOASSAY, AS UNIVERSAL INDICATOR OF TOXICITY
Cortés Y, Pardo F, Perich F and Quiroz A
BEHAVIOURAL RESPONSES OF CLOVER ROOT BORES (Hylastinus obscurus) 47

(COLEOPTERA: SCOLYTIDAE) TO FATTY ACID IDENTIFIED FROM YOUNG AGED RED
CLOVER ROOTS (Trifolium pratense)
Manosalva L, Pardo F, Perich F, Mutis A, Parra L, Ortega F and Quiroz A
EVIDENCE OF SEXUAL PHEROMONE IN THE RASPBERRY WEEVIL Aegorhinus superciliosus 48

(COLEOPTERA: CURCULIONIDAE)
Mutis A, Parra L, Manosalva L, Palma R, Lizama R, Pardo, F, Perich F and Quiroz A
RESISTANCE TO PYRETHROIDS IN Haematobia irritans (DIPTERA: MUSCIDAE) AND 49

DETECTION OF KDR MUTATION IN CHILE
Oyarzún M.P and Figueroa C
ELECTROANTENNOGRAPHIC RESPONSE OF CLOVER ROOT BORER Hylastinus obscurus 50

(COLEPTERA: SCOLYTIDAE) TO COMPOUNDS IDENTIFIED IN VOLATILES FROM
CONSPECIFIC AND ITS HOST
Palma R, Mutis A, Manosalva L, and Quiroz A
BIOLOGICAL AND CHEMICAL FACTORS ASSOCIATED IN THE INTERACTION BETWEEN 51

Aegorhinus superciliosus (COLEOPTERA: CURCULIONIDAE) AND ITS HOST Vaccinium
corymbosum L.
Parra L, Mutis A, Perich F, Pardo F and Quiroz A
ANTIFUNGAL ACTIVITY OF VOLATILES EMITTED BY MICELIAL CULTURES OF 52

CHILEAN SAPROPHYTIC FUNGI
Schalchli, H., Hormazabal, E. and Quiroz, A.
SEMIOCHEMICALS INVOLVED THE MATING BEHAVIOUR OF Artemia SPP. 53
Tapia C, Quiroz A and Gajardo G
ESTUDIO DE COMPUESTOS VOLÁTILES, CAPACIDAD ANTIOXIDANTE Y 54

ANTIBACTERIANA DEL ACEITE ESENCIAL DE TEPA (Laureliopsis philippiana (LOOSER)
SCHODDE). BASES PARA SU POTENCIAL USO Y APLICACIÓN
Toledo D, Mutis A, Hormazabal E, Scheurmann E and Quiroz A
SIMPOSIO 3. MICROBIOLOGÍA Y RIZOSFERA 55

ARBUSCULAR MYCORRHIZAE IN AGRICULTURAL AND FOREST ECOSYSTEMS OF 57

CHILE.
F. Borie, R Rubio, A. Seguel & P.Cornejo.
ARBUSCULAR MYCORRHIZAL FUNGI AND ITS ROLE IN STABILIZING ECOSYSTEMS 58

POLLUTED BY MINING ACTIVITIES
Pablo Cornejo, Sebastián Meier, Cecilia García & Fernando Borie
PAPEL DE LAS MICORRIZAS ARBUSCULARES EN LA PROTECCIÓN DE LA PLANTA 59

FRENTE A PATÓGENOS
M. J. Pozo, J. A. López-Raéz, S. C. Jung, C. Azcón-Aguilar
EFECTIVIDAD DE RESIDUOS AGROINDUSTRIALES BIOTRASFORMADOS Y SU 60

INTERACCIÓN CON INOCULANTES MICROBIANOS PARA SU USO EN REVEGETACIÓN
R. Azcón
MYCORRHIZAS OF TEMPERATE FORESTS IN SOUTH-CENTRAL CHILE 61

Godoy, R., Etcheverría, P. & Boy, J.
CONSTRUCTION OF VECTORS TO IMPROVE BOTH PHOSPHATE AND PHYTATE 62

UTILIZATION BY RHIZOBACTERIA
Fernández, M.T., Gonzáles, T., Fraga, R., Rodríguez, H. and Goire, I.
PHYTATE-DEGRADING BACTERIA AND THEIR BIOTECHNOLOGICAL POTENTIAL IN 63

AGRICULTURE
Jorquera, M.A. and Mora, M.L.
THE ECOLOGY OF MICROBIAL COMMUNITIES IN THE PLANT RHIZOSPHERE 64

Crowley, D.E.
DETECTION AND QUANTIFICATION OF INDOLE ACETIC ACID IN RHIZOBACTERIA 69

STRAINS ISOLATED FROM VOLCANIC SOILS GRASSLANDS OF SOUTHERN CHILE
Acuña J, Jorquera M and Mora M.L
EFFECT OF THE APPLICATION OF NATURAL PRODUCTS PROMOTERS OF THE VEGETAL 70

GROWING AS STIMULATORS OF THE ARBUSCULAR MYCORRHIZAL IN EXTENSIVE
CROPS OF THE AGRICULTURAL INTEREST IN THE CENTRAL-SOUTHERN ZONE OF
CHILE
Castillo C.G
ARBUSCULAR MYCORRHIZAL FUNGI AND GLOMALIN AS SOIL ORGANIC MATTER 71
STABILIZATION AGENTS OF SOIL IN A MEDITERRANEAN AGROECOSYSTEM AT
CENTRAL CHILE
Curaqueo, G., Acevedo, E., Rubio, R., Borie, F
ABIOTIC STRESS AFFECTING THE MYCORRHIZAL OF FOREST ECOSYSTEMS FROM 72

CENTRAL SOUTHERN CHILE, AN OVERVIEW
Etcheverría P and Godoy R
NOVEL BACTERIA AND MICROBIAL COMMUNITIES INVOLVED IN SIMAZINE 73

DEGRADATION IN AGRICULTURAL SOILS OF CENTRAL CHILE
Hernández M, Jonquera M, Moore E and Seeger M
ISOLATION AND SCREENING OF POTENTIAL PLANT GROWTH PROMOTING 74

RHIZOBACTERIA FROM PASTURES IN CHILEAN VOLCANIC SOIL UNDER NITROGEN
FERTILIZATION
Martínez O, Jorquera M and Mora M.L
DIFFERENT PATTERNS OF LMWOA EXUDATION IN METALLOPHYTE AND 75

AGRICULTURAL PLANTS AT INCREASING COPPER LEVELS
Meier S, Alvear M, Borie F, Aguilera P and Cornejo P
TWO PHYTASE PRODUCING BACTERIA WITH BIOTECHNOLOGICAL POTENTIAL FOR 76

USE IN ENHANCING P BIOAVAILABILITY IN SOILS AND MANURES
Menezes D, Jorquera M and Mora M.L
SCREENING AND ISOLATION OF PHOSPHATE SOLUBILIZING AND MINERALIZING 77

FUNGI FROM SOILS OF SOUTHERN CHILE
Morales A, Alvear M and Borie F
MICROBIAL BIOFERTILIZERS: PREPARATION OF MYCORRHIZAL INOCULANTS ON 78

POROUS INERT MATRICES
Rubio R, Castillo C, Borie F and Castillo C.G
CHANGES IN THE DENSITY OF MYCORRHIZAL FUNGI PROPAGULES ASSOCIATED TO 79

Oenothera affinis IN COPPER POLLUTED SOILS
Seguel, A., Meier, S., García, C., Borie, F., y Cornejo, P
SIMPOSIO 4. SUELOS, FISIOLOGÍA Y NUTRICIÓN VEGETAL 81

SOIL-PLANT INTERACTIONS IN VOLCANIC SOILS 83

María de La Luz Mora G.
ABIOTIC STRESS EFFECTS ON PHYSIOLOGICAL AND BIOCHEMICAL TRAITS IN 86

BLUEBERRY (Vaccinium corymbosum L.) CULTIVARS
Miren Alberdi, Marjorie Reyes-Díaz, Alejandra Ribera, María de la Luz Mora
CARBOXYLATION ACTIVITY OF RUBISCO IN Arabidopsis thaliana UNDER CD AND CU 89

TOXICITY - ROLE OF CARBONIC ANHYDRASE AND RUBISCO ACTIVASE IN
ADAPTATION TO STRESS CONDITIONS
A. Siedlecka, O. Moskvin, G. Samuelsson, Z. Krupa, A.G. Ivanov
BIOACCUMULATION AND BIOTRANSFORMATION OF HEAVY METAL(LOID)S IN 90
RELATION TO BIOREMEDIATION: USING A CASE STUDY INVOLVING BACTERIAL-
INDUCED LEAD IMMOBILIZATION.
Jinhee Park, Nanthi Bolan and Ravi Naidu
FACTORS AFFECTING THE MOBILITY AND BIOAVAILABILITY OF (MICRO) NUTRIENTS, 93

HEAVY METALS AND METALLOIDS IN SOIL ENVIRONMENTS
A. Violante
MULTICOMPONENT ADSORPTION OF CU AND ZN ON ALLOPHANIC CLAY 99

Jara A, Pezo C and Mora M.L
NUTRIENT DYNAMIC IN RESPONSE TO NITROGEN AND PHOSPHOROUS FERTILIZATION 100

IN A PERENNIAL RYEGRASS PASTURE OF SOUTHERN CHILE
A. A. Jara, R., Demanet, E. Stohmann, M. González, E. Cantero and M. L. Mora
ANTIOXIDANT ACTIVITY AND TOTAL PHENOLIC AND ANTHOCYANINS CONTENTS IN 101

HIGHBUSH BLUEBERRY FRUITS AT DIFFERENT MATURITY STAGES
Ribera A, Reyes M, Alberdi M, Verdugo M, Mora M.L
EFFECT OF NITROGEN FERTILIZATION ON SOIL PHOSPHOROUS DYNAMICS AND 102

LOLIUM PERENNE L. PRODUCTION AND NUTRITION UNDER GREENHOUSE CONDITIONS
A.E. Ribera, A.A. Jara, E. Stohmann, M. González, M. Reyes-Díaz, M. Alberdi and M.L. Mora.
IDENTIFICACIÓN DE ARCILLAS EN SUELOS DE LA REGIÓN DE ANTOFAGASTA Y SU 103

USO COMO ADSORBENTES DE METALES PESADOS
Fariña C, Miranda N, Agüero P, Alarcón N and Fuentes B
IRRIGACIÓN DE UN SUELO SALINO CON AGUA RESIDUAL PROVENIENTE DE LA 104

INDUSTRIA CERVECERA: EFECTO SOBRE LA CONDUCTIVIDAD Y EL PH
Miranda N, Fariña C, Agüero P, Alarcón N and Fuentes B
DISPONIBILIDAD Y MOVILIDAD DE NITRÓGENO EN UN ANDISOL Y SU INFLUENCIA 105

SOBRE LA PRODUCCIÓN DE Lolium perenne
Paredes C., y Mora M.L.2
DIFFERENTIAL ANALYSIS OF GENE EXPRESSION BY CDNA-AFLP IN HIGHBUSH 106

BLUEBERRY (Vaccinium corymbosum L.) UNDER ALUMINUM TOXICITY
Inostroza C, Aquea F, Reyes M, Alberdi M, Mora M.L, and Arce-Johnson P
EFFECT OF AL/CA RELATION ON PHOTOSYNTHETIC CAPACITY OF HIGHBUSH 107

BLUEBERRY CULTIVARS (Vaccinium corymbosum L.)
Meriño C, Reyes M, Alberdi M and Mora M.L
IMPROVING PROTEIN EXTRACTION METHODS FROM DIFFERENT PLANT ORGANS OF 108

AGRICULTURAL CROPS FOR ENHANCED PROTEOMIC ANALYSIS
Wulff C, Caruso I, Greco B, Ribera A.E and Mora M.L
IDENTIFICATION OF PROTEINS INVOLVED IN THE ADAPTATION RESPONSES TO 109

ALUMINUM STRESS IN ROOT TISSUES OF RYEGRASS GROWN IN DIFFERENT
CONDITIONS OF SULFUR FERTILISATION
Wulff-Zottele C, Stohmann E, Mora M.L..
GENETIC AND PHYSIOLOCICAL CHARACTERIZATION OF THE DROUGHT RESPONSE IN 110
TOMATO
Greco B., De Stefano R., Reyes-Diaz M., Wulff-Zotelle C., Di Matteo A., Mora Gil M. L. and Barone
A.
GREENHOUSE GASES EMISSIONS FROM AGRICULTURAL SOILS AND MITIGATION 111

STRATEGY
Muñoz C, Paulino L, Stolpe N, Ovalle C, Suescún F and Zagal E
THE EFFECT OF AL/CA RATIO ON NITROGEN, MEAN RELATIVE GROWTH RATE AND 112
RUBISCO CONTENT IN VACCINIUM CORYMBOSUM L.
Alarcón E, Reyes M and Alberdi M
SYNTHETIC NANOPARTICLES LIKE ALLOPHANE: POTENTIAL USE AS CATALYST 113

HETEROGENEOUS IN THE FENTON LIKE REACTION FOR ORGANIC POLLUTANT
REMOVAL
Garrido E.G and Mora M.L
COSTS OF CONSTRUCTION AND CONVERSION EFFICIENCY OF DRY MATTER OF 114

BLUEBERRY (VACCINIUM CORYMBOSUM L.) ORGANS
Jorquera E and Franck N
METABOLIC AND MORPHO-ANATOMICAL ANALYSIS IN SYNTHETIC 115

AUTOTETRAPLOIDS OF WILD POTATO SPECIES
Caruso I, Aronne G, Aversano R, Dal Piaz F, De Micco V, Fasano C, Garramone R, Lepore L,
Scognamiglio N and Carputo D
SOIL WETTABILITY: A COMPARISON OF METHODS FOR ITS EVALUATION 116

Ramirez J
NATURAL AND NANOPARTICLES FROM AN ANDISOL AND ITS APPLICATION IN 117

ATRAZINE CONTROLLED RELEASE FORMULATIONS
Cea M and Mora M.L
STRUCTURAL CHARACTERIZATION OF NATURAL NANOMATERIALS: POTENTIAL USE 118

TO INCREASE THE PHOSPHORUS MINERALIZATION
Calabi M, Jara A, Bendall J, Welland M and Mora M.L
EFFECT OF GYPSUM APPLICATION ON PHOTOSYNTHETIC PERFORMANCE OF Vaccinium 119

corymbosum L.
Reyes M, Meriño C, Mora M.L and Alberdi M
AN ASSESSMENT OF THE SE BENEFITS FOR PASTURES SPECIES GROWING IN 120

SOUTHERN CHILE
Cartes P, Gianfreda L, Jara A, Shene C, Pinilla L, Rosas A, Mora M.L
EFECT OF CLIMATE VARIABILITY ON CROP SYSTEM 121

Orrego R and Ávila A
MANGANESE TOXICITY IN BLUEBERRY (Vaccinium corymbosum L.) CULTIVARS : ITS 122

EFFECTS ON SOME PHOTOCHEMICAL AND BIOCHEMICAL PARAMETERS OF LEAVES
Millaleo R, Reyes M, Mora M.L and Alberdi M
POTENTIAL CARBON MINERALIZATION IN VOLCANIC SUBSOIL OF A PRISTINE 123

TEMPERATE RAIN FOREST OF SOUTHERN CHILE
Neculman R, Panichini M, Matus F and Godoy R
EFECTO DE TOXICIDAD POR MANGANESO Y RADIACIÓN UV-B SOBRE LA EFICIENCIA 124
FOTOQUÍMICA DEL FOTOSISTEMA II (PSII) DEL ARÁNDANO ALTO (Vaccinium corymbosum
L.)
Rojas Y, Reyes M, Alberdi M and Mora M.L
EFFECTS OF DIFFERENT AGRICULTURAL MANAGEMENT ON SOIL QUALITY IN AN 125

IMPORTANT AGRICULTURAL AREA OF SOUTHERN ITALY
Scotti R., Sultana S., Scelza R., Marzaioli R., D’Ascoli R., Rao M.A.
CHARACTERIZATION OF GARDERMOEN AIRPORT SOIL CONTAMINATED BY DE-ICING 126

COMPOUNDS AND POSSIBLE BIOREMEDIATION STRATEGY
Scotti R., Rao M.A., Colarieti M.L., Toscano G., Cavalca L., Andreoni V., Gianfreda L., Greco G. Jr.
RESPUESTA FOTOPROTECTORA DEL APARATO FOTOSINTÉTICO DE ALGAS PARDAS 127

FRENTE AL INCREMENTO DE LA RADIACIÓN UV Y TEMPERATURA.
Edgardo Cruces, Iván Gomez
129
SIMPOSIO 5. BIOTECNOLOGÍA AMBIENTAL Y MANEJO DE CONTAMINANTES
WHITE-ROT FUNGI FOR THE BIOREMEDIATION OF CONTAMINETD ENVIRONMENTS 131

WITH RECALCITRANT ORGANIC COMPOUNDS
M. C. Diez
NITROGEN REMOVAL IN HIGH LOADED INDUSTRIAL WASTEWATERS 133

C. Bornhardt, C. Antileo
MOBILITY AND TRANSFORMATION OF PESTICIDES IN SOIL: AGRONOMICAL AND 138

ENVIRONMENTAL IMPLICATIONS
Palma, G., Briceño, G., Aguilera, P. Candia, O. Mora, M.L. Demanet. R.
CONTRIBUTION OF SAPROPHYTIC AND MYCORRHIZAL FUNGI TO THE FORESTRY AND 141

AGRICULTURE PLANTS SPECIES BY AMENDMENT WITH SEWAGE SLUDGE IN
GREENHOUSE TRIALS.
Arriagada, C, Lagos, C, Cornejo, P, Pacheco P.
USE OF INDUSTRIAL WASTE FROM PULP MILLS IN AGRICULTURE 143

Gallardo F.
ROLE OF ENZYMES IN THE REMEDIATION OF POLLUTED ENVIRONMENTS 144

Liliana Gianfreda
145
PRESENTACIONES PANELES
EFFECT OF RHIZOSPHERE ON THE DEGRADATION OF PENTACHLOROPHENOL BY 147

BIOAUGMENTATION OF Anthracopyllum discolor
Urrutia C, Paredes C, Rubilar O and Diez M.C
BIOESTIMULATION, BIOAUGMENTATION AND ENZYMATIC BIOREMEDIATION IN 148

SPIKED SOIL WITH POLYCYCLIC AROMATIC HYDROCARBONS
Acevedo F and Diez M.C
USE OF BIOBED SYSTEM FOR CHLORPYRIFOS DEGRADATION 149

Tortella G, Rubilar O, Cea M, Avendaño M, Fernandez S and Diez M.C
EFFECT OF DAIRY MANURE AMENDMENTS ON ATRAZINE AND DIMETHENAMID 150
LEACHING IN A SOUTHERN CHILEAN ANDISOL
Candia C, Briceño G, Aguilera P, Demanet R, Mora M.L, and Palma G
EFFECT OF INCUBATION TIME OF A BIOBED ON CHLORPYRIFOS DEGRADATION 151

Fernández S, Tortella G and Diez M.C
ENZYME ACTIVITIES AND DYE DEGRADATION BY COMPLEX PELLETS OF WHITE-ROT 152

FUNGUS Trametes versicolor
Elgueta S, Cuevas R, Rubilar O and Diez M.C
PRODUCTION OF AN ECOFERTILIZER USING BIOCHAR AS RAW MATERIAL 153

González M.E, Diez M.C and Navia R
INFLUENCE OF SOYA LECITHIN ON THE ENZYMATIC SYSTEM OF WHITE-ROT FUNGI 154

Anthracophyllum discolor
Bustamante M, González M.E, Cartes A and Diez M.C
DEGRADACIÓN DE METHYL ORANGE UTILIZANDO NANOPARTÍCULAS DE FE- 155

IMOGOLITA COMO UN CATALIZADOR HETEROGÉNEO DEL TIPO FENTON
Arancibia N, Escudey M, Manquián K and Molina M
PELLETIZATION OF WHITE-ROT FUNGUS Anthracophyllum discolor FOR LIGNIN 156

DEGRADATION IN AIRLIFT REACTOR
Rubilar O, Tortella G, Elgueta S, Gianfreda L and Diez M.C
CARACTERIZACIÓN DEL POTENCIAL CONTAMINANTE DE PURINES GENERADOS EN 157

PLANTELES BOVINOS
Morales E.
EFFECT OF DAIRY MANURE RATE AND THE STABILIZATION TIME OF AMENDED SOILS 158
ON ATRAZINE DEGRADATION
Paula Aguilera, Gabriela Briceño, Maribel Candia, Maria de la Luz Mora, Rolando Demanet, Graciela
Palma
SIMPOSIO 6. VALORIZACIÓN DE RESIDUOS, BIOENERGÍA Y ENERGÍAS 159

RENOVABLES
WASTE TO ENERGY, BIOENERGY & RENEWABLE ENERGY 161

Rodrigo Navia
ADVANCES IN BIOTECHNOLOGY PROCESS TO PRODUCE BIODIESEL 162

Ciudad, G., Azócar, L., Muñoz, R., Reyes. I., Wick, Y. L Heipieper, H., and Navia, R.
GLYCEROL AS RAW MATERIAL FOR POLYESTER MANUFACTURE AND THEIR USE IN 163
BIODIESEL REFINING
Toro C., Hidalgo P., Sanhueza S., Catril C., Navia R.
MICROALGAE BIOFUELS 164

Rivas M., Bahamondes C., Bazaes J., González L., Marticorena P., Sepúlveda C., Silva F., Varas R.,
Vargas P., Riquelme C.
TURBINAS EÓLICAS DE BAJA POTENCIA: UNA APUESTA PARA APOYAR LA 165
INDEPENDENCIA ENERGÉTICA EN SECTORES PRODUCTIVOS AISLADOS
R. Hunter A., B. Pavez L.
IMPROVING FATTY ACID METHYL ESTERS PRODUCTION YIELD IN A LIPASE- 169

CATALYZED PROCESS BY USING WASTE FRYING OILS AS FEEDSTOCK
Azócar L, Ciudad G, Heipieper H, Muñoz R and Navia R
PETROLEUM COKE FLY ASHES AND THEIR POTENTIAL REUSE 170

González A, Moreno N and Navia R
EVALUACIÓN DE LA ACTIVIDAD METANOGÉNICA EN LODOS ANAEROBIOS POR 171

MÉTODO MANOMÉTRICO
Valdés F and Jeison D
CONFERENCIA DE CLAUSURA/CLOSING LECTURE 173
UTILIZATION OF COAL COMBUSTION PRODUCTS (CCPS) FOR ENVIRONMENTAL

REMEDIATION
Nanthi Bolan, Ravi Naidu and Balaji Seshadri
PROGRAMA GENERAL
Lunes 23, Mañana
8:30-9:30. Inscripción de participantes y entrega de materiales. Colocación de paneles salas 1 y 2.

9:30-9:45. Palabras de Bienvenida, Autoridades Universitarias.
9:45-10:00. Palabras de recepción y presentación del evento. Dra. María de la Luz Mora, Directora
Doctorado y Magíster en Ciencias de Recursos Naturales y Directora Núcleo Científico y Tecnológico en
Biorecursos, BIOREN-UFRO.
Simposio 1. Avances en Biotecnología Agroalimentaria (Moderadores: Dra. Carolina Shene; Dra. Mónica
Rubilar).
10:00-10:30. Dra. Carolina Shene, Universidad de La Frontera, Chile: “Producción microbiana de lípidos”.
10:30-11:00. Dra. Mónica Rubilar, Universidad de La Frontera, Chile: “Actividad biológica de compuestos
polifenólicos y sus aplicaciones en la industria de alimentos”.
11:00-11:20. Coffe-break
11:20-12:00. Dr. Roberto Quevedo, Universidad de Los Lagos, Chile: “Determinación de la vida útil de
alimentos aplicando visión computacional: Un sistema de registro en líneas de proceso para la
trazabilidad”.
12:00-12:40. Conferencia. Dr. Rodrigo Borquez, Departamento de Ingeniería Química, Universidad de
Concepción : “Innovaciones Tecnológicas en Bioprocesos”.
Almuerzo: Gran Hotel Pucón
Lunes 23, Tarde
Simposio 2. Ecología Química como Herramienta Biotecnológica (Moderadores: Dr. Andrés Quiroz; Dr.
Christian Fuentes).
14:40-14:50. Dr. Andrés Quiroz, Universidad de La Frontera, Chile: “Chemical Ecology: An
interdisciplinary science”.
14:50-15:30. Conferencia. Dr. Rufus Isaacs. Michigan State University, USA: “Integrated pest management
in berries”.
15:30-15:50. Dr. Claudio Ramírez. Universidad de Talca: “Biotechnology of plant protection”.
15:50-16:10. Dr. Christian Figueroa. Universidad Austral de Chile: “Ecological and evolutionary genomics
of herbivore insects”.
16:10-16:30. Dr. Eduardo Fuentes. Universidad de Talca, Chile: “Insecticide resistance of fruit pests”.
16:30-16:50. Dr. Alejandro Urzúa. Universidad de Santiago, Chile: “Pharmacologycal and physiological
properties of extracts and pure compounds used in Popular Medicine”.
16:50-17:40. Sesión de Paneles de Alumnos y Postgraduados del Programa de Doctorado en Ciencias de

Recursos Naturales. Salas 1 y 2. Coordinadores: Dra. Carolina Shene. Dr. Andrés Quiroz, Dr. Christian
Figueroa, Dr. Claudio Ramírez, Mg. Fernando Perich.
17:40-18:10. Se discutirán y evaluarán las presentaciones de los Alumnos y Postgraduados del Programa de
Doctorado en sus respectivas líneas de investigación, asociadas a los simposios 1, 2 y 3.
i
Martes 24, Mañana
Simposio 3. Microbiología y Rizosfera (Moderadores Dr. Milko Jorquera; Dr. Fernando Borie).
9:00-9:30. Dr. Fernando Borie, Universidad de La Frontera, Chile: “Micorrizas arbusculares en ecosistemas
agrícolas y forestales de Chile”
9:30-9:50. Dr. Pablo Cornejo, Universidad de La Frontera, Chile: “Hongos micorrícico arbusculares y su rol
en la estabilización de ecosistemas afectados por actividades mineras”
9:50-10:20. Dra. Concepción Azcón-Aguilar, Estación Experimental del Zaidin (CSIC), España: “Hongos
formadores de micorrizas arbusculares en biocontrol de patógenos”.
10:20-10:50. Dra. Rosario Azcón, Estación Experimental del Zaidin (CSIC), España: “Efectividad de
residuos agroindustriales biotrasformados y su interacción con inoculantes microbianos para su uso en
revegetación”.
10:50-11:10. Coffe-break.
11:10-11:40. Dr. Roberto Godoy, Universidad Austral de Chile: “Micorrizas en bosques templados de la
zona Centro-Sur de Chile”.
11:40-12:00. Mg. María Teresa Fernández, ICIDCA, Cuba: “Construcción de vectores para mejorar la
utilización de fosfato y fitato por rizobacteria”.
12:00-12:20. Dr. Milko Jorquera, Universidad de La Frontera, Chile: “Bacterias degradadoras de fitato y su
potencial biotecnológico en agricultura”.
12:20-13:00. Conferencia: Dr. David Crowley, University California, USA: “The ecology of bacterial
communities in the plant rhizosphere”.
Martes 24, Tarde
Simposio 4. Suelos, Fisiología y Nutrición Vegetal (Moderadores: Dra. María de la Luz Mora; Dra. Miren
Alberdi).
14:45-15:00. Dra. María de la Luz Mora, Universidad de La Frontera, Chile:”Interacción suelo-planta en
suelos volcánicos”.
15:00-15:20. Dra. Miren Alberdi, Universidad de La Frontera, Chile: “Abiotic stress effects on physiological
and biochemical traits of Blueberry (Vaccinium corymbosum L.) cultivars”.
15:20-15:50. Dr. Alexander Ivanov, University of Western Ontario, Canada: “Carboxylation activity of
Rubisco in Arabidopsis thaliana under Cd and Cu toxicity - role of carbonic anhydrase and Rubisco activase
in adaptation to stress”.
15:50-16:20. Dr. Nanthi Bolan. University of South Australia: “Bioaccumulation and biotransformation of
heavy metal(oid)s in relation to bioremediation - using a case studies involving bacterial induced lead
immobilization”.
16:20-16:50. Conferencia. Dr. Antonio Violante, Università Federico II, Italy: “Factors Affecting the
Mobility and Bioavailability of (Micro)Nutrients, Heavy Metals and Metalloids in Soil Environments”.
16:50-17:40. Sesión de Paneles de Alumnos y Postgraduados del Programa de Doctorado en Ciencias de

Recursos Naturales. Salas 1 y 2. Coordinadores: Dra. Liliana Gianfreda, Dra. Rosario Azcón, Dr. David
Crowley, Dr. Fernando Borie, Dr. Nanthi Bolan.
17:40-18:10. Se discutirán y evaluarán las presentaciones de los Alumnos y Postgraduados del Programa de
Doctorado en sus respectivas líneas de investigación, asociadas a los Simposios 4, 5 y 6.
ii
Miércoles 25, Mañana
Simposio 5. Biotecnología Ambiental y Manejo de Contaminantes (moderadores Dra. María Cristina Diez;
Dr. Cristian Bornhardt).
9:00-9:30. Dra. María Cristina Diez, Universidad de La Frontera, Chile:“Hongos de pudrición blanca en la
biorremediación de ambientes contaminados con compuestos orgánicos recalcitrantes”.
9:30-10:00. Dr. Cristian Bornahrdt, Universidad de La Frontera, Chile:”Eliminación de nitrógeno en aguas
residuales de alta carga”.
10:00-10:30. Dra. Graciela Palma, Universidad de La Frontera, Chile: “Movilidad y transformación de
pesticidas en suelo: Implicancias agronómicas y ambientales”.
11:20-11:50. Dr. César Arriagada, Universidad de La Frontera, Chile: “Contribuciòn de hongos ligninoliticos
al crecimiento de especies de interés silvoagropecuario”.
11:50-12:20. Mg. Felipe Gallardo, Universidad de La Frontera, Chile: “Uso de residuos industriales
provenientes de plantas de celulosa en la agricultura”.
12:20-13:00. Conferencia: Dra. Liliana Gianfreda, Università Federico II, Italy: “El rol de las enzimas en la
remediación de la contaminación ambiental”.
Miércoles 25, Tarde
Simposio 6. Valorización de Residuos, Bioenergía y Energías Renovables (Moderador: Dr. Rodrigo Navia).
14:45-15:00. Dr. Rodrigo Navia. Universidad de La Frontera, Chile: “Valorización de Residuos, Bioenergía y
Energías Renovables”.
15:00-15:20. Dr. Gustavo Ciudad. Universidad de La Frontera, Chile: “Avances en procesos biotecnológicos
para la producción de biodiesel”.
15:20-15:40. Dr. Claudio Toro. Universidad de La Frontera, Chile: “Glicerol como materia prima para la
elaboración de poliésteres y su uso en la refinación de biodiesel”.
16:40-16:10. Dra. Mariella Rivas. Universidad de Antofagasta, Chile: “Microalgas como fuente de energía
renovable”.
16:10-16:30. Dr. David Jeison. Universidad de La Frontera, Chile: “Biogás como fuente de energía
renovable”.
16:30-16:50. Dr. Renato Hunter. Universidad de La Frontera, Chile: “Turbinas eólicas de baja potencia: una
apuesta para apoyar la independencia energética en sectores productivos aislados”.
16:50-17:00. Ronda de consultas.
Finalización del evento.
17:20-17:50. Conferencia de Clausura. Dr. Nanthi Bolan. University of South Australia: “Utilization of
coal combustion products for environmental management”.
17:50-18:00. Consideraciones finales y palabras de clausura. Dra. María de la Luz Mora, Directora
Doctorado y Magíster en Ciencias de Recursos Naturales (UFRO).
iii
iv
1º Workshop y 4º Curso Internacional: Avances en Ciencia y Tecnología de Recursos Naturales
Simposio 1.
Avances en Biotecnología Agroalimentaria
Presentaciones Orales
Moderadores: Dra. Carolina Shene; Dra.Mónica Rubilar.
1. Dra. Carolina Shene, Universidad de La Frontera, Chile: “Producción

microbiana de lípidos”.
2. Dra. Mónica Rubilar, Universidad de La Frontera, Chile: “Actividad biológica

de compuestos polifenólicos y sus aplicaciones en la industria de alimentos”.
3. Dr. Roberto Quevedo, Universidad de Los Lagos, Chile: “Determinación de la

vida útil de alimentos aplicando visión computacional: Un sistema de registro
en líneas de proceso para la trazabilidad”.
4. Rodrigo Bórquez, Departamento de Ingeniería Química, Universidad de

Concepción: “Technological innovation in bioprocess”.
1
2
PRODUCCIÓN MICROBIANA DE LÍPIDOS

C. Shene1,2, A. Leyton, B. Quilodrán3, I. Hinzpeter4, M. Rubilar1,2
1
Departamento de Ingeniería Química. Universidad de La Frontera
2
Unidad de Tecnología y Procesos del Centro de Genómica Nutricional Agro-acuícola.
Universidad de La Frontera
3
Departamento Gobierno y Empresa, Universidad de Los Lagos, casilla 557, Puerto Montt, Chile.
4
Departamento Recursos Naturales y Medio Ambiente, Universidad de Los Lagos, casilla 557,
Puerto Montt, Chile.
Los ácidos grasos poli-insaturados (AGPI) entre los que se encuentran los ácidos araquidónico
(ARA, C20:4n-6), eicosapentaenoico (EPA, C20:5n-3), docosapentaenoico (DPA, C22:5n-3) y
docosaexaenoico (DHA, C22:6n-3) son componentes de membranas y algunos de ellos están
involucrados en el desarrollo neuronal y de la retina (Uauy et al., 2001). Existe abundante
evidencia clínica que demuestra los efectos benéficos de los AGPI omega-3 en el tratamiento de
diversas enfermedades (coronarias, hipertensión, psoriasis, agresión/depresión, desórdenes
inflamatorios y del sistema autoinmune, y cáncer).
La principal fuente de AGPI son los aceites de pescado, recurso que es utilizado principalmente por
la industria acuícola. Debido a algunas de sus características (olor y sabor, contaminación metales
pesados, dioxinas y el efecto de los procesos térmicos asociados a la eliminación de aromas) los
aceites de pescado no serían un producto adecuado para consumo humano, lo que sumado al
estancamiento de los niveles de producción ha motivado la búsqueda de nuevas fuentes de AGPI
omega-3.
Actualmente, la única alternativa corresponde a lípidos microbianos, producidos por un número
reducido de bacterias, hongos y microalgas. Los microorganismos oleaginosos son aquellos que
pueden acumular lípidos en niveles superiores al 20-25% del peso de la biomasa seca. La
acumulación de lípidos ocurre en medios de cultivo deficientes en algún nutriente, generalmente N.
La capacidad de la maquinaria celular para suministrar en forma continua acetil-CoA y NADPH,
moléculas precursoras de síntesis de AG, explicaría la acumulación de lípidos en estos
microorganismos (Ratledge, 2004).
Muchas especies de bacterias marinas, especialmente aquellas que viven en ambientes fríos y de
elevada presión, acumulan lípidos con alto contenido de EPA y DHA (Nichols et al., 1993). Poseen
la ventaja de producir solamente un ácido graso, a diferencia de la compleja mezcla en aceites de
pescado y de microalgas; la desventaja radica en las bajas concentraciones de lípidos (Nichols et al.,
2002). Las bacterias productoras de AGPI pertenecen a los géneros Shewanella, Colwelia y
Moritella de la división Proteobacteria. Diversos hongos se han estudiado para la producción de AG
de cadena larga entre los cuales se encuentran aquellos del género Mortierella pertenecientes a la
clase Phycomycetes. Mortierella ramannianna, M. Isabellina y Mucor circinelloides son
productores de ácido γ–linolénico, M. alpina y M. elongata se caracterizan por ser productores de
EPA (Certik & Shimizu, 1999) y la cepa Pythium acanthicum produce DPA (Singh & Ward, 1998).
Especies como Pythium y Mortierella producen AA.
Mayores productividades de AGPI se han obtenido en el cultivo de distintas cepas de
traustoquitridios, microorganismos relativamente abundantes en diversos ambientes marinos. Se han
descrito 5 géneros de traustoquitridios: Schizochytrium, Althornia, Ulkenia, Japonochytrium y
Thraustochytrium. La importancia de estos microroganismos es que un alto porcentaje de los lípidos
corresponde a DHA. La razón por la cual producen DHA no está clara, aun cuando se sabe que el
3
DHA en los fosfolípidos tiene un importante papel en la mantención de la integridad de la

membrana (Stillwell & Wassall, 2003). No obstante, solo el 5% de los lípidos en traustoquitridios se
encuentra en los fosfolípidos mientras que entre un 70 a 98% de los lípidos se encuentra en los
triacilgliceroles en forma de lípidos de reserva (Nakahara et al., 1996; Yaguchi et al., 1997; Ashford
et al., 2000). Las distintas cepas de traustoquitridios producen perfiles específicos de AGPI lo que
permite su clasificación (Yokochi et al., 1997). Los diferentes perfiles de AGPI se atribuyen a
distintas enzimas y/o regulaciones responsables de la biosíntesis de AG.
La información reportada demuestra una enorme variabilidad en los rendimientos de biomasa,
lípidos y DHA dependiendo de las condiciones y composición del medio de cultivo. A nivel
comercial algunos autores reportan concentraciones superiores a los 200 g/L de biomasa con un
contenido de lípidos del 40% (Bailey et al., 2003). La mayor velocidad de crecimiento de algunas
cepas de Schizochytrium permite obtener una mayor productividad (con concentraciones de biomasa
de 200 g/L, DHA de 40-45 g/L en ciclos de 90-100 h) en comparación a los cultivos de
Crypthecodinium sp. (De Swaaf et al. 2003) considerado uno de los mejores productores de DHA.
Los trabajos realizados previamente por Hinzpeter et al., (2009) han permitido caracterizar cepas de
traustoquitridios aislados desde la zona costera de Puerto Montt. De cuarenta y seis cepas que
presentaron características morfológicas descritas para traustoquitridios, sólo 6 cepas produjeron
lípidos en los que se detectó DHA. A través del cultivo la cepa seleccionada como mejor productora
(M12-X1) en medio suplementado con glutamato monosódico fue posible incrementar en un 71% la
concentración de DHA. Desde trabajos similares realizados en la zona de Puerto Saavedra se han
aislado 8 cepas que producen de DHA, la mayoría de ellas pertenecientes al género
Thraustochytrium (Leyton et al., 2009). Quilodran et al., (2009) han evaluado la productividad de
cultivos de cepas nativas (Thraustochytriidae sp. M12-X1 y C41) en medios de cultivo formulados
con sustrato de bajo costos (residuos de la industria de alimentos) alcanzando valores de 55 mg/(L
día) en experimentos en matraces. Tres son las variables claves en la caracterización de la
productividad de aceites microbianos (concentración de biomasa, contenido de lípidos en la biomasa
y contenido del AGPI en los lípidos). De estas tres, la concentración de biomasa ha sido el factor
limitante de los cultivos de las cepas nativas. Mejores resultados [550 mg/(L día)] se han obtenido
con un cepa del género Ulkenia, la que es capaz de producir concentraciones de biomasa de hasta 14
g/L. Trabajos están siendo conducidos para mejorar la productividad del cultivo de cepas nativas en
operaciones de cultivo por lote alimentado en las cuales es posible prolongar la etapa de
acumulación de lípidos.
Agradecimientos: a la Dirección de Investigación de la Universidad de La Frontera a través de los

convenios de Desempeño I, II y GAP.
Referencias
Ashford, A. Barclay, WR. Weaver, CA. Giddings, TH. Zeller, S. 2000. Electron microscopy may
reveal structure of docosahexaenoic acid-rich oil within Schizochytrium sp. Lipids 35: 1377-1387.
Bailey, RB., DiMasi, D., Hansen, JM., Mirrasoul, PJ., Ruecker, CM., Veeder, GT., Kaneko, T.,
Barclay, WR. 2003. Enhanced production of lipids containing polyenoic fatty acid by very high
density cultures of eukaryotic microbes in fermentors. US Patent 6607900
Certik, M., Shimizu, S. 1999. Biosynthesis and regulation of microbial polyunsaturated fatty acid
production. J. Biosci Bioeng. 87: 1-14.
4
De Swaaf, ME., Sijtsma, L., Pronk, JT. 2003. High-cell-density fed-batch cultivation of the
docosahexaenoic acid producing marine alga Crypthecodinium cohnii. Biotechnol Bioeng 81:666–
672.
Hinzpeter, I. 2008. Identificación de microorganismos del orden thraustochytriales provenientes de
la zona costera de Puerto Montt, Chile y determinación de la capacidad de producción de ácido
docosahexaenoico. Tesis para optar al grado de Doctor en Ciencias de Recursos Naturales.
Universidad de La Frontera.
Leyton A., Rubilar M., Shene C. Producción de ácidos grasos poli-insaturados de cadena larga por
microorganismos marinos nativos. XV Congreso Latinoamericano de Nutrición. Santiago de Chile,
15 y 19 de Noviembre, 2009
Nakahara, T. Yokochi, T. Higashihara, T. Tanaka, S. Yaguchi, T. Honda, D. 1996. Production of
docosahexaenoic and docosapentaenoic acids by Schizochytrium sp. isolated from Yap Islands. J
Am Oil Chem Soc 73: 1421-1426.
Nichols, DS. Nichols, PD. McMeekin, TA. 1993. Polyunsaturated fatty-acids in antarctic bacteria.
Antarctic Science 5: 149-160.
Quilodrán, B. Hinzpeter, I., Quiroz, A., C. Shene. 2009. Evaluation of liquid residues from beer and
potato processing for the production of docosahexaenoic acid (C22:6n-3, DHA) by native
thraustochytrid strains. World J Microbiol Biotechnol. DOI 10.1007/s11274-009-0115-2.
Ratledge, C. 2004. Fatty acid biosynthesis in microorganisms being used for single cell oil
production. Biochimie 86: 807-815.
Singh, A., Ward, OP. 1998. Docosapentaenoic acid (C22:5, v-3) production by Pythium acanthicum.
J Ind Microbiol Biotechnol 20: 187–191.
Stillwell, W. Wassall, SR. 2003. Docosahexaenoic acid: membrane properties of a unique fatty acid.
Chem Physics of Lipids 126: 1-27.
Uauy, R. Hoffman, DR. Peirano, P. Birch, DG. Birch, EE. 2001. Essential fatty acids in visual and
brain development. Lipids 36: 885-895.
Yaguchi, T. Tanaka, S. Yokochi, T. Nakahara, T. Higashihara, T. 1997. Production of high yields of
docosahexaenoic acid by Schizochytrium sp. strain SR21. J Am Oil Chem Soc 74: 1431-1434.
5
ACTIVIDAD BIOLÓGICA DE COMPUESTOS POLIFENÓLICOS Y SUS

APLICACIONES EN LA INDUSTRIA DE ALIMENTOS
M. Rubilar1,2 , C. Gutiérrez2, C. Jara1, Villarroel, M. 1,2, C. Shene1,2
1
Departamento de Ingeniería Química. Universidad de La Frontera
2
Unidad de Tecnología y Procesos del Centro de Genómica Nutricional Agro-acuícola. Universidad
de La Frontera
Los compuestos fenólicos forman un grupo heterogéneo de fitoquímicos biológicamente activos.

Estos compuestos se caracterizan por poseer en su estructura al menos un anillo aromático unido a
uno o varios grupos hidroxilo. Esta disposición química es especialmente propicia para la pérdida de
los átomos de hidrógeno presentes en el grupo hidroxilo, que actúa como un excelente capturador de
electrones o radicales libres de otras especies. Sin embargo, la actividad antirradicalaria no es la
única vía antioxidante que poseen los polifenoles. Estos pueden actuar además quelando iones
metálicos, inhibiendo la oxidación lipídica y enzimas (Halliwell et al., 1995).
Los radicales libres juegan un rol principal en el proceso oxidativo que ocurre en el cuerpo humano
y en los alimentos con alto contenido lipídico. Aproximadamente un 2% del oxígeno consumido por
un organismo normal va a la formación de especies reactivas de oxígeno (ROS), de las cuales varias
son radicales libres (Chance et al, 1979). Estos se generan como subproductos del metabolismo
normal. También se producen cuando un organismo está expuesto a situaciones de enfermedad,
estrés ambiental, radiación ionizante, drogas con propiedades oxidantes o xenobióticos. Cuando la
generación de ROS sobrepasa las numerosas barreras de defensa antioxidantes del organismo, se
produce un aumento en el daño por lesión química de las estructuras biológicas y a este proceso se
le denomina estrés oxidativo (Torres et al, 2002). El cual es en definitiva un desbalance entre la
producción de radicales libres y las defensas antioxidantes y es el causante de una variedad de
condiciones patológicas que incluyen disfunciones cardiovasculares, inflamaciones,
arterioesclerosis, diabetes, carcinogénesis y enfermedades neurodegenerativas (Manian et al, 2007).
Es por esta razón que el cuerpo humano necesita la incorporación constante de antioxidantes
exógenos a través de la dieta para mantener un equilibrio entre antioxidantes y ROS
(Anagnostopoulou et al, 2006).
Es así como un gran número de estudios demuestran que una dieta rica en frutas y verduras tiene un
efecto preventivo sobre el cáncer y otras enfermedades debido a su alto contenido en sustancias
antioxidantes tales como flavonoides y compuestos fenólicos, entre otros. Sin embargo, las
tendencias actuales de alimentación en el mundo consideran mayoritariamente alimentos
procesados, con altas dosis de azúcar, grasas saturadas y sal. Este cambio en la dieta ha propiciado
un aumento de las enfermedades crónicas no transmisibles que hoy constituyen la primera causa de
muerte en países desarrollados o en vías de serlo. Frente a este problema la industria de los
alimentos ha desarrollado los alimentos funcionales, que se definen como alimentos que pueden
proporcionar beneficios para la salud más allá de la nutrición básica, es decir, alimentos similares en
apariencia a los alimentos convencionales que son consumidos como parte de una dieta normal, pero
que se han modificado para ayudar en funciones fisiológicas más allá del suministro de los
requerimientos de un nutriente (Bech-Larsen & Grunert, 2003). En este contexto, los compuestos
polifenólicos, así como otros compuestos bioactivos, pueden ser empleados en la industria de
alimentos funcionales.
Los compuestos polifenólicos además presentan efectos inhibitorios sobre numerosas enzimas, entre
las que se destacan α-amilasa y α-glucosidasa. Estas enzimas se encuentran presentes en el proceso
6
de digestión de carbohidratos que realiza el cuerpo humano en el tracto digestivo. La enzima α-

amilasa actúa sobre polisacáridos como el almidón, específicamente hidroliza los enlaces internos α-
1,4-glicosídicos produciendo glucosa, maltosa o dextrinas. Por su parte la enzima α-glucosidasa,
cataliza el paso final de la absorción de la glucosa al intestino. Por tanto, inhibidores naturales de
estas enzimas, como los polifenoles, podrían ofrecer un atractivo enfoque terapéutico para el
tratamiento de hiperglucemia postprandial, contribuyendo al tratamiento de diabetes tipo II
(Apostolidis et al, 2006). McDougall et al. (2005) concluyen que los polifenoles provenientes de
extractos de plantas pueden causar efectos parecidos a la insulina en el sentido de reducir los niveles
de glucosa en la sangre, que se producen después del consumo de alimentos. Ciertos polifenoles
pueden inducir directamente la secreción de insulina por las células pancreáticas o bien como
inhibidores de enzimas α-glucosidasa y α-amilasa.
Hasta ahora se han realizado estudios in vitro sobre potenciales inhibidores de α-glucosidasa
empleando compuestos presentes en alimentos y plantas como el arándano, pimienta, extractos de
soja, orégano, entre otros (Apostolidis et al., 2006; Subramanian et al., 2008, Ali et al., 2006,
Schafer & Hogger, 2007). Se han realizado estudios en matrices vegetales regionales tales como
frutos, hojas, tallos de murta y maqui. Los resultados muestran que el extracto crudo de hoja de
maqui, además de su importante contenido polifenólico y actividad antioxidante, presenta un alto
efecto inhibitorio de enzimas, pudiendo asegurar un mínimo de efectos secundarios en terapia de
hiperglicemia postprandial. Luego, el extracto crudo de hoja de maqui puede ser una alternativa para
el tratamiento de diabetes mellitus tipo II (Poo, 2008, Jara, 2009).
En plantas, los compuestos polifenólicos actúan como mecanismos de defensa frente a
enfermedades. Se ha informado que algunos compuestos fenólicos tienen actividad antimicrobiana
frente a un amplio espectro de microorganismos. Entre los distintos usos posibles, su aplicación en
la industria de alimentos estaría basada en la necesidad de reemplazar aditivos sintéticos, los que
actualmente están siendo cuestionados por sus efectos en la salud del hombre.
En relación a la actividad antimicrobiana de plantas nativas, recientemente se ha informado que los
extractos crudos de hojas de murta son más efectivos que los de bayas debido al mayor contenido de
compuestos polifenólicos. A su vez, estos estudios han mostrado que los extractos de hoja inhiben el
crecimiento de organismos patógenos tales como Klebsiella pneumoniae ATCC 13883, Pseudomona
aeruginosa ATCC 27853 y Staphylococcus aureus ATCC 25923 y no muestran actividad frente a
bacterias lácticas ni frente a hongos (Shene et al, 2009). Entre los microorganismos patógenos
sensibles a los extractos de murta, S. aureus es de importancia en temas alimentarios puesto que esta
bacteria puede llegar fácilmente a los alimentos a través de los manipuladores. Al emplear técnicas
de separación como extracción líquido-líquido de los extractos crudos de hoja de murta se ha
favorecido la actividad antimicrobiana de extractos solubles en la fracción orgánico acuosa. Se ha
demostrado así, un mayor poder inhibitorio frente a S. aureus superando la inhibición del extracto
crudo (Rubilar et al, 2009).
En el trabajo desarrollado con matrices vegetales provenientes de plantas nativas de Chile se ha
buscado establecer el potencial bioactivo de estas especies, puesto que esta información entrega una
herramienta importante para definir las diferentes áreas de aplicación a la industria de alimentos,
sean estas como aditivos antioxidantes, antimicrobianos o hiploglicemiantes que pueden ser
empleados en la formulación de alimentos funcionales.
Finalmente para el futuro la investigación en el área de alimentos está apoyada en la convicción de
que es posible hacer un mejor uso de los recursos de la naturaleza para diseñar una alimentación
sana.
7
Agradecimientos: a la Dirección de Investigación de la Universidad de La Frontera a través de los

convenios de Desempeño I, II y GAP 2009.
Referencias
Anagnostopoulou, M. A., Kefalas, P., Papageorgiou, V. P., Assimepoulou, A. N., & Boskou, D.
2006. Radical scavenging activity of various extracts and fractions of sweet orange peel (Citrus
sinensis). Food Chemistry, 94, 19–25.
Ali, H., Houghton, P., Soumyanath, A. 2006. Amylase inhibitory activity of some Malaysian plants
used to treat diabetes; with particular reference to Phyllanthus amarus. J. Ethnopharm., 107:449-455.
Apostolidis, E., Kwon, Y., Shetty, K. 2006. Potential of cranberry-based herbal synergies for
diabetes and hypertension management”. Asia Pac. J. Clin. Nutr., 15 (3):433-441.
Bech-Larsen T. & Grunert K.G., 2003. The perceived healthiness of functional foods: A conjoint
study of Danish, Finnish and American consumers' perception of functional foods. Appetite 40(1),9-
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Chance B, Sies H, Boveris A. 1979. Hydroperoxide metabolism in mammalian organisms. Physiol.
Rev. 59:527-605.
Halliwell, B.; Aeschbacht, R.; Loligert, J.; Aruoma, O.I. 1995. The characterization of Antioxidants.
Food Chem. Toxic., 33(7), 601-617
Jara C, Shene C. Villarroel M, Rubilar M. 2008. Extractos de maqui (Aristotelia chilensis) como
antioxidantes e inhibidores de enzimas α-glucosidasa y α-amilasa. XVIII Congreso de la Sociedad
Chilena de Nutrición, bromatología y toxicología, Segunda Reunión de las Sociedades de Nutrición
del Cono Sur y España, Puerto Varas, Chile, 26 al 29 de Noviembre
Manian, S., Manian, R., Anusuya, N., Siddhuraju, P. 2007. The antioxidant activity and free radical
scavenging potential of two different solvent extracts of Camellia Sinensis (L.) O. Kuntz, Ficus
Bengalensis L. and Ficus Racemosa L. Food Chemistry. 107, 1000-1007.
McDougall,G.J., Shpiro, F., Dobson, P., Smith, P., Blake, A., Stewart, D. 2005. Different
polyphenolic components of soft fruits inhibit alpha-amylase and alpha-glucosidase. J. Agric. Food
Chem., 53(7):2760-2766.
Poo Y, Villarroel M, Shene C, Rubilar M. 2008. Capacidad inhibitoria de extractos de murta (Ugni
molinae Turcz.) sobre enzimas a-glucosidasa y a-amilasa. XVIII Congreso de la Sociedad Chilena
de Nutrición, bromatología y toxicología, Segunda Reunión de las Sociedades de Nutrición del
Cono Sur y España, Puerto Varas, Chile, 26 al 29 de Noviembre
Rubilar, Gutiérrez, Villarroel & Shene C, 2009. Influence of separation conditions on antimicrobial
activity of polyphenolic fractions from murta leaves extracts. CyTA Journal of Food. Aceptada
Schäfer, A., Höger, P. 2007. Oligomeric procyanidins of French maritime pine bark extract
(Pycnogenol®) effectively inhibit α-glucosidase. Diab. Res. Clin. Prac., 77:41–46.
Shene, C., Reyes, A., Villarroel, M., Sineiro, J., Pinelo, M., & Rubilar, M. 2009. Plant location and
extraction procedure strongly alter the antimicrobial activity of murta extracts. European Food
Research and Technology, 228 (3), 467 -475.
Subramanian, R., Asmawi, Z., Sadikun, A. 2008. In vitro α-glucosidase and α-amylase enzyme
inhibitory effects of Andrographis paniculata extract and andrographolide. A. Bioch. Pol.,
55(2):391-398.
Torres J. L., Lozano C., Julia L., Sanchez-Baeza, F. J., Anglada M., Centelles J. J. and Cascante M.
2002. Cysteinyl-flavan-3-ol Conjugates from Grape Procyanidins. Antioxidant and Antiproliferative
Properties. Bioorganic & Medicinal Chemistry 10, 2497–2509
8
ADVANCED TECHNOLOGY IN FOOD COLOR:

DESCRIPTION OF THE KINETIC ENZYMATIC BROWNING IN FRUIT SLICES USING
NON-UNIFORM COLOR INFORMATION FROM DIGITAL IMAGES
Roberto Quevedo
a
FITOGEN program, Department of Science and Food Technology, Universidad de Los Lagos, Av.
Fushlocher 1305, Osorno - Chile.*Corresponding author, e-mail: rquevedo@ulagos.cl
1.1.1. Abstract
A novel methodology called Fractal Browning Indicator (FBI) that describes the enzymatic
browning kinetic based in the use of irregular pattern of colors from banana slices images, is
presented. It uses the fractal Fourier texture images value in a selected area, in order to calculate a
fractal dimension (FD), which represents the complexly of the color distribution. In the procedure,
colors from digital images were first transformed to L*a*b* space color using a transformation
function (quadratic model) in order to derivate three color channels, lightness (L*), redness (a*),
and a yellowness (b*). In the results, lightness and yellowness parameters decreased during the
browning kinetic when their respective FD values increased, indicating a major complexity in the
distribution of the colors in select analyzed area during the kinetic. Redness color (a*) not showed
any statistical variation. Empirical power law model was suitable to correlated enzymatic browning
kinetic data also for FBI and for traditional method (when a L* mean was used). However,
enzymatic browning rates using FBI method, were between 8.5 to 35 times higher than rates
calculated with traditional. In general, FBI method appears to register some additional information
during the enzymatic browning kinetic that traditional method can not record.
Keywords: enzymatic browning; banana slices; computer vision systems, Fourier fractal texture
Introduction
Browning of raw fruits is a major problem in the food industry and is believed to be one of the
main causes of quality loss during post-harvest handling and processing. In fact, when fruits are cut,
the cut surface turns brown; it reduces not only the visual quality but also results in undesirable
changes in flavour and loss nutrients due to enzymatic browning (Luo & Barbosa, 1997). Browning
can cause deleterious changes in the appearance and organoleptic properties of the food market,
value, and in some cases, complete exclusion of the food product from certain markets (McEvily,
Iyengar & Otwell, 1992). The control of cut-surface browning is critical to maintaining the quality
and safety of fresh-cut produce.
Traditionally, enzymatic browning has been quantified using browning indicators trough of
biochemical index, for example using the polyphenol oxidase activity (Hosoda, Inoue, Iwahashi,
Sakaue, Tada & Nagata, 2005; Murata, 2001; Osanai, Motomura & Sakurai, 2003; Sannomaru,
Katayama, Kashimura & Kaneko, 1998; Sharon & Kahn, 1979; Waliszewski, Pardio & Ovando,
2007) or physical indicators such the color surface (Elshimi, 1993; Kang, Oh, Go, Kim, Park &
Kim, 2004; Lambrecht, 1995; Lozano de Gonzalez, Barrett, Wrolstad & Durst, 1993; Lozano,
Drudisbiscarri & Ibarzribas, 1994; Lu, Luo, Turner & Feng, 2007; Luo & Barbosa, 1997; Molme,
Buta & Newman, 1999; Shengmin, Yaguang, Ellen Turner & Hao Feng, 2007). In the case the
physical indicators based in color, L*a*b* space or CIELab has been the color model more
9
extensively used; especially the L* value, which has been used as an browning indicator in fruits
(Luo & Barbosa, 1994, 1997; Luo & Patterson, 1994; Luo & Barbosa, 1995; Parpinello, Chinnici,
Versari & Riponi, 2002; Pristijono, Wills & Golding, 2006; Sapers & Douglas, 1987; Sapers &
Ziolkowski, 1987; Severini, Baiano, De Pilli, Romaniello & Derossi, 2003; Soliva-Fortuny, Elez-
Martinez, Sebastian-Caldero & Martin-Belloso, 2002; Valentines, Vilaplana, Torres, Usall &
Larrigaudiere, 2005).
During the description of browning kinetic in bananas, using color information, generally a color
mean value is assumed in the analyzed region. That is, an average of the L*, a* or b* values is
calculate from colorimeters devices or CVS in the analyzed area (Yoruk, Yoruk, Balaban &
Marshall, 2004). But, such it is observed for banana slices during browning, colors development an
non-uniform color patterns and color values are not homogenous in the region. The texture image
analysis has been suggested in order to quantify non-homogenous gray color from images, using the
entire gray intensities information from the image without segments it. This has been possible
because the image texture reflects the changes in intensity pixel values, which might contain
information about the color components and the geometric structure of objects (Du & Sun, 2004;
Gonzales-Barron & Butler, 2008a, b, c; Quevedo, Carlos, Aguilera & Cadoche, 2002; Quevedo,
Mendoza, Aguilera, Chanona & Gutierrez-Lopez, 2008b; Zheng, Sun & Zheng, 2006). Because
texture image analysis can be applied to gray color information, the hypothesis of this research is
that texture image (fractal Fourier texture) also can be used to describe irregularities from a non-
homogenous color channels (L*, a* or b*) during enzymatic browning in banana slices. The objects
of this research are: i) to apply the fractal texture Fourier on banana slices images in order to
describe the enzymatic browning (called here “fractal browning indicator” or FBI) and ii) to
compare the FBI with the traditional indicator browning method based in a color mean value.
Materials and methods
1.1.2.1.1.1 Computer vision system

A computer vision system (CVS) described by Quevedo (2008a; 2008b) was used to capture the
images (1700 x 850 pixels RGB colour). Briefly, samples were illuminated using four fluorescent
lamps TL-D deluxe, natural daylight, 18 W/965 (Philips, Santiago, Chile) with a color temperature
of 6500 K (D65, standard light source commonly used in food research) and a colour-rendering
index (Ra) close to 95%. Lamps (60 cm long) were arranged in the form of a square, 35 cm above
the sample and at an angle of 45º in relation with the sample. Additionally, light diffusers covering
each fluorescent lamp and electronic ballasts ensured a uniform illumination system.
1.1.2.1.1.2 Banana slices samples

Bananas (Musa Cavendish) of uniform size and color were chosen at ripening stage 5 (yellow
peel with green tip) were used in the experiments; they were peeled and cut into eight equal slices (2
cm x 2 cm) using a sharp stainless steel knife. Ten slices from each of twenty five different bananas
respectively, was pooled together and put inside the CVS under the camera and pictures were taken
every three or four minutes during 300 minutes. Experiments were replicated (3 times) in a room
temperature 10ºC. The photographed images were stored in TIFF format. Images corresponded to 2
x 2 cm of the samples having 5 mm of thickness
10
1.1.2.1.1.3 Image texture analysis

A methodology similarly to the development by Quevedo et al., (2008b) for banana images, was
used with three modifications: First, color images were transformed from RGB space color to
L*a*b* space color using the model proposed by Leon et al., (2006) and used recently by Quevedo
et al., (2008a). These transformation functions allowed to obtain three color channels from an
image: the L* channel, the a* channel and the b* channel. Second, plotting pixel coordinates (x, y)
against the corresponding color levels in the z-axis (Quevedo et al., 2002) from each color channel,
were done. Thus, a surfaces intensity from L* channel (SIL*), from a* channel (SIa*), and from b*
channel (SIb*) can be obtained. Fractal Fourier method (Chan, 1995; Quevedo et al., 2008b; Russ,
1994) was used to compute the fractal dimension for a 2-D from each color channel image.
1.1.2.1.2 Model browning kinetic
In order to express a rate browning kinetic, the power law model (Corradini & Peleg, 2004,
2006) was applied to the kinetic data:
C
= exp( k ⋅ t n ) (1)
Ci
Where C/Ci is the conversion fraction of a component with respect to the initial value; k is a rate
parameter and n is a parameter called “shape factor”. When k is positive, the equation describes
exponential isotherm growth and when negative, an exponential isotherm decay. We preferred to use
this model instead a empirical pre-determine zero or first order model, found for some experimental
brown kinetic in fruits. In fact, the equation describes a zero or first empirical order kinetic when n
is 0 or 1, respectively. When FBI method was applied to the browning data, C was substituted by
the respective FD and a positive k was assumed in the Equation 1. When the traditional
methodology was applied, C was substituted by L*, a* or b*, and a negative k was assumed. Model
was adjusted to data using a nonlinear least squares regression. Differences in k values calculated by
the FBI and traditional method, for each color parameter, were established using a t test at 95%
confidence level.
Results and discussion.
In Figure 1, SIL* obtained by one selected area from a banana slice image is shown. The intensity
level and distribution of the L* values are the product of reflection of light when it impinges the
sample surface under the standard setting conditions used here (illuminants, spatial position of
sample respect to the camera, distance the camera to the samples, among other factors) which were
kept constant during all experiments. The plane superimposed in Figure 1 corresponding to the
average of these L* values, and it is traditionally used when enzymatic kinetic is described in food
research. The idea here, is to use a parameter (FDL*) that represent the entire form and distribution
of these L* values over the area, instead to use the average of these L* values. Application the
fractal Fourier texture to this SIL*, a FDL* value equal to 2.50 was obtained; when the average of
these L* values corresponded to 45.5. Highlighting that a FDL* value per se, has not a physical
significance here; it is only a way to represent how the spatial L* values are distributed on the area
analyzed (Russ, 1994). But, it will acquire relevance when the same area will be analyzed during the
kinetic. Similar analysis can be applied for the other color parameters.
In Figure 2, a gallery of three selected images, with their respective SIL*; at 0, 108 and 287
minutes during the enzymatic browning for one banana slices image, is shown. During the
11
enzymatic browning, some regions inside the same area appeared to be more brown than other
regions, such has been noted for banana slices (Molme et al., 1999). It turned SIL* to be more
irregular, showed by an increment of its FDL* value. In other words, when the same area is analyzed
during the kinetic, distribution of L* values on the area appears more heterogeneous, and non-
uniform color patterns emerges on images. This phenomena is not quantified when a average of L*
values is used. With respect to the average of the L* values, it decreased during the kinetic such as it
has been proved that occurs during enzymatic browning in fruit slices (Lu et al., 2007; Luo &
Barbosa, 1994, 1997; Sapers & Douglas, 1987; Wang, Tang, Quan & Zhou, 1994; Yoruk et al.,
2004).
Description of enzymatic browning for one banana slice using a L* mean value (upper), b*
mean value (middle) and FDL* or FDb* value (down) as browning indicators, in another banana
slices, is presented in Figure 3. Again, the average of L* values decreased between 47 to 41 during
enzymatic browning kinetic, confirming that banana slices surface turn brown (Luo & Barbosa,
1997; Molme et al., 1999; Vamosvigyazo, 1995; Yoruk et al., 2004); when simultaneous FDL*
increased from 2.50 to 2.90 showing a growing in the irregularity of SIL*. b* value decreased
between 31 to 21 during enzymatic browning kinetic such has been noted for banana slices during
browning (Molme et al., 1999), when its FDb* value increased from 2.40 to 2.90. In general, an
increment in the FD values as an indicating that L* and b* color parameters on images decreased in
a non-homogenous way during the browning kinetic. In our results, redness color (a*) value not
showed any statistical variation during browning.
In Table 1, rate parameters kinetic (k) and shape factors (n) when the equation 1 was fitted to the
fractional conversion data, are presented. We found Pearson correlations in all fitted model, higher
to 0.94, which give us and idea that the power law model could be used to describe the enzymatic
browning kinetic in banana slices, such has been postulated by Corradini and Peleg (Corradini &
Peleg, 2004) and when fractional conversion data is used.
From Table 1, and when traditional method was applied, rate parameters for the enzymatic
browning ranged between -0.0001 to -0.0002 min-1. When a b* mean value was used, rate parameter
ranged from -0.0002 to -0.0007 min-1 not statistical differences were detected in between rate
parameters. With respect to rate parameters derived by FBI method, they ranged from 0.002 to
0.012 min-1 when FDL* was used, and from 0.002 to 0.01 when FDb* was used. Also, not
statistical differences were detected in their rate parameters. However, more relevant in this research
was the magnitude of the rate parameter k estimated by FBI method, when was compared with
traditional method. FBI method produced a major k value that traditional method in all cases. In
fact, when FBI method was applied based in L* color, k parameter was 35 times higher (in absolute
value) that k obtained by traditional method. In the case of b* color, the extension was 8.5 times
higher respectively. This extension was minor that found using L* color, indicating that the way of
how the color decreasing during the kinetic was different depending the parameter color used.
The calculated values of the enzymatic browning rate parameters in this study could be different
from other investigations since the degree to which bananas turn brown depends not only of the
cultivars but also of other factors such as the endogenous phenolic and polyphenol oxidase content
among others (Amiot, Tacchini, Aubert & Oleszekz, 1995); In fact, it has been demonstrated that
phenolic composition varies greatly with cultivar, stage of maturity and postharvest storage
conditions (Amiot, Tacchini, Aubert & Nicolas, 1992; Oleszek, Amiot & Aubert, 1994; Ranadive &
Haard, 1971; Wrolstad, Heatherbell, Durst, Hsu, Spanos & Yorgey, 1988). However, Amiot et al.
(1992) and Soliva-Fortuny, Elez-Martinez, Sebastian-Caldero, & Martin-Belloso (2004) concluded
12
that for pears picked at dates close to the commercial maturity stage, the susceptibility to browning
was not greatly different.
Another important result is the shape factor (n) obtained. Although power law model is only a
empirical model (no a theoretical model), shape factor can be seen an indicator of how near is the
empirical kinetic with respect to the zero-order or first-order models, depending is the n factor is
close to 0 o 1 respectively. Traditionally first-order kinetic has been attributed to the enzymatic
browning in fruit slice, (Luo & Barbosa, 1994, 1997; McEvily et al., 1992; Nicolas, Richardforget,
Goupy, Amiot & Aubert, 1994; Vamosvigyazo, 1995). Our results confirmed that browning kinetic
for banana slices following a empirical first-order (n shape factor were near to 1) when traditional
methods was applied with L* or b* color. However, when FBI was applied trough the application of
FD, not a first-order was established. In fact, for L* and b* color, order kinetic were minor to 1
(0.82 and 0.61 respectively). These differences in the order of the model could be due to that FBI
method describes no only the changes in the intensity colors, but register the changes in the
distribution of these intensity color during the browning kinetic; changes that the traditional method
(assuming a L* mean) can not register.
(Aquí quiero decir: Las diferencias encontradas entre ambos métodos referidas al orden del
modelo, pueden ser atribuidas al hecho de que el método FBI registra no solo los cambios en los
valores de las intensidades de los colores (L* o b*), sino que además registra los cambios en la
distribución de tales intensidades de colores que ocurren durante el pardeamiento. Cosa que el
método tradicional no puede registrar.)
Conclusion.
A methodology called FBI that describes enzymatic browning kinetic based in the use of
irregular pattern of colors from digital banana slices images is presented. It uses the fractal Fourier
texture images in order to calculated a fractal dimension (FD) value in a selected area in the image
which represents the complexly of the color distribution for lightness or yellowness color (L* and
b*). When traditional color method was used as a browning indicator (a L* or b* mean value),
lightness and yellowness parameters decreased during the kinetic. In the case of FBI method, their
respective FD increased, indicating a major complexity in the distribution of the colors in select
analyzed area during the enzymatic browning kinetic.
Empirical power law model was suitable to correlated enzymatic browning kinetic data also for
FBI and for traditional method. However, enzymatic browning rates using FBI method, was
between 8.5 to 35 times higher that rates calculated with traditional method. Empirical non first-
order model (n lower that 1) was established for samples for FBI method when a first-order for
traditional method. In general, FBI method appears to register some information during the
enzymatic browning kinetic that traditional can not register, (the changes in the distribution of the
intensity colors on the surface during the browning kinetic), such it is actually used in food research.
FBI method open a great potential to be applied not only in banana but also to fruits and foods
where pattern colors have non-homogenous color surface during any kinetic.
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15
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Figure 1.- SIL* of one selected area from

a banana slice image. Plane represents
the average of L* values at 0 min in the
kinetic.
16
Figure 2.- Three selected images with

their SIL* (left) during the enzymatic
browning for a one banana slice. Number
followed by the arrow shows the L* mean
value of same region respectively.
Figure 3.- Kinetic enzymatic browning

for a banana slice measured by the FBI
method (down) and traditional method
(upper and middle).
17
TECHNOLOGICAL INNOVATION IN BIOPROCESS

Profesor: Rodrigo Bórquez Yáñez. Dr.
Departamento de Ingeniería Química. Facultad de Ingeniería. Universidad de Concepción.

rborquez@udec.cl
COMBINED OSMOTIC AND MICROWAVE-VACUUM DRYING OF BIOMATERIALS

Dehydration is one of the most effective methods of preserving fruit. The moisture content of
finished products ranges from 1-5%. Because of such a low moisture content, dried fruits are not
readily prone to microbial spoilage and undesirable enzymatic reactions. Raw fruits are usually
pretreated with an antioxidant and preservative before dehydration. Soaking raw fruits in a sugar
solution before drying has been beneficial in retarding enzymatic browning reaction. Moreover, the
moisture content of the fruit significantly drops before drying because of the osmotic pressure effect,
thereby reducing drying time.
Vacuum drying allows water to vaporize at a lower temperature than at atmospheric pressure, and
fruits are dried without exposing to high temperature. Moreover the absence of air during
dehydration diminishes oxidation reactions. Because of these advantages, color, taste, and flavor of
dried products are improved. From these techniques, microwave vacuum drying in industrial food
processing has several advantages over convective drying, for example, fast heating, high energy
efficiency, and almost uniform moisture distribution in products.
Microwave heating is an important technique in industrial drying and food processing because it has
several advantages over convective heating, for example, fast heating, high energy efficiency, and
uniform moisture distribution in products.
Microwaves are electromagnetic waves in the frequency range 300 MHz to 300 GHz (equivalent to
a wavelength of 1-0.01 m) generated by a magnetron type vacuum tube. Electromagnetic energy at
915 and 2450 MHz can be absorbed by water containing materials or other substances, such as
carbon and some organics, and converted to heat. Because waves can penetrate directly into the
material, heating is volumetric (from inside out) and provides fast and uniform heating throughout
the entire product. The quick energy absorption by water molecules causes rapid water evaporation
(resulting in higher drying rates of food), creating an outward flux of rapidly escaping vapour. In
addition to improving the drying rate, this outward flux can help to prevent the shrinkage of tissue
structure, which prevails in most conventional air drying techniques. Hence better rehydration
characteristics may be expected in microwave dried products.
Two strategies have been proposed in order to effectively apply microwaves for drying: 1) create a
vacuum in the dryer to lower the drying temperature; 2) apply microwave energy in a pulsed manner
to maximize drying efficiency since continuous heating does not accelerate the rate of water
removal when critical moisture content is reached.
Because of vacuum conditions the product temperature is mainly controlled by water pressure,
which helps to save temperature sensitive ingredients like vitamins or colors.
Also, cellular structure is preserved better when osmotic treatment is used. Microwave-vacuum
drying of osmotically pretreated fruits combines the benefits of both unit operations: high-quality
products in terms of color, taste, vitamin C, structure and volume are obtained.
IMPINGEMENT DRYING OF BIOMATERIALS

Impingement dryers belong to a new class of drying equipment, where two or more gas-solid
streams are impacted against, giving rise to high relative velocities between gas and solid phases, in
18
strong turbulent regime, thus enhancing rates of transfer and reducing drying time. Most of the
moisture removal occurs in the impact front.
Impingement jet flow systems provide high local heat and mass transfer rates over the impact
surfaces, which has considerable advantages in food drying where long exposure times will damage
the products. As stated also by the application of impingement systems allows higher rates of drying
of granular materials, pastes, solutions and suspensions. The efficiency of the process increases due
to considerable increase of the relative velocity between phases, oscillatory motion of particles, and
turbulence in the impingement zone. Radial impingement jet systems have also been described as an
interesting alternative to conventional drying and freezing methods, which enhance heat transfer and
therefore quality product, and which is achieved in horizontal cylindrical chambers, with tangential
gas inlet, and axial solids motion.
As a result of the strong interaction of gas and solid phases, the following physical effects are
present in the impingement zone: mixing of streams with changes in velocities and directions of
flows; penetration of solid particles within the opposed gas stream with reverse and oscillatory
motion; collision of particles causing some disintegration. As a consequence heat and mass transfer
rates are enhanced due to the increased gas-solid relative velocity in a strong turbulent motion.
From available data from literature it is observed that heat transfer coefficients for impingement
systems are higher than the corresponding heat transfer coefficients for pneumatic systems operating
under similar hydrodynamic conditions. For example, the heat transfer coefficient for a coaxial
impingement dryer is about 850 W/m2K, whereas for pneumatic dryers heat transfer coefficients
range from 300 to 520 W/m2K. Also the volumetric heat transfer coefficient is higher, 260 to
112,500 W/m3K in impingement dryers, while for spray dryers is about 1,000 to 6,000 W/m3K, and
for spouted beds it is of the order of 5,800 W/m3K.
The most import applications of impingement systems in the drying of biomaterials are the
following: drying of pharmaceutical products in counter-current and corotational systems; drying of
microorganisms in counter-current and counter-rotational beds; drying of residual sludges in waste
water treatment in coaxial systems; and drying of aminoacids such as lysine in moving impingement
zone.
The use of superheated steam as drying medium in impingement systems has potential benefits to
consumers and industry which have been detailed by many authors. In air drying the moist air must
be replaced by fresh air, whereas in superheated steam drying the moisture removal becomes a part
of the drying medium, and superheated steam is preferred instead of hot air because of process
economy (exhausted steam recovered or reused). Superheated steam is also desirable since it is a
non-reacting drying agent to process biological materials, as oxygen and nitrogen are almost absent,
particularly in systems working with a slight overpressure to avoid the entrance of air. The
disadvantage of superheated steam is that the processed material is subjected to the saturation
temperature of water vapor at the working steam pressure, and it is assumed to stay at that
temperature as the moisture content of solids is above its critical moisture. In many cases this
saturation temperature in superheated steam drying may result higher than the wet bulb temperature
of humid air in air drying, and for temperature sensitive products biological or chemical damage
may be increased. This effect is of less extent at short residence time of the processed material..
MEMBRANE SEPARATION OF BIOMATERIALS

Membranes have been major tools in the purification, fractionation and concentration of food
products for more than 25 years. Several industrial applications of membrane operations have been
developed for fruit, vegetable and sugar juices, beverages and dairy products. In comparison with
19
conventional techniques, membranes often offer a cleaner, energy-efficient way for the production
of high-quality food.
Ultrafiltration (UF) is a pressure-driven process using a semi-permeable membrane to separate
macromolecules or colloids from liquids and is based on a simple sieving mechanism. Solute
dimensions in relation to the membrane’s pore size distribution determine whether or not a solute
molecule can pass through the membrane.A major limiting factor in UF processes is permeate flux
decline with time due to solute polarisation near the membrane surface and membrane fouling.
Fouling is generally attributed to macromolecule or colloid accumulation on the membrane surface,
and adsorption and precipitation of smaller solutes in the membrane pores.
An alternative treatment for fish processing wastewaters is the use of membrane separation
processes, in particular UF and NF. The advantages of these processes over the conventional
coagulation / flocculation process are the good quality of the permeate which can be recycled into
the fish processing plant, as well as the simultaneous recovery and concentration of soluble proteins.
One of the interesting features of UF and NF in comparison with other concentration processes lies
in the fact that water separation is energy efficient, since it is carried out without phase change.
Preliminary testing is essential to identify suitable operating conditions (transmembrane pressure,
crossflow velocity and volume reduction factor), which will determine the membrane type and
pumps specifications, two important parcels of the investment cost.
Concentration polarisation is due to the membrane permselectivity. Solutes are dragged to the
membrane surface by convective transport of the solvent, some of which may pass through the
membrane, whereas rejected solutes accumulate in the membrane vicinity and may form fairly
viscous and gelatinous layers. The formation of a gel-layer or secondary membrane reduces the
permeation flux and may also hinder the passage of low molecular weight solutes.
Membrane fouling results from the deposition of submicron particles, as well as crystallisation,
precipitation and adsorption of solutes on the membrane surface or inside its pores. Fouling may be
irreversible (e.g. proteins adsorption) and its extent mainly depends on the membrane nature and
feed characteristics. The first means for controlling this phenomenon is an adequate feed pre-
treatment and a careful choice of the membrane type. Moreover, a module design providing suitable
hydrodynamic conditions for the particular application should be chosen.
Physical and chemical properties of the substances present in the feed also play a role on the
membrane performance. For instance, particles size distribution determines the occurrence of
fouling inside the membrane pores. Moreover, the solute geometry also affects the separation
performance, as well as its electrical charge, as this property may determine the solute affinity for
the membrane (adsorption).
At present, there is no general theory to predict accurately the performance of UF / NF processes.
Nanofiltration can be envisaged as a “tight” ultrafiltration, though it is also similar to reverse
osmosis as for the role of electrostatic forces in salts separation, which are negligible for common
ultrafiltration.
SPRAY DRYING OF PROBIOTICS

The term “probiotic bacteria” refers by definition to a live microorganism that, when consumed in a
certain number, produces additional health benefits beyond the basic nutritional ones. These
bacteria are usually included in functional-type foods, such as yogurt, which help maintain intestinal
bacteria that collaborate in maintaining good digestion. Since they are inoffensive, they can be
introduced in distinct live environments (humans or animals) to favor the presence of beneficial
colonies and prevent the presence of pathogenic bacteria.
20
Bacteria’s superficial properties are intimately related with their capacity to adhere to different
substrates. Additionally, bacterial adhesion to a tissue is a fundamentally important factor for its
subsequent colonization and is a highly important characteristic in the selection of probiotic
microorganisms. An approximate measurement of bacterial adhesive properties is the study of its
hydrophobic characteristics. Within the lactobacillus genus, the production of antimicrobial
substances of diverse types has been described. The organic acids, hydrogen peroxide (H2O2) and
the bacteriocines have been widely studied.
Many studies have been performed to test or to discover different types of medicinal use of probiotic
bacteria. Among the potential benefits, which depend on each strain are: colon cancer prevention,
lactose intolerance, increased immunological functions, and infection prevention.
A therapeutic dose of live microorganisms (108 cells daily) can assure probiotic effects in the
consumer. This aspect can be considered in the production of this preparation type as well as in the
conservation of strain’s viability and original probiotic properties over time.
In Chile, salmon industry is the second most important one. In order to keep this place, salmon
industries are forced to assure a product of high quality which has to satisfy the demanding
requirements of the most industrialized countries. Salmons for exportation must have a specific size
and color, and be able to resist diseases. In order to avoid opportunistic infections, salmon farmers
use yearly around 100 ton of antibiotics, but the knowledge of the adverse effects that they have, e.g.
pathogen resistance, has caused that, in the last three years, some salmon shipments had been
rejected at their arrive in United States, United Kingdom and Japan.
Conscious of the economic and social problem that this mean for future business, there is interest to
find a natural way to protect the salmon farms, assuring a health product free of traces of
undesirable substances. Currently, some works have been published and researchers think that the
response to problems in aquaculture could be subsaned with the use of probiotics.
Probiotic cultures for applications are supplied in frozen or dried form. Dried cultures are commonly
produced by freeze drying, however, because is an expensive process in terms of time and energy,
scale up is difficult. An alternative method of preservation is spray drying, whose production rates
are higher and the operative cost lower. The principal disadvantage of this process is related with
loss of viability, so, works are oriented to improve the treatments before spray drying as a way to
reduce the percentage of mortality, which is produced principally by thermal and dehydration
inactivation.
Among techniques oriented to protect lactic acid bacteria before spray drying, use of prebiotics ,
microencapsulation and pre-adaptation to non-lethal stress have been widely studied .
Concerning pre-adaptation of bacteria to non-lethal stressful situations there are three kinds of
situations: acidic, thermal and osmotic stress. Among these, the last one has been profusely studied
but the way in which bacteria are protected is not already clear, although, there have been founded
overproduction of heat shock proteins and production of compatible solutes.
Spray drying can use up to 50% less energy than lyophylization and its application is generally used
in the production of dehydrated enzymes, detergents, coffee extracts, and isolated proteins as well as
a production method for culture of lactic acid bacteria and dehydrated probiotic bacteria. The
principal advantages of spray drying is that it is less expensive and faster than traditional techniques,
such as freezing and lyophylization, used to preserve lactic cultures.
Two principal mechanisms have been proposed as being responsible for viability deterioration or
loss during convective drying of bacterial culture: deactivation due to dehydration and deactivation
due to temperature. It is not clear which of these mechanisms is more damaging because they
generally occur simultaneously. These mechanisms affect a large number of cellular components,
21
including DNA, RNA, proteins, cytoplasmatic membrane, and cell walls. Of these cell components,
cytoplasmatic membrane is considered to be the most affected by dehydration because there is a loss
of several intracellular components when the membrane is damaged.
The short residence time of spray drying implies that the air must enter at a high temperature in
order to achieve the necessary heat for water evaporation. The bacteria survival rate is strongly
related with the air dryer’s exit temperature due to the cooling effect produced by moisture
evaporation in the constant velocity drying stage. A lower exit temperature will result in a higher
survival rate and higher product moisture.
Another process parameter that strongly influences the survival rate is the size of atomized particles
since a higher solid concentration in the cellular suspension fed into the dryer results in larger size
particles. The drying time increases resulting in diminished bacteria survival due to the higher
contact time with the hot air. Additionally, a higher concentration in the dryer feed reduces the
energy cost due to a lower quantity of water to be evaporated.
Other factors that impact on the drying survival are: the bacterial strain species, the growth
conditions, the incorporation of protective additives, cellular concentration and pH, rehydration
conditions, and the strain’s stress conditions prior to the drying stage. Even when all these factors
cannot be directly manipulated in the drying stage, they can have a deep impact on microorganism
survival and the conservation of probiotic properties during storage.
RESEARCH GRANTS:
FONDECYT: 1990399/ 1020434/ 1060919
FONDEF: D01I1121/ D04I1326
INNOVACHILE 05CT6PPT-13
22
Simposio 1.
Avances en Biotecnología Agroalimentaria
Presentación de Paneles
23
24
INMOVILIZACIÓN DE PAPAÍNA EN ALUMINOSILICATOS NANOTUBULARES

SINTÉTICOS.
Nicolás Arancibia-Miranda , Karen Manquián1, Rodrigo Contreras2, Gustavo Zúñiga3.
1
1
Programa de Doctorado y Magíster en Ciencias de los Recursos Naturales, Depto. Ciencias
Químicas, Universidad de La Frontera, Av. Francisco Salazar 01145, Casilla 54-D, Temuco, Chile.
E-mail: naranci@ufro.cl
2
Programa de Doctorado en Biotecnología, Facultad de Química y Biología, Universidad de
Santiago de Chile, Av. B. O’Higgins, 3363 Santiago, Chile.
3
Facultad de Química y Biología, Universidad de Santiago de Chile, Av. B. O’Higgins, 3363
Santiago, Chile.
El aumento en la aplicación de los bionanocompositos ha impulsado el desarrollo de nuevos

nanomateriales [1]. Es así como la imogolita un aluminosilicato nanotubular (ANT) surge como una
alternativa novedosa para inmovilizar proteínas, debido a las características fisicoquímicas de este
ANT (morfología, area superficial y punto isoelectrico). La papaína es una proteasa derivada de la
papaya (Carica papaya L.). Sus aplicaciones se han centrado fuertemente en la industria
alimenticia, médica y medioambiental por lo que sus potencialidades resultan interesantes de
ampliar y potencia r[2,3]. El objetivo de este trabajo es determinar los parámetros críticos de
inmovilización de papaína en imogolita en dos estados (gel y sólido). La síntesis de la imogolita se
realizó según lo descrito por Denaix, et al., 1999, obteniéndose una razón Al/Si de 2 [4]. La
imogolita fue caracterizada por TEM, DRX, IR y PZC. La inmovilización de la papaína fue
realizada en un stock de ANT de 100 mg en suspensión a la cual se le adicionó 4ml de 0.1M buffer
fosfato (pH 8.0) con agitación a 30º C por 5min. A partir de los ensayos de inmovilización existen
claras diferencias entre los dos estados físicos de la arcilla, por una parte la gran hidratación de la
ANT cuando esta gelificado favorece la adsorción de la papaína, debido a que existen mayor
cantidad de grupos disponibles para la formación del complejo arcilla/enzima. En estado sólido el
número de grupos disponibles de hidratación se reduce por lo que adsorción disminuye. Estas
diferencias generan comportamientos de actividad diferentes.
[1] Tamás Szabó, Raluca Mitea, Hugo Leeman, Gnanasiri S. Premachandra, Cliff T. Johnston,
Márta Szekeres, Imre Dékány and Robert A. Schoonheyd.(2008) Clays and Clay Minerals. 56; 5;
494-504.
[2] Gray, J.J. (2004) . Current Opinion in Structural Biology, 14, 110-115.
[3] Zhao, X.S., Bao, X.Y., Guo, W., and Lei, F.Y. (2006) Materials Today, 9, 32-39.
[4] L.I. Denaix Laurence, Bottero Jean Yves, (1999) Colloids and Surfaces A: Physicochem. Eng.
Aspects 158, 11.
25
SOLVENT EXTRACTION OLIGOSACCHARIDES OF LUPINE FLOUR

Medina H1., Shene C2.
2
Departamento of Chemical Engineering, 1Processes and Technology Unit of the Center for
Nutritional Genomics Agro-aquaculture and Food Biotechnology Center and Bio separations,
Universidad de la Frontera. hmedina@bengala.cl, cshene@ufro.cl
In the search for maximization of their revenues aquaculture industry is in a constant need for cost-
efficient diets and highly digestive raw materials. Low-cost raw materials correspond to those of
plant origin that are used for partial replacement of fish derive products (meal and oil). Since these
raw materials contain antinutritional factors (ANF) such as saponins, phytates, phytoestrogens,
oligosaccharides, etc., is necessary to improve their nutritional quality through processing. In lupine
meal, some ANFs are non-starch polysaccharides, from the raffinose family, that could be
eliminated or reduced through solvent extraction. In this work preliminary results on the
optimization of the extraction process are presented. We investigated the effect of enzymatic
pretreatment (Viscozyme L) on the extraction yields of oligosaccharides and carbohydrates in the
lupin meal, expressed as total sugars (TS). The effect of other factors (temperature and time) was
also determined. To optimize the process conditions in the extraction the surface response
methodology (SRM) was used. Statistical analysis indicates that the control factors explain 70% of
the response due to factor variability. Optimal conditions were an enzyme to substrate ratio equal to
0.08, extraction temperature of 40° C and extraction time carried out by 1.5 h. Preliminary results
indicated that under optimal conditions 25% of the substrate was extracted as soluble compounds;
the extracted lupin meal contained 90% protein (dry basis).
Acknowledgments: This work was carried out with funding provided by Center for Genomics and
Nutrition Agro-aquaculture.
26
INCORPORACIÓN DE FLAVONOIDES AGLICONA Y GLICOSILADOS EN UNA

MATRIZ BIOPOLIMÉRICA DE CARBOXIMETILCELULOSA SÓDICA: ESTRUCTURA
QUÍMICA Y PERMEABILIDAD AL VAPOR DE AGUA
Silva-Weiss A. (1), Ihl M. (2), Bifani V. (2)
(1)
Programa de Doctorado en Ciencias de recursos Naturales, acsilva@ufro.cl,(2) Departamento de
Ingeniería Química, Universidad de La Frontera, Temuco- CHILE
Los polifenoles, metabolitos secundarios derivados de las plantas, son conocidos por su
actividad biológica (antioxidante, antimicrobiano y/o antiflamatorio, entre otros). Ellos pueden
modificar las propiedades físicas de las matrices biopoliméricas que los contienen, como se ha
demostrado en particular al incorporar extracto acuoso de hojas de murta en matrices de gelatina de
pescado y de carboximentilcelulosa. El extracto acuoso de hojas de murta (Ugni molinae Turcz),
planta endémica de Chile, posee capacidad antioxidante y en su composición se encuentran
principalmente ácidos fenólicos como ácido gálico, además de flavonoides aglicona y glicosilados,
entre los que se encuentra quercetina. El objetivo de este trabajo fue evaluar el efecto de la
incorporación de flavonoides (quercetina y rutina) y extracto acuoso de hojas de murta, en la
permeabilidad al vapor de agua (PVA) y estructura química de matrices biopoliméricas de
carboximetilcelulosa (CMCNa). Para esto se formularon soluciones filmogénicas de CMCNa (2%
p/p), glicerol (20 g/100 g CMCNa) y aceite de girasol (25 g/100 g CMCNa), incorporando
quercetina o su correspondiente glicósido quercetina-3-O-rutinosido (rutina), en una concentración
de 0,8 g/100 g CMCNa, o extracto de hojas de murta en una proporción de 50 mL por gramo de
CMCNa (concentración extrato100 mg/mL).
La espectroscopia infrarroja por transformada de Fourier (FTIR) fue empleada para analizar
variaciones sobre la estructura química de las matrices con quercetina, rutina o extracto de hojas de
murta de siete ecotipos (A, B, C, D, E, F, G). El espectro de CMCNa en polvo muestra la banda
característica de este polisacárido a 1605 cm-1, correspondiente a la presencia de grupos carbonilos
(COO-). Todas las matrices estudiadas presentaron una banda a 1737 cm-1, la cual indica la
presencia de grupos carboxílicos, siendo esta banda menos intensa en matrices sin glicerol. Al
comparar los espectros de las matrices biopoliméricas con extracto, quercetina o rutina se observó
que las que contenían extracto de hojas de murta de los siete ecotipos fueron similares, encontrando
en todas una banda nueva y aguda a 2335 cm-1. El mismo efecto se ha observado al incorporar ácido
ferúlico como agente de entrecruzamiento en matrices de almidón-quitosano, a una longitud de 2362
cm-1. Esta banda no se presentó en matrices con quercetina ni rutina, ni en el control, indicando que
los polifenoles aislados, ya sean glicosilados o aglicona, no ocasionan este cambio en la estructura a
2335 cm-1.
Según los antecedentes, serían los ácidos fenólicos presentes en el extracto acuoso de hojas de
murta, como el ácido gálico y/o sus derivados, los que modifican la estructura de la matriz
biopolimérica de CMCNa. La PVA es reducida con la presencia de quercetina y extractos de hojas
de murta, presentando tanto quercetina como el ecotipo B (1,47 y 1,51 × 10-10 g/ m s Pa,
respectivamente) una disminución significativa de la permeabilidad (p <0,05) con respecto al control
(2,21× 10-10 g/ m s Pa.), mientras que los flavonoides glicosilados como rutina tienden a incrementar
la PVA, alcanzando un valor de 2,56 × 10-10 g/ m s Pa.
Agradecimientos: Beca de Doctorado Nacional 21070302 y AT 24090134, CONICYT; Proyectos
INNOVA-CORFO 06CN12PAT-57 y DIUFRO DI 08-2009, Universidad de La Frontera.
27
MECHANICAL PROPERTIES, CHEMICAL INTERACTIONS AND STRUCTURE OF

STARCH/CMC/MONTMORILLONITE NANOCOMPOSITE FILMS
Silva-Weiss A1., Garrido E.G.1, Mora M.L.2, Bifani V.3, Theng B.K.4
(1)
Postgraduate Program on Sciences of Natural Resources, acsilva@ufro.cl, (2) Scientific and
Technological Bioresource Nucleus and (3) Department of Chemical Engineering, Universidad de La
Frontera, Temuco, Chile. (4) Landcare Research, New Zealand.
The assembly of inorganic solids with dimensions on the scale nanocomposites to natural polymers,
is a very advantageous strategy for the development of new materials with functional properties and
structural interest on various fields of application. Thus, impact on science and technology are as
relevant as plastic called "green", materials for tissue engineering, controlled release systems drug or
as active coating of food for increasing their shelf life.
This work shows the study of mechanical properties of starch-based films (3.5 or 3.0 % w/v)
plasticized with 0.2 [g glycerol/g polymer dry] (S film), incorporating 0.5 %w/v sodium
carboxymethylcellulose (CMC, SD 0.7-0.9) (SC film) or nano montmorillonite (Mn) 0.02 [g
nanoclay/g polymer dry] (SMn film) or both CMC and Mn (SCMn film). Films were prepared by
casting and the interactions of its components were observed through Fourier Transform Infrared
Spectra (FTIR) and Scanning Electronic Microscopy (SEM).
The FTIR of the films studied shows characteristic absorption bands of starch, CMC and
montmorrillonite powder reported for the other authors. The OH-stretching vibration for both SC
and SMn based films were centred at 3336 cm-1 and it were broader than for SCMn and S based
films. This behavior was attributed to a wide distribution of hydrogen-bonded hydroxyl groups. The
band at 1643 cm-1 assigned to δ (O-H) bending of water for the starch and starch film appeared as a
shoulder at 1638 cm-1 in SC and SCMn spectres. In the SC and SCMn appeared a typical band of
CMC at 1605 cm-1 which was displaced a lower frequency (1591 cm-1), this band confirmed the
presence of COO- groups present in the carboxymethylcellulose .
The surface of SCMn was continuous compared with SC and SMn. All films and nanocomposite
shows cross-section structures very different. In case of cross-section of SC shows a structure like
honeycomb in some sections, but in SCMn shows montmorrillonite layers and basal spacing
between layers are clearly seen.
The thickness of the films were found to range from 84 to 104 µm. The tensile strength and
deformation at break values of the starch matrix increased significantly (p<0.05) by the addition of
both CMC and Mn clay, being tensile strength of SCMn 28.04 ± 0.24 MPa and deformation at
break 3.06 ± 0.11 mm. However, when it includes only CMC or Mn, the tensile strength decreased
in relation to the starch film. The elongation values of the films are a measure of the flexibility of the
film and was increased with the addition of CMC and CMC/Mn in the starch film.
Acknowledgements: CONICYT scholarship 21070302 and AT 24090134, Project INNOVA-

CORFO Nº06N12PAT-57 and Postgraduate Program on Sciences of Natural Resources,
Universidad de La Frontera, Temuco – Chile.
28
ZEBRAFISH (Danio rerio) LARVAL REARING: PROGRESS IN ESTABLISHING A

MODEL ORGANISM FOR STUDIES OF NUTRITION AND GROWTH APPLIED TO
CULTURED FISHES.
Ulloa P.1, Lizama C.2, Araneda C.2, Neira R.2, Iturra P3.
1
Programa de Doctorado en Ciencias de Recursos Naturales Universidad de La Frontera. 2Facultad
de Ciencias Agronómicas Universidad de Chile. 3Facultad de Medicina Universidad de Chile.
pilarelizabeth@gmail.com.
Zebra fish (Danio rerio) is a model organism widely used in studies of developmental biology,
physiology, molecular genetics and toxicology. Some of their main advantages are: small size, short
generation interval (12-14 weeks), high fecundity and fertility, and transparent eggs during
embryogenesis. Nowaday, zebrafish has been also proposed as a possible model organism for
nutrition and growth studies in fish. However, standardized protocols for larval rearing have not
been fully established, difficulting the production of large numbers of adult fish; a necessary
requirement for applying the zebrafish as a model in aquaculture. Larvae survival depends upon
food supply and physical-chemical water parameters, such as temperature (T°), pH, hardness (DH),
dissolved oxygen (O2) and nitrogen wastes (NO3-/NO2-). Levels of dissolved oxygen, and fish
density in larval rearing have not been determined yet. The aim of this study is to determine the
optimal conditions for zebra fish larval rearing from 8 to 25 days post fertilization (dpf). To assess
the physical-chemical parameters of water on larval survival under conditions of aeration and no
aeration we used two wild-type strains obtained from University of Chile, where pair mating were
used to produce 24 full-sib families (n = 6373). Eggs were incubated in 90 mm Petri dishes at 28 ° C
until 7 dpf. Larvae were reared by families in separate tanks without water recirculation from 8 to 25
dpf and constant photoperiod (14 h light: 10 h dark). Larvae were fed to satiety three times daily
(9:00,13:30 and 17:30 h) with commercial food Seramicron® and Gold Micro protein®, and were
kept at a density of 10 cm3 · fish-1 without aeration and 22 cm3· fish-1 with constant aeration. Under
aeration condition the length (mm), weight (mg) and condition factor [K = weight / length3 · 100]
were recorded in a sample (20%) of fishes per family (n = 619) at 28 dpf. Water physic-chemical
conditions recorded along larvae rearing period (17 days) in 24 tanks were: T° 25.71 ± 1.25 °C (20.0
min – 27.7 max), pH 7.67± 0.13 (7.26 – 8.10), conductivity 282.6 ± 41.3 µ s · cm-2 (214 - 573), NO3-
69.64 ± 82.73 mg · L-1 (0 – 250), NO2- 5 ± 4.92 mg · L-1 (0 - 10). Results indicate a highly
significant reduction in larvae mortality from a 52.81% in fish without aeration v/s 4.95% mortality
in fish with constant aeration (χ21gl = 1852.87, P-value <0.000001). A higher mortality was observed
from 12 to 15 dpf and decreased dramatically after that period. Family differences in mortality at 25
dpf were confirmed. The average weight of larvae at 28 dpf was 1.65 ± 0.77 mg (0.40 – 4.4), the
length 5.29 ± 0.66 mm (3.5 - 7.0) and the condition factor 1.11 ± 0.44 (0.32 to 2.66). There were
significant differences in these three variables between families after Kruskall-Wallis test (P-value =
0.00001). It was conclude that for optimal larval rearing of zebra fish is important to consider the
density of fish per tank (about 22 cm3 / fish) and a constant aeration from 9 dpf onwards.
Proyecto Fondecyt 1060623 Consorcio Aquainnovo SA- INNOVA 206-5047
29
OPTIMIZATION OF AN AQUEOUS EXTRACTION OF MUCILAGE FROM FLAXSEED

CAKE
M. Verdugoa, M. Rubilarb,c
a
Ph.D. Program in Sciences of Natural Resources, Universidad de La Frontera, Casilla 54-D,
Temuco, Chile, e-mail: mverd001@yahoo.es; bCenter of Food Biotechnology and bioseparations
BIOREN, Universidad de La Frontera, Casilla 54-D, Temuco, Chile; cUnidad de Tecnología y
Procesos CGNA, Universidad de La Frontera, Casilla 54-D, Temuco, Chile
Recently, flaxseed (Linum usitattisimum L.) has become very important in human nutrition due
to the recognized potential health benefits that some of its components offer. The compounds with
biological activity include alfa-linolenic acid, lignans, and dietary fiber (1). Flaxseed has
traditionally been used as an oilseed. In the cold extraction process, flaxseeds are pressed to produce
oil; residual oilcake is primarily used as a rich-protein livestock feed. Some attempts have been
made to develop other uses for the flaxseed oilcake. A number of studies have attempted to purify
and characterize the protein fraction and the mucilage from flaxseed.
The mucilage comprises the outer layers of hull and is rich in water soluble carbohydrates. Main
factors that affect purity and yield of mucilage extraction process are pH, temperature and water to
seed ratio, among others (2). This soluble dietary fiber has drawn much attention mainly because of
its health benefits such as reduction of diabetes risk, and total cholesterol. Other potential food
applications, as thickener and emulsifier agent, are due to its physical properties (3).
The main objective of this study was to optimize the aqueous extraction process to produce
mucilage of high purity and high yield by using flaxseed oilcake as raw material.
The extraction of mucilage will be development according to Diederichsen et al. (3), who
described an aqueous extraction process from flaxseed. In the present study the methodology was
adapted for mucilage extraction from the oilcake that was defatted, dried, ground and sieved. Two
particle size (0.3>d: fine fraction; 0.3<d<0.425: coarse fraction) were used; experimental levels for
pH, temperature, water:solid ratio were 2.0-7.0, 20-85ºC, and 20-60, respectively. The preliminary
mucilage extractions, from fine and coarse fractions, were made at pH 4.2, 5.4 y 6.6, temperature of
85ºC and water:solid ratio of 40. The results have proved that coarse fraction has a remarkable
influence on both purity and yield of crude mucilage extracted, while the effect of pH on extraction
yield was not significant, however its effect on purity remains to be evaluated. Up to now, results
are agree with previous works (4) for instance extraction yield was higher from meal than from
flaxseed and protein content shows the same behavior. Therefore, in future experiments the coarse
fraction from the oilcake will be used as raw material for the production of high purity mucilage.
Acknowledgment: Authors acknowledge the financial support given by Conicyt through Fondecyt
project 1090516 and technical support provided by GAP Dirección de Investigación at Universidad
de La Frontera.
References
(1) Kozlowska, J., Muñoz G. A. and Kolodziejczyk P. P. (2008). International Conference on Flax
and Other Bast Plants(ISBN #978-0-9809664-0-4).
(2) Cui, W., Mazza, G., Oomah, B.D. and Biliaderis, C.G. (1994). Lebensm. Wiss. u. Technol. (Food
Science + Technology) 27:363-369.
(3)Diederichsen, A., Raney, J.P. and Duguid, S.D. (2006). Crop science, vol. 46.
(4) Fedeniuk, R.W. and. Biliaderis C. G. (1994). J. Agric. Food Chem. Vol 42: 240-247.
30
Simposio 2.
Ecología Química como Herramienta Biotecnológica
Moderadores: Dr. Andrés Quiroz; Dr. Christian Fuentes.
1. Dr. Andrés Quiroz, Universidad de La Frontera, Chile: “Chemical Ecology: An

interdisciplinary science”.
2. Dr. Rufus Isaacs. Michigan State University, USA: “Integrated pest

management in berries”.
3. Dr. Claudio Ramírez. Universidad de Talca: “Biotechnology of plant

protection”.
4. Dr. Christian Figueroa. Universidad Austral de Chile: “Ecological and

evolutionary genomics of herbivore insects”.
5. Dr. Eduardo Fuentes. Universidad de Talca, Chile: “Insecticide resistance of

fruit pests”.
6. Dr. Alejandro Urzúa. Universidad de Santiago, Chile: “Pharmacologycal and

physiological properties of extracts and pure compounds used in Popular
Medicine”.
31
32
CHEMICAL ECOLOGY: AN INTERDISCIPLINARY DISCIPLINE

Andrés Quiroz
Laboratorio de Ecología Química, Universidad de La Frontera
The origin of new disciplines is directly related to the convergence of complementary interests
of classical ones. Chemical ecology is the product of an association between biologists-ecologists
and natural products chemists jointed by common scientific questions and empowered by
complementary expertise. This common scientific perspective, coming from the fact that all the
organisms release chemical signals and that all, depending on their respective ways, responds to the
chemical emissions of others. Nature, in accord with this construct, is a dynamic assemblage of vast
complexity, driven by interactions that are, for the most part, mediated by molecules. The natural
products chemists contribute with chemical knowledge for the identification of these chemical
signals. In this scenario, chemical ecology was born in the 1950s, coinciding with the developing of
improved techniques and instruments, very useful for chemical structural elucidation. In their origin,
the objective of Natural Products Chemistry was the discovery of molecules of use, substances of
medical, industrial, and agricultural interest. Its expansion into the domain of chemical ecology
represented new goals, related with solving fundamental problems rather than applied significance,
but the challenge was immense and the potential significance of the findings enormous. These
interactions allowed identifying the first sex pheromone 50 year ago. Bombykol, (E, Z)-10,12-
hexadecadien-ol, was isolated from the silkworm moth, Bombyx mori (L.). The elucidation of the
structure spanned 20 year and required a half million female abdomen.
OH
H3C
Bombyx mori (L.) Bombykol
The seminar will review part of the Chemical Ecolgy history and the main results obtained from
the Chemical Ecolgy group of the Universidad de La Frontera.
33
APPLICATION OF INSECT CHEMICAL ECOLOGY TO BLUEBERRY AND GRAPE

PRODUCTION IN THE GREAT LAKES REGION OF THE UNITED STATES
Rufus Isaacs, Luis Teixeira, Keith Mason, and Steve Van Timmeren
Berry Crops Entomology Lab, Department of Entomology, Michigan State University. 202 CIPS
Building, East Lansing, MI 48824, USA. Email: isaacsr@msu.edu, URL: www.isaacslab.msu.edu
Insect pests are a major impediment to production of fruit crops in the Great Lakes region of the
United States. This region is the leading producer of blueberry in the world, with ~8,000 Ha and it
also produces a significant volume of juice from native grapes (Vitis labrusca). Together, these
crops constitute the dominant berry crops grown in this region and each has a spectrum of insect
pests that are managed by growers to meet the high quality standards of national and international
trade. To assist farmers in meeting these targets, our program works to understand pest insect
biology and management and to then use this understanding to develop tools that growers can
employ within their Integrated Pest Management programs for fruit crop production with minimal
pesticide inputs. As an example of how insect pheromones are used to improve insect pest
monitoring and control, I will highlight the use of pheromone-baited monitoring traps that are being
used with a degree-day model to improve timing of insecticides for control of a fruitworm moth pest
in blueberry. This project links insect biology, weather monitoring programs, and new reduced-risk
pesticides to improve integrated pest management in blueberry. In the grape system example, we
have been working to create an effective delivery and implementation program for SPLAT-GBM, a
wax-based matrix that contains the sex pheromone of grape berry moth. Our recent trials at the small
plot and full vineyard scale will be described, including 2009 data on the performance of this
product for control of this key pest of grape vineyards in eastern North America.

34
BIOTECHNOLOGY OF CROP PROTECTION

Claudio C. Ramirez
Instituto de Biología Vegetal y Biotecnología

Universidad de Talca
With the advent of a plethora of new techniques assisting molecular biology, the molecular basis of
plant-pest interactions has been revealed and their findings are suitable to enhance crop protection.
In this talk, recent advances in plant-pest interaction, both from the plant side and pest side, are
reviewed. Main advances are focused in the plant-insect interaction, where most of the research and
biotechnologies are aimed to increase plant defense against insects. The development of plants with
inserted transgenes expressing compounds which are toxic to insects is a well-know strategy.
Alternative strategies are focused on deciphering natural plant defenses mechanisms against
herbivores insects. Studies using this approach have shown that insect damage elicits the expression
of genes which affect levels of primary and secondary compounds of plant metabolism. Feeding
modes among pest insects appears as a critical feature to consider when studying plant response to
insect damage. Many studies have shown large difference in plant responses to chewing and
phloem-feeding insects. One of the main challenges of the research in this area is the identification
of specific insect-derived elicitors of plant defenses. How this and other novel findings are suitable
to be implemented as biotechnological tools are discussed and exemplified with different plant-
insect systems.
35
ECOLOGICAL AND EVOLUTIONARY GENOMICS OF HERBIVORE INSECTS

Christian Figueroa
Instituto de Ecología y Evolución, Universidad Austral de Chile, Casilla 567, Valdivia , Chile
Email: christianfigueroa@uach.cl
The recent advent of evolutionary genomics is allowing studying how the genome of an organism
responds to the environment, through the identification of the molecular targets on which natural
selection act. Aphids are ubiquitous phytophagous insects that frequently constitute important pest
species. They are good models to study (i) insect specialization processes mediated by the host-
plant, and (ii) plant resistance to insect attack. A first step in genomics of plant-aphid interaction is
the identification of an initial set of candidate genes and gene products putatively involved in this
adaptive process. Through functional genomic and proteomic studies we are now able to identify (1)
regulated genes and proteins in the aphid according to the host-plant, and (2) regulated defensive
compounds and proteins of response to insect attack in the plant.
36
INSECTICIDE RESISTANCE IN PEST OF FRUITS IN CHILE

Eduardo Fuentes-Contreras
Facultad de Ciencias Agrarias, Universidad de Talca, casilla 747, Talca, Chile. efuentes@utalca.cl
Development of insecticide resistance is a frequent problem in agricultural pests from several crops
worldwide. During the last fifty years the numbers of cases and pest species which have been
reported as resistant to insecticides have increased substantially, while the number of available
insecticides to control them has decreased. Also, the knowledge on the molecular basis and
mechanisms of insecticide resistance has shown remarkable progress, including the identification of
genes and mutations responsible of insecticide resistance. Similarly, the development of suitable
methodologies for the identification of insecticide mechanisms in large samples, have allowed the
study of the structure and dynamics of insecticide resistant populations in the field. This historical
progress is analyzed with two main pest of fruit crops in Chile: the codling moth, Cydia pomonella
(Lepidoptera: Tortricidae), and the green peach aphid, Myzus persicae (Hemiptera: Aphididae).
These pests have been studied with a multidisciplinary and collaborative approach during the last
decade in Chile. For the codling moth we have found resistance to the organophosphate insecticides
azinphosmethyl and chlorpyriphos in some stages of the life cycle. This resistance was associated
with a significant increase in the activity of glutathione-S-transferases, which are well known
detoxifying enzymes. No mutations in the acetylcholinesterase (AchE) and a low frequency (<5%)
of knock down resistant (kdr) mutations in the sodium channel have been found. For the green peach
aphid we have found low to mid levels of esterase activity, which provide resistance to
organophosphate and carbamate insecticides. The frequency of AchE and kdr mutations in field
populations was also detected at low levels (5-10%). In summary, we have found that the frequency
of insecticide resistant individuals of the studied pests was rather low in relation with similar studies
performed in other countries, although insecticide sprays were as frequent as in other fruit producing
temperate areas of the world. An analysis of neutral genetic variability using microsatellites was
performed for the codling moth and is in progress with the green peach aphid. These studies have
found high levels of gene flow and very little population differentiation at different spatial scales of
analysis. Such results suggest the existence of “susceptibility refugia” in the agroecosystems of
central Chile, which could explain the low frequency of insecticide resistant individuals of the
studied pest species.
37
PHARMACOLOGICAL AND PHYSIOLOGICAL PROPERTIES OF EXTRACTS AND

PURE COMPOUNDS USED IN POPULAR MEDICINE
Alejandro Urzúa
Faculty of Chemistry and Biology, Laboratory of Chemical Ecology, University of Santiago, Casilla
40, Correo 33, Santiago Chile. E-mail: alejandro.urzua@usach.cl
Introduction
Species of the genera Haplopappus, Pseudognaphalium and Aristeguietia, with reputed antibacterial
properties, are well represented in Northern and Central Chile. Like many of the plant species of that
particular geographic area shows exudates biosynthesised in specialised glands (trichomes)
populating the surface of all aerial structures of the plants. In addition, other specialise structures
produce the waxy coating and additional secretory structures may also be involved. The
antimicrobial properties of the extracts, obtained from the plants by immersion in CH2Cl2, were
tested against Gram positive and Gram negative bacteria. They all proved to be inactive against the
latter, but some exhibited an interesting activity against Gram positive microorganisms.
Each extract was fractionated by CC and a bio-directed antibacterial experiment lead to the
isolation of several diterpenoids. The structures of the compounds were elucidated by 1 and 2D
NMR (COSY, HSQC, HMBC and NOESY) and HRMS.
Results and Discussion
Kaurenoic acid (1) and its 3-β-hydroxy derivative 3 were obtained from Pseudognaphalium vira
vira. Methyl kaurenoate (2), was prepared by methylation of kaurenoic acid with diazomethane.
Salvic acid (4) and its acetylated derivative 5 were obtained from Aristeguietia salvia. 18-Acetoxy-
cis-cleroda-3-en-15-oic acid (6) was obtained from Haplopappus uncinatus. 18-Acetoxy-cis-
cleroda-3-en-15-oic acid methyl ester (7) was prepared by methylation of 18-Acetoxy-cis-cleroda-3-
en-15-oic acid with diazomethane.
CO2R
R2
CO2R1
CO2H
OR
AcO
R1 = H , R2 = H (1) R = H (4) R = H (6)

R1 = Me, R2 = H (2) R = Ac (5) R = Me (7)
R1 = H, R2 = OH (3)
Diterpenoids isolated from Pseudognaphalium vira vira, Aristeguietia salvia and
Haplopappus uncinatus
Two substituted derivatives, 8 and 9, of cis-cleroda-3,13(Z), 8(17)-trien-15-oic acid, were

obtained from Haplopappus foliosus . 18-Acetoxy-cis-cleroda-3,13-Z-dien-15-oic acid (10) was
38
obtained from Haplopappus deserticola. 13-epi- Sclareol (11) was obtained from
Pseudognaphalium heterotrichium and Pseudognaphalium cheiranthifolium.
OH
CO2H H CO2H
H
RO OH
AcO
(10) (11)
R = H (8)
R = Ac (9)
Diterpenoids isolated from Haplopappus foliosus, Haplopappus uncinatus, Pseudognaphalium
heterotrichium and Pseudognaphalium cheiranthifolium.
Minimum inhibitory amounts (MIA) of terpenoids against Bacillus cereus and

Staphylococcus aureus.
MIA values (µg)
Compound B. cereus S. aureus
1 0.16 0.32
2 ia ia
a
3 i ia
4 12.5 25.0
5 2.5 5.0
6 0.625 1.25
7 i i
8 0.625 1.25
9 0.625 1.25
10 6.25 12.5
11 3.2 25.0
a
MIC value > 25, considered inactive.
In seeking to establish structure-activity relationships from the MIA values lipophilicity seemed
an important variable, as we hypothesized that the bacterial lysis by these diterpenoids was due to
their insertion and disruption of the lipophilic cell membrane. However, although in a given series
the activity seemed to increase with the molecular lipophilicity, it was clear that other factors were
also important. Increasing the lipophilicity of the parent acids 1 or 6 by esterification of the
carboxylic function (compounds 2 and 7, respectively) led to a suppression of the activity. This was
an indication that a hydrogen-bond-donor (HBD) group strategically positioned in the molecule was
also an important requirement for activity. In fact, in addition to a lipophilic decalinic ring system,
all active compounds 1, 5, 6 and 8-11 possessed an HBD group. However, the analysis of this
second structural requirement was complicated by the observation that some compounds meeting
this requirement were either inactive or showed a very low activity. This was the case, for example,
of compounds 3 and 4, where the presence of two HBD groups actually led to a reduction or
39
suppression of the antimicrobial activity, if compared with the corresponding analogs 1 and 5,
where only one such group was present.
Two reasons may explain these observations. The first stems from the requirements of these
amphiphilic molecules. Optimum interaction with an amphipathic membrane would require a polar,
HBD substituent on a lipophilic skeleton. The presence of a second HBD group in the molecule
would reduce the lipophilicity of the hydrophobic moiety, rendering more difficult its interaction
with the hydrophobic region of the membrane. This argument, however, cannot explain the same
activity of compounds 8 and 9, since the latter, with a more hydrophobic decalinic fragment, should
be more active than the former.
A second explanation, based on intramolecular interactions between the two hydrophilic groups,
which might compete with intermolecular hydrogen-bonds between the HBD groups and the cell
membrane, may be invoked, In fact, X-ray diffraction analysis of ent-3ı -hydroxy-16-kaurenoic acid
(3) revealed strong intramolecular hydrogen-bonds between the carboxyl and the hydroxyl functions
of this compound. This argument, however, should apply where this intramolecular interaction is
favoured by the proximity of the hydrophilic groups. This is not always the case for some of the
analyzed molecules.
As stated above, whatever the causes of such reductions in activity, the picture that emerges at
this stage is still complex. Interaction of these terpenoids with cell membranes is probably an
important or a facilitating factor for their observed activity, but clearly other structural aspects must
operate in each particular case. Further investigation is required on the specific mode of action of
these compounds in order to rationalize these apparently contradictory observations.
In search of further support to our model, we simulated the insertion of kaurenoic acid (1) and its
derivatives 2 and 3 into a phosphatidylcholine (POPC) bilayer. The results are shown in Figures 1-3.
Figure 1 shows that kaurenoic acid (1) orients itself in the bilayer interface, with the hydrophobic
decalinic ring moiety surrounded by the hydrocarbon chains of the lipid. The hydrophilic carboxylic
group of 1 projects itself away from the hydrophobic region, interacting with the phosphorilated
groups of the micellar system through hydrogen-bonds like the one depicted, with an CO2H---O=P
distance of 1.91 Å
Figure 1. Mode of insertion of one molecule of kaurenoic acid (1) into a POPC bilayer,
showing the hydrogen-bond interaction between the carboxylic group of the diterpenoid and a
phosphoryl oxygen atom of the surfactant.
40
Figure 2. Mode of insertion of one molecule of methyl kaurenoate (2) into a POPC bilayer
Figure 3. Mode of insertion of one molecule of the ent–3-ı -hydroxy kaurenoicacid 3 into
a POPC bilayer. The –CO2H----O=P hydrogen-bond is now larger (3.11 Å) than the one
depicted in Figure 1 (1.91 Å).
In summary, an analysis of the activity of several antimicrobial terpenoids and of some of their
derivatives led to the identification of some structural requirements for their action. These structural
features included a substituted decalinic system, capable of insertion into a lipophilic region, and a
hydrophilic fragment possessing one hydrogen-bond-donor group, capable of interactions with
hydrogen-bond-acceptor groups in the membrane. Although in the case of the studied compounds,
there is no direct evidence for this model, simulations of the insertion of compound 1 and two of its
derivatives, 2 and 3, into a phopholipid bilayer were in full agreement with this proposal.
41
42
Simposio 2.
Ecología Química como Herramienta Biotecnológica
Presentación de Paneles.
43
44
MÉTODOS PARA DETERMINACIÓN DE CAPACIDAD ANTIOXIDANTE EN PLANTAS

NATIVAS CHILENAS
ALFARO, S. (1), SCHEUERMANN, E. (2), QUIROZ, A (3)
(1) Universidad de La Frontera, Programa de Doctorado en Recursos Naturales, Facultad de

Ingeniería, Ciencias y Administración, Casilla 54-D, Temuco Chile E-mail: s.alfaro01@ufromail.cl
(2) Universidad de La Frontera, Departamento de Ingeniería Química, Facultad de Ingeniería,
Ciencias y Administración, Scientifical and Technological Bioresources Nucleus (BIOREN), Casilla
54-D, Temuco Chile
(3) Universidad de La Frontera, Universidad de La Frontera, Departamento de Ciencias Químicas,
Facultad de Ingeniería, Ciencia y Administración, Casilla 54-D, Temuco, Chile
En Chile existe una flora nativa de gran riqueza botánica, cuyas especies particulares han sido
utilizadas desde tiempos remotos por nuestros pueblos indígenas quienes les atribuyen propiedades
curativas de sus extractos. El boldo (Peumus boldo Mol.), murtilla (Ugni molinae Turcz), matico
(Bluddeja globosa Hope) y calafate o michay (Berberis darwinii) representan algunas de estas
especies nativas de interés. El objetivo de este trabajo fue establecer un ranking con respecto a la
capacidad antioxidante de estas plantas nativas en base a dos métodos de medición de actividad
antirradicalaria. Se utilizaron hojas y frutos de murtilla cultivada, obtenidos de una estación
experimental de INIA-Carillanca en Puerto Saavedra, Región de La Araucanía. Las hojas de boldo y
matico fueron recolectadas en el jardín de la Universidad de La Frontera y las hojas de calafate se
recolectaron en el sector cordillerano de la Región de La Araucanía. Se determinó el contenido de
humedad según AOAC (1990). Las muestras de hojas y frutos se secaron y molieron antes de su
extracción con dos solventes: mezcla agua/etanol (50:50%) y metanol. Los extractos se utilizaron
para medir la actividad antirradicalaria por medio de prueba de inhibición del radical libre DPPH y
prueba TEAC modificado utilizando el radical libre ABTS. Los resultados de ambas pruebas fueron
expresados como la concentración efectiva del extracto (EC50%) en equivalentes Trolox® por gramo
de materia seca (m.s.). Se determinó el siguiente ranking en orden decreciente de la capacidad
antioxidante en base a la prueba TEAC: boldo hoja > calafate hoja > murtilla hoja > matico hoja >
murtilla fruto. Con la prueba DPPH, el ranking fue: boldo hoja > murtilla hoja > calafate hoja >
matico hoja > murtilla fruto. Con respecto al método utilizado existen diferencias en cuanto a que
con la prueba TEAC se obtienen valores menores de EC50% en µ M Trolox®/g m.s. comparados con
los obtenidos por la prueba de DPPH, lo cual concuerda con la literatura y que normalmente es
atribuido al radical libre y tipo de solvente utilizado en la extracción. Se concluye que por ambos
métodos de medición, las hojas de boldo presentan la mayor capacidad antioxidante entre las
especies nativas evaluadas en este estudio.
45
LETHAL AND NEUROTOXIC EFFECTS OF EXTRACTS FROM THE RED MARINE

ALGA Ceramium rubrum (HUDSON) (RHODOPHYTA, FLORIDEOPHYCEAE)
DETERMINED BY THE Artemia franciscana (KELLOG, 1906) BIOASSAY, AS
UNIVERSAL INDICATOR OF TOXICITY
Cortés, Ya; Pardo, F.b; Perich, F.b and Quiroz, A.b
a
Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera, Temuco
4811230, Chile
b
Laboratorio de Ecología Química, Departamento de Ciencias Químicas, Universidad de La
Frontera, Temuco 4811230, Chile
The oceans are a unique source of natural products. Many marine animals and plants have evolved
potent defense mechanisms against several pathogens, such as parasites, bacteria, viruses and fungi.
In the last 30 years, a small number of organisms have already yielded more than 12,000 novel
chemicals, with hundreds of new compounds still being discovered every year. These investigations
have yielded several bioactive metabolites that have been successfully developed by the
pharmaceutical industry. In this study, we investigated lethal and neurotoxic effects of two extracts
and three fractions from the red marine macroalga Ceramium rubrum in a short-term bioassay using
brine shrimp, Artemia franciscana. Tests were carried out with algae collected in Maullín (Los
Lagos Region, Chile) between December and February 2009. Total extract of C. rubrum was
obtained by exhaustive extraction with chloroform-methanol (1:1) and aqueous extract was obtained
according to the methodology described by Serkedjieva (2004). Three fractions of increasing
polarity were obtained from the total extract by liquid-liquid extraction: ethyl acetate, n-butyl
alcohol and aqueous. Extracts and fractions were concentrated in a rotory evaporator to obtain the
respective residues. The A. franciscana lethality was performed according to the methodology
described by Meyer et al. (1982). This bioassay consisted in placing 10 nauplii in an assay tube with
seawater and the required test concentration. The samples were incubated at 25 °C in darkness and
the mortality was recorded after 24 hours of exposure. Percent mortalities were used for estimating
the lethal concentration required for obtaining 50% of survival (LC50). The results allowed calculate
a LC50 between 12-17 ppm and 28-33 ppm for aqueous and total extracts, respectively. However, it
is necessary to improve the statistical level of the results (p < 0.03). On the other hand, the fractions
resulted not active at concentrations of 32 ppm, but the LC50 value will be determined. This result
would indicate that the fractions are less active than the total and aqueous extracts. Future work will
include two neurotoxic evaluations on A. franciscana: behavioral bioassays and acetylcholine
concentrations. For behavioral assays, 60 individuals will be placed into Petri dishes and exposed to
the LC50 doses for 24 hours. The survivors will be used to study the effect of compound on their
locomotor behavior with the computerized videotracking system Ethovision 3.1 (Noldus
Technologies-Netherlands). The acetylcholine concentrations will be determined according to the
methodology reported by Venkateswara et al. (2007). This experiment will consist in placing 200
nauplii of A. franciscana will be exposed to the LC50 doses for 24 hours, and other 200 will be
maintained into a control treatment by the same time. It is expected that the LC50 of extracts affect
the nauplii locomotor behavior after 24 hours of exposure, coinciding with the acetylcholinesterase
activity.
46
BEHAVIOURAL RESPONSES OF CLOVER ROOT BORES (Hylastinus obscurus)

(COLEOPTERA: SCOLYTIDAE) TO FATTY ACID IDENTIFIED FROM YOUNG AGED
RED CLOVER ROOTS (Trifolium pratense).
Manosalva L1, Pardo F1, Perich F1, Mutis A1, Parra L1, Ortega F2, Quiroz A1.
1
Laboratorio de Química Ecológica, Departamento de Ciencias Químicas, Universidad de La
Frontera, Casilla 54-D, GL-Temuco, Chile. 2Instituto de Investigaciones Agropecuarias, CRI-
Carillanca, Casilla 58-D, GL-Temuco, Chile. loretomanosalva@gmail.com
The red clover root borer, Hylastinus obscurus (Marsham), is one of the most serious pests of red
clover, Trifolium pratense L., throughout the world (Steiner and Alderman, 2003). Both larvae and
adults of this scolytid bore and feeds into the roots causing a significant reduction in both production
levels and persistence of red clover stands within two years after sowing (Cuevas and Balocchi,
1983; Steiner and Alderman, 2003). Pesticides have not been successful in controlling borer
infestations and at present there is no control for clover root borers other than crop rotation
(Aguilera et al., 1996). In a previous study performed with root extracts from 1.5 and 2.5-year-old
red clover plants, we determined that H. obscurus was attracted to root volatiles of 1.5-year-old
extracts but not to those from 2.5-year-old extracts (Tapia et al., 2007). In both root extracts were
identified methyl benzoate and E-2-hexenal that attracted H. obscurus and limonene that was
repellent towards borers. Quantification of the active compounds in root materials of red clover
revealed significant variation in their concentration depending on age of clover plants, which
allowed us to explain the loss of attraction of 2.5-year-old clover roots towards the borer. Thus, the
objectives of this study were to analyze, qualitatively and quantitatively, by GC-MS the long-chain
free fatty acid content in root extracts from 9-month-old red clover obtained by solvent extraction
and also to test the behavioural response of H. obscurus towards root extracts as well as to free fatty
acids therein identified in olfactory using an automated system in the detection and recording of the
insect behaviour. GC-MS analysis and Kovats indices allowed identification of lauric acid, palmitic
acid, oleic acid and stearic acid as the main long-chain free fatty acids in the root extract. In a four-
arm olfactometer the clover root extract significantly attracted both male and female of H. obscurus.
When the four pure identified fatty acids and their blend at ratio found in the root extract were
assayed at 10 µ g/mL on the insect, lauric acid, palmitic acid and oleic acid elicited an attractant
behavioural response from female individuals, whereas all substances tested were inactive on
conspecific individual males. The results suggest the long-chain free fatty acids presents in 9-month-
old red clover roots might play a role as close-range olfactory cues in host-finding process by female
H. obscurus.
FONDECYT 1070270
47
EVIDENCE OF SEXUAL PHEROMONE IN THE RASPBERRY WEEVIL Aegorhinus

superciliosus (COLEOPTERA: CURCULIONIDAE).
Mutis, A.1,2; Parra, L.1,2; Manosalva, L.1,2; Palma, R.1,2; Lizama, R2, Pardo, F3; Perich, F3 and
Quiroz, A2,3
1
Doctorado y Magister en Ciencias de Recursos Naturales, Universidad de La Frontera, Temuco,
Chile.
2
Frontera, Temuco, Chile.
3
Departamento de Ciencias Químicas, Universidad de La Frontera, Temuco, Chile.
Aegorhinus superciliosus (Guérin) (Coleoptera: Curculionidae), is the principal pest of blueberry

and raspberry in the South of Chile. In this work, are showed electrophysiological, and behavioral
responses of A. superciliosus to the semiochemicals released from adult individuals, and field effect
of the most active compound. Volatiles released from females significantly attracted conspecific
males in olfactometric bioassays. However, female volatiles did not attract other females, and male
volatiles attracted neither males nor females. Gas chromatographic and mass spectral (GC-MS)
analysis of volatile extracts of females revealed the presence of limonene, and α-pinene.
Electroantennogram recording indicated that A. superciliosus females have the ability to smell R-
limonene, and males to smell both R-limonene and α-pinene. However, olfactometric bioassays with
pure compounds indicated that only R-limonene is capable of evoking a response, as evidenced by
the attraction to the area developed by the stimulus males. The field results reinforce the laboratory
assay data, the results shown that R-limonene caught more weevils than control treatment,
meanwhile the number of males fallen in the traps was larger than females.
Key Words: Aegorhinus superciliosus, electroantennogram, behavioral bioassay, R-limonene, Field

assays.
48
RESISTANCE TO PYRETHROIDS IN Haematobia irritans (DIPTERA: MUSCIDAE) AND

DETECTION OF kdr MUTATION IN CHILE
María Paz Oyarzún, Christian Figueroa
Instituto de Ecología y Evolución, Facultad de Ciencias, Universidad Austral de Chile, Isla Teja
S/N, Valdivia, Chile. mpoyarzun@uach.cl
The horn fly its and important biting fly species that parasites cattle in Chile and other countries
throughout America. Control of this pest has been relied in chemical insecticides, mainly synthetic
pyrethroids and organophosphates (OP). However, in the last years, farmer’s complaining of control
failures with pyrethoids have been alerted of development of resistance in horn fly populations.
Whenever a good strategy of pest control is required, it is important to know features modulating
population’s susceptibility to insecticides. Nevertheless, so far none survey has been made in our
country, in order to determine susceptibility of horn flies to most common insecticides used in field.
The objective of this survey is to determine susceptibility of horn fly populations in Chile to
cypermethrin and diazinon using a filter paper bioassay technique, and to detect mechanisms of
target-insensitivity underlying resistance using polymerase chain reaction assays (PCR) to detect the
presence of kdr allele (associated to pyrethroid resistance) and HiαE7 esterase allele (associated to
OP resistance). Metabolic mechanisms are been currently investigated as well. In summer 2009,
horn flies were sampled from Holstein Friesian cattle at eight ranches in southern Chile. Sampled
populations exhibited from 49 to 390 fold cypermethrin resistance than reference susceptible
laboratory population (Kerrville, USA), whereas all populations were highly susceptible to diazinon
and no mutant HiαE7 esterase allele were detected. Allele kdr was present in all horn fly
populations (allelic frequency 61% average). Two ranches where horn fly is not under control with
insecticides had the lower allelic kdr frequency (43 y 47%). When individuals of horn fly population
were genotyped as resistant (RR) or susceptible (SS) homozygotes or heterozygotes (SR), the higher
frequency of SS and SR genotype was found on same populations from untreated cattle, whereas
populations under highly pyrethroids chemical control had the higher frequency of RR individuals.
These preliminary results, demonstrate for the first time that horn fly populations in Chile are
resistant to pyrethroids, likely by kdr mutation. Results of ongoing research will aid to design
effective strategies of control within the framework of integrate pest managemt involving still
effective insecticides and non-selective treatments.
49
ELECTROANTENNOGRAPHIC RESPONSE OF CLOVER ROOT BORER Hylastinus

obscurus (COLEPTERA: SCOLYTIDAE) TO COMPOUNDS IDENTIFIED IN VOLATILES
FROM CONSPECIFIC AND ITS HOST.
Palma, R.1;, Mutis, A.1; Manosalva, L.1 and Quiroz, A.2
1
Programa de Doctorado y Magister en Ciencias de Recursos Naturales, Universidad de La
Frontera, Temuco, Chile
2
Laboratorio de Ecología Química, Universidad de La Frontera, Temuco, Chile.
3
Departamento de Ciencias Químicas, Universidad de La Frontera, Temuco, Chile. rpalma@ufro.cl
The electroantennographic activity of Hylastinus obscurus was studied as to compounds identified

in its environment as to others reported previously by their activity in different scolytid species.
Thus, adult individuals were collected from field. And their heads were separated from the body and
mounted on micromanipulators in order to connect the indifferent electrode to the base of the head,
and the recording electrode to the tip of one antenna. The electrodes consisted of silver wires inside
glass capillaries filled with an electrolytes solution. These preparations were exposed to a stream of
humid and charcoal-purified air, while the stimuli were applied using disposable syringes as odor
cartridges. The results shown all tested compounds elicited responses larger than clean air either to
males and females, suggesting those semiochemicals are recognized by the antennae. The largest
response were obtained by E-2-hexenal, ethanol and hexanal, either by males and females; which
are distinguishable from those obtained by terpenic compounds, such as α-pinene, β-pinene y
limonene. Finally, this is the first physiological approach about the olfaction ability of this borer.
Acknownledgment: Fondecyt 1070270, Conicyt AT24090126
Keywords: Electroantennography, volatiles, Hylastinus obscurus.
50
BIOLOGICAL AND CHEMICAL FACTORS ASSOCIATED IN THE INTERACTION

BETWEEN Aegorhinus superciliosus (COLEOPTERA: CURCULIONIDAE) AND ITS HOST
Vaccinium corymbosum L.
L. Parra1,2, A. Mutis1,2, F. Perich3, F. Pardo3 and A. Quiroz2,3
1
Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera, Temuco, Chile
2
Frontera, Temuco, Chile.
3
Departamento de Ciencias Químicas, Universidad de La Frontera, Temuco, Chile.
Corresponding author: aquiroz@ufro.cl
The objective of this study was to evaluate the role of host volatiles in the relationship between
Vaccinium corymbosum L. and Aegorhinus superciliosus (Guérin), the principal pest of blueberry in
the south of Chile. Volatiles from the aerial part of different phenological stages of the host were
collected on Porapak Q and analyzed by coupled gas chromatography-mass spectrometry (GC-MS).
Several chemical groups were identified including green leaf volatiles, aromatic compounds and
terpenes. The olfactometric responses of A. superciliosus towards different odor sources were
studied in a four-arm olfactometer. Blueberry shoots at the phenological stages of fruit set and blue-
pink elicited the greatest behavioral responses from weevils. Five compounds (2-nonanone,
eucalyptol, R- and S- limonene and 4-ethylbenzaldehyde) elicited an attractant behavioral response
from A. superciliosus. However, only with eucalyptol was obtained differential response between
females and males Subsequently, EAG tests confirmed the biological activity of insect toward these
compounds. In field test, no significant differences in the capture of A. superciliosus. The results
suggest the host location behavior of A. superciliosus is mediated in part by volatiles derived from
V. corymbosum. This work has identified a number of compounds with which it is possible to
develop a lure for control the principal pest of blueberry in southern Chile.
Financial support: CONICYT (Project AT 24080090) and FONDECYT (Project 1070270)
51
ANTIFUNGAL ACTIVITY OF VOLATILES EMITTED BY MICELIAL CULTURES OF

CHILEAN SAPROPHYTIC FUNGI
Schalchli, H. 1, Hormazabal, E. 2 and Quiroz, A.2
1
Doctorado en Ciencias de Recursos Naturales. Laboratorio de Química Ecológica. Departamento
de Ciencias Químicas, Universidad de La Frontera. Casilla 54-D. Temuco, Chile.
hschalchli@ufro.cl. 2 Laboratorio de Ecología Química. Departamento de Ciencias Químicas,
Universidad de La Frontera. Casilla 54-D. Temuco, Chile.
Fungi produce and emit Volatile Organic Compounds (VOCs) including several classes of
chemical substances (e.g. hydrocarbons, alcohols, ketones, aldehydes, ethers, esters, terpenes,
terpene derivatives, and a variety of sulphur and nitrogen compounds). These compounds have been
studied for a variety of purposes, including as natural aromatic flavouring, indicators of fungal
contamination and biocontrol agents (BCAs) of agricultural pest plants. The biological activity of
volatiles produced by fungi has been tested on several plant pathogenic organisms, such as fungi,
bacteria, nematodes and insects. The antifungal activity of these volatile metabolites on plant
pathogenic fungi shows a potential application in biological control strategies of fungal plant
diseases. The aim of this study was to evaluate the antifungal activity of volatile metabolites emitted
by saprophytic fungi growing in the temperate native forest of the South of Chile on the mycelial
growth of plant pathogenic fungi that causes important fungal diseases (Botrytis cinerea, Fusarium
sp. and Mucor miehei). The results shown that volatiles emitted by the twelve strains of saprophytic
fungi tested inhibit the mycelial growth of B. cinerea, Fusarium sp. and M. miehei, evaluated by bi-
compartmented Petri dish assays. Except volatiles of Trametes versicolor, volatiles emitted by this
specie stimulated the mycelial growth of Fusarium sp. about 1.4%. The inhibition, expressed in
percentage of inhibition in relation to the control, was dependent of the volatile-producing fungus.
Shizophyllum commune and Marasmiellus alliodorus inhibited 24,9 % ± 1,4 the micelial growth of
B. cinerea and, Aleuria aurantia inhibited 19,6% ± 3,9 and 22,2% ± 1,3 the micelial growth of
Fusarium sp. and M. miehei, respectively. We conclude that volatiles emitted by S. commune, M.
alliodorus and A. aurantia were the most bioactive volatile-producing fungi on pant pathogenic
fungi growth tested in this study. However, it is necessary to evaluate the effect of the increment of
biomass of volatile-producing fungus on the bioactivity of VOCs emitted by these fungi and to
identify the VOCs involved in antifungal activity.
Keywords: antifungal activity, VOCs, saprophytic fungi.
52
SEMIOCHEMICALS INVOLVED THE MATING BEHAVIOUR OF Artemia spp.

Tapia C1, Quiroz A1, Gajardo G2.
1
Doctorado en Ciencias de Recursos Naturales, Laboratorio de Ecología Química, Universidad de
La Frontera. Temuco, Chile. E-mail: c.tapia02@ufromail.cl
2
Laboratorio de Genética & Acuicultura, Universidad de los Lagos. Osorno. Chile
Microcrustaceous Artemia is of great importance in terms of food for aquaculture (FAO, 1986). The
genus is composed by di-, tri-, tetra- and pentaploid parthenogenetic population and eight bisexual
species. However, it is still questioned the existence and characterization of new populations
(Triantaphyllidis, et al., 1998). The study of semiochemicals of crustacean was initiated in 1960, but
the species studied are few (Stebbing et al 2003) and there is no knowledge of the existence of
pheromones in brine shrimp. This information can be important for several areas, in the fisheries for
being used as baits and in aquaculture for the manipulation of the mating behaviour (Bauer, 2004).
This work will focus in the study of the semiochemicals involved in the mating behaviour of
Artemia spp. In a first step was standardized the methodology for studying the mating behavior in
Artemia spp. mediated by semiochemicals. This study was based in the work reported by Pino-
Marambio et al. (2008); in a second step, it was evaluated the biological activity elicited by
chemical compounds released from Artemia spp. on conspecifics of the same and different sex.
The experiment consisted in placing a glass Y tube arena on a top of a light box, above which a
digital camera was placed for tracking the movement of Artemia during bioassays. The Y tube was
divided in three zones: Leg, Control arm and Stimulus arm. For each experiment, a single Artemia
was introduced into the base of the leg. The movements during 10 min were recorded by frame
grabber into a computer, and the entire tracks were analyzed using behavioural tracking software
(Ethovision 3.1, Noldus Technologies). A flask (5 L) containing six alive organisms in artificial
seawater (Sera, Aquarium salt) and salinity 35 ‰. was connected by means of Teflon tubing to one
of the Y-tube arms, another flask with artificial seawater was connected to the control arm. Water
from both flask flowed thought the Y-tube at30mL/min.
The results indicate that the study of semiochemicals involved in intraspecific relationships
between Artemia individuals can be developed using the Ethovision tracking software. Moreover,
preliminary results would indicate that the mating behavior in Artemia could be mediated by
pheromonal semiochemicals.
53
ESTUDIO DE COMPUESTOS VOLÁTILES, CAPACIDAD ANTIOXIDANTE Y

ANTIBACTERIANA DEL ACEITE ESENCIAL DE TEPA (LAURELIOPSIS PHILIPPIANA
(LOOSER) SCHODDE). BASES PARA SU POTENCIAL USO Y APLICACIÓN.
1
Toledo, D., 1Mutis, A., 2Hormazabal, E., 3Scheurmann, E. y 2Quiroz, A.
1
Doctorado en Ciencias de Recursos Naturales. Universidad de la Frontera. Temuco, Chile.
dayand.toledo@gmail.com. 2Laboratorio de Ecología Química, Departamento de Ciencias
Químicas. Universidad de la Frontera. Temuco, Chile. 3Departamento de Ingeniería Química.
Universidad de La Frontera. Temuco, Chile.
El interés por la flora nativa como fuente de compuestos activos ha aumentado considerablemente
en el último tiempo, debido a la tendencia mundial de reemplazar el uso y consumo de compuestos
sintéticos que provocan efectos secundarios negativos para la salud de los seres humanos y/o para el
medio ambiente, por extractos o compuestos naturales. Uno de los extractos de mayor uso en la
medicina natural, lo constituyen los aceites esenciales, los cuales son una mezcla compleja de
compuestos químicos volátiles de origen natural, son aromáticos y provienen en su mayoría de
especies vegetales. El campo de utilización de los aceites esenciales es muy amplio, siendo aplicable
industrialmente en diversos sectores. En la industria farmacéutica, alimenticia, cosmética y de los
perfumes, tienen un rol protagónico, ya que una variada gama de estos productos tienen como base
irremplazable esencias naturales, las cuales actúan como saborizantes, aromatizantes o exhibiendo
variadas actividades biológicas, entre las que destacan la actividad antioxidante y antimicrobiana. El
método de extracción de aceites esenciales más utilizado y económico es la destilación por arrastre
de vapor, en este proceso se obtiene como producto secundario el hidrolato que es la fracción
soluble de las moléculas aromáticas de la esencia. La actividad biológica de la especie arbórea
nativa Laureliopsis philippiana, ha sido asociada a la presencia de aceite esencial en las hojas. Esta
especie de follaje siempreverde y de extensa distribución en Chile despide una agradable fragancia y
ha sido utilizado medicinalmente por el pueblo mapuche. Esta investigación se fundamenta en el
estudio del aceite esencial de tepa (Laureliopsis philippiana) desde un punto de vista químico y
biológico para contribuir al conocimiento científico de la flora vascular Chilena y proporcionar
información útil para los pequeños y medianos propietarios de bosque nativo, con la finalidad de
incentivar la diversificación de productos no madereros y promover que la industria alimenticia,
farmacéutica y cosmética utilicen como materia prima al aceite esencial de tepa, para de esta manera
dar valor agregado a esta especie forestal nativa. A la fecha existen muy pocas referencias de
estudios relacionados a la composición química y propiedades biológicas de esta especie. En esta
investigación se obtuvo aceite esencial de tepa por medio de hidrodestilación, con un rendimiento
promedio de 0,43%. Se identificaron 22 compuestos que representan el 99,7% del aceite en estudio,
siendo linalol (56,1%), eucaliptol (15,4%) y α-terpineol (4,84%), los principales componentes de
naturaleza monoterpénica. La actividad antibacteriana será evaluada por difusión en agar y se
determinará la concentración mínima inhibitoria (CMI) y bactericida (CMB) sobre las cepas. Hasta
la fecha se tiene como resultado la CMI de las cepas, siendo para E.coli (ATCC 25922) 6 µg/mL, E.
aerogenes (ATCC 13048) 20 µ g/mL, S.aureus (ATTC 25923) 32 µ g/mL y S.epidermidis (ATCC
12228) 35 µ g/mL. Se espera que estos resultados promuevan al aceite esencial de tepa como materia
prima para la producción de fitofármacos y para dar valor agregado a los productos que se elaboran
en la actualidad.
54
Simposio 3.
Microbiología y Rizosfera
Moderadores Dr. Milko Jorquera; Dr. Fernando Borie.
1. Dr. Fernando Borie, Universidad de La Frontera, Chile: “Micorrizas

arbusculares en ecosistemas agrícolas y forestales de Chile”
2. Dr. Pablo Cornejo, Universidad de La Frontera, Chile: “Hongos micorrícico

arbusculares y su rol en la estabilización de ecosistemas afectados por
actividades mineras”
3. Dra. Concepción Azcón-Aguilar, Estación Experimental del Zaidin (CSIC),

España: “Hongos formadores de micorrizas arbusculares en biocontrol de
patógenos”.
4. Dra. Rosario Azcón, Estación Experimental del Zaidin (CSIC), España:

“Efectividad de residuos agroindustriales biotrasformados y su interacción
con inoculantes microbianos para su uso en revegetación”.
5. Dr. Roberto Godoy, Universidad Austral de Chile: “Micorrizas en bosques

templados de la zona Centro-Sur de Chile”.
6. Mg. María Teresa Fernández, ICIDCA, Cuba: “Construcción de vectores para

mejorar la utilización de fosfato y fitato por rizobacteria”.
7. Dr. Milko Jorquera, Universidad de La Frontera, Chile: “Bacterias

degradadoras de fitato y su potencial biotecnológico en agricultura”.
8. David Crowley, University California, USA: “The ecology of bacterial

communities in the plant rhizosphere”.
55
56
ARBUSCULAR MYCORRHIZAE IN AGRICULTURAL AND FOREST ECOSYSTEMS

OF CHILE.
F. Borie, R Rubio, A. Seguel & P.Cornejo.
BIOREN-Universidad de La Frontera. P.O.Box 54-D Temuco. Chile. E mail: fborie@ufro.cl
Acidity and scarcity of available phosphorus alone or together are the most serious limitations of
agricultural productivity worldwide specially in developing countries where food production is
essential. It has been estimated that more than 30% of the word total land have both negative
characteristics. It is recognized that plant growth in acid soils (pH< 5.5) is generally depressed by a
complex of severe conditions including excessive H+ ions, Al and/or Mn phytotoxicity together with
some essential mineral nutrients deficiencies such as P, Ca, Mg and Mo. However, it is accepted
that the main deleterious effect is assigned to free Aluminum, which mainly negatively affect fine
roots growth with a concomitant reduction in nutrient acquisition. There is a lot of studies which
demonstrate that such adverse effects can be alleviated by applying some agricultural practices all
of them focused to diminish Al activity. Calcitic and dolomitic liming, compost addition and
phosphate fertilizer application are habitually used by worldwide farmers for reducing Al
phytotoxicity. Liming and phosphate fertilization are the main strategies used by local farmers from
Southern Chile for overcoming toxic effects. Other strategy is to use Al-tolerant crop genotypes.
Arbuscular mycorrhiza (AM) is a widespread terrestrial symbiosis that help plants in nutrient
acquisition, especially P, Ca, Mg, Cu and Zn being the most important nutritional effect the increase
in P uptake. If mycorrhizal plants increase the efficiency in absorption, transporting and transferring
nutrients like P, Ca and Mg all of them antagonist to Al injury of plant roots, AM symbiosis could
be playing a key role in decreasing Al toxicity. Such a role conferring Al tolerance to their plant
hosts have been confirmed in the ninety’s by some researchers working with tropical plants and
even with crops growing in temperate ecosystems. More recently, it have been concluded thet some
AM fungal strains give higher Al tolerance to plants through a higher organic root which decrease
the concentration of free Al on their root zones. Such studies have provided evidences that variation
among AM fungal species in Al resistance conferred to plant hosts is associated with the root
exudation of Al binding organic acids and highlight the potential importance that AM fungal
diversity can play in decreasing the adverse conditions for plant growth in acidic soils. In addition,
at the end of ninety’s it have been postulated that AM fungal strains produce a glycoprotein named
glomalin which have shown to bind Fe in the molecule and recently we have demonstrated that
glomalin extracted from Andisols have also Al together Fe. Glomalin production as well as organic
acid exudated by mycoorhizal roots s would be interesting ways of decreasing phyototoxic Al.
In this work we discuss the role played by mycorrhizal fungi in acidic soils from Southern
Chile in terms of phosphate nutrition but mainly focused to deep in the knowledge of the potential of
some native mycorrhizal strains in conferring higher Al-tolerance to some cereal crops.
57
ARBUSCULAR MYCORRHIZAL FUNGI AND ITS ROLE IN STABILIZING

ECOSYSTEMS POLLUTED BY MINING ACTIVITIES
Pablo Cornejo*, Sebastián Meier, Cecilia García & Fernando Borie
Center of Amelioration and Sustainability of Volcanic Soils. Scientific and Technological

Bioresource Nucleus BIOREN-UFRO, Universidad de La Frontera, Avda. Francisco Salazar 01145,
P.O.Box 54-D, Temuco, Chile. *pcornejo@ufro.cl
The activities associated with cooper (Cu) mining, the main economic activity in Chile, have
affected several natural ecosystems, producing a lot of environmental effects difficult to solve in the
near future. In central Chile large areas oriented to agricultural activities have been affected,
reducing their stability and productivity. The most visible aspect of this adverse effect has been the
reduction of stability, diversity, structure and plant cover of the different ecosystems, either natural
or agroecosystems. It is well known that the main trouble caused by mining in natural ecosystems is
the discharge of metals (such as Hg, Pb, Cd, Cr, As, Zn or the same Cu, among others), which are
toxic for the majority of organisms. While this is one of the main reasons for the loss of biodiversity
in natural ecosystems, it is also a mechanism for the selection of tolerant/resistant organisms to this
kind of abiotic stress. This kind of pollution has been scarcely studied in our country, and even very
few studies have been carried out related to the role which the soil microorganisms can be playing
under environmental stress conditions originated by high Cu levels.
However, the use of soil microorganisms such as arbuscular mycorrhizal fungi (AMF) which
have shown a recognized detoxification capacity, appears as an ecologically, environmentally and
economically more feasible alternative to be established on a large scale in recovery/restoration
programs that involve the use of plants in soils (and ecosystems) who have been affected by the
deposition of materials enriched with Cu.
The studies carried out in our group show an important AMF diversity associated with endemic
metallophytes (in terms of species richness and community structure). The molecular identification
revealed the existence of ecotypes of Acaulospora aff. lacunosa that are found throughout the Cu-
polluted ecosystem studied, and most particularly in the rhizosphere of Oenothera affinis. In vivo
studies also have shown that colonization with AMF ecotypes present in Cu-polluted soils increase
the Cu concentration in endemic metallophyte species, especially in root. For this reason, its
combined use could be a tool to be focused in phytostabilization of soils affected by the deposition
of Cu-enriched particles. In addition, glomalin (Glomalin-Related Soil Protein, GRSP), a protein
produced by AMF and released to soil in large quantities, has shown a high ability to immobilize
this element in its molecule. Recent results suggest that the amount of Cu immobilized by GRSP can
be proportional to the level of soil pollution. Therefore, the selection of adapted AMF ecotypes and
with high glomalin production capacity could be an alternative for the stabilization of Cu in soils
with high levels of this element. The usefulness of the different mechanisms present in AMF
colonized plant to reduce toxic Cu levels in soil, as well as future prospects of its implementation
will be discussed in the presentation.
Acknowledgements: This research is supported by Fondecyt, Grant 11080131 (Initiation Into

Research) and Grant 3070052 (Postdoctoral Research), from Comisión Nacional Científica y
Tecnológica, Chile, and the “Programa de Formación de Investigadores Postdoctorales para su
Inserción en la Academia” from Universidad de La Frontera and MINEDUC, Chile.
58
PAPEL DE LAS MICORRIZAS ARBUSCULARES EN LA PROTECCIÓN DE LA PLANTA

FRENTE A PATÓGENOS
M. J. Pozo, J. A. López-Raéz, S. C. Jung, C. Azcón-Aguilar
Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del

Zaidín, CSIC, Profesor Albareda 1, 18008 Granada, España
Las micorrizas arbusculares son simbiosis mutualistas que se establecen entre hongos microscópicos
del suelo y raíces de la mayoría de las plantas. En esta asociación el hongo coloniza biotróficamente
la raíz de la planta, se extiende por el cortex radical y penetra en las células del mismo originando
las estructuras que dan nombre a la simbiosis, los arbúsculos. El establecimiento de la simbiosis
implica el intercambio de una serie de señales de reconocimiento entre ambos simbiontes y la
entrada en funcionamiento de programas genéticos complejos. Estos programas conducen a cambios
en la organización y el metabolismo celular que facilitarán, en último extremo, la colonización de la
planta por el microsimbionte y la acomodación física y funcional de ambos organismos. Como
consecuencia de la formación de la simbiosis se producen una serie de cambios en la fisiología de la
planta que resultan en una mejora del estado nutricional de la misma, alteraciones en los niveles
hormonales, incrementos en la actividad fotosintética, etc… Las plantas micorrizadas presentan en
general un mayor grado de tolerancia a estreses ambientales, tanto de tipo biótico (patógenos) como
abiótico (estrés hídrico, salino, exceso de metales pesados, etc..). En relación al efecto de la
simbiosis en interacciones patogénicas, cabe destacar que las plantas micorrizadas presentan
normalmente un mayor grado de tolerancia/resistencia frente al ataque de determinados patógenos,
especialmente de aquellos que atacan el sistema radical. Esta protección se ha demostrado efectiva
frente a nematodos, hongos, oomicetos y determinadas bacterias. Entre los mecanismos implicados
en este mayor grado de protección cabe mencionar el mejor estado nutritivo de las plantas
micorrizadas, la competición por carbohidratos y sitios de colonización entre el hongo de la
micorriza y el patógeno, la compensación de los daños provocados por el patógeno, la inducción de
cambios en la arquitectura radical de la planta, cambios en las poblaciones microbianas de la
rizosfera y activación de los mecanismos de defensa de la planta. También se han puesto de
manifiesto la existencia de efectos de protección a nivel sistémico, es decir, mediados por la planta.
Los estudios del efecto de las micorrizas sobre enfermedades que afectan a la parte aérea de la
planta son bastante mas escasos y menos concluyentes, con resultados que apuntan hacia la
protección frente a determinados patógenos, pero también a una mayor susceptibilidad o
agravamiento de la enfermedad frente a otros. En esta presentación se discutirán los efectos de las
micorrizas arbusculares sobre las susceptibilidad o resistencia de la planta a distintos tipos de
organismos perjudiciales, con especial énfasis en los mecanismos implicados en la respuesta de las
plantas micorrizadas frente a los distintos tipos de patógenos.
59
EFECTIVIDAD DE RESIDUOS AGROINDUSTRIALES BIOTRASFORMADOS Y SU

INTERACCIÓN CON INOCULANTES MICROBIANOS PARA SU USO EN
REVEGETACIÓN
R. Azcón
Departamento de Microbiología del Suelo y Sistemas Simbióticos. Estación Experimental del Zaidín
(CSIC). Profesor Albareda nº 1, 18008 Granada, Spain.
La principal característica que presentan las zonas expuestas a estreses medio ambientales como la
falta de agua o exceso de metales pesados es una limitada cubierta vegetal y una alta degradación
del suelo. Una estrategia efectiva para su recuperación es el establecimiento de especies vegetales ya
que evitan la erosión e incrementar la calidad del suelo. Para ello, tanto el uso de microorganismos
asociados a las raíces vegetales como la aplicación de residuos orgánico biotransformados
contribuyen al mejoramiento de las condiciones suelo/planta. Los microorganismos rizosféricos,
particularmente los formadores de la asociación micorriza, resultan casi imprescindibles para el
crecimiento de las plantas en situación de estrés ya que no solo son estimuladores de la nutrición y
el desarrollo vegetal sino que además atenúan los efectos nocivos del estrés ambiental. Los residuos
orgánicos biotransformados mejoran la calidad del suelo y su interacción con el inóculo microbiano
provoca los mayores estímulos tanto en el crecimiento y nutrición vegetal como en la fertilidad y
estructura del suelo. Las bacterias rizosféricas estimuladoras del desarrollo vegetal también
incrementan su supervivencia y actividad en presencia de la enmienda que supone una fuente
energética para el metabolismo microbiano. Por ello, la interacción de microorganismos
estimuladores resulta más estable y efectiva en presencia de la enmienda orgánica. El residuo
biotransformado estimuló el desarrollo extraradical del micelio micorrízico, la estructura y
biodiversidad de la comunidad microbiana del suelo y además, incrementó la formación de
agregados y actividades enzimáticas rizosféricas. Estos efectos son particularmente relevantes
cuando el estrés ambiental provoca un desarrollo vegetal muy limitado. Se ha determinado que el
efecto de la micorrización disminuyendo la translocación de metales de suelo contaminado a la
planta es potenciada por la enmienda. En condiciones de estrés hídrico la interacción enmienda-
micorriza afectó los valores de agua disponible en el suelo y estimuló el desarrollo vegetal a un nivel
equiparable al de las plantas crecidas sin limitación hídrica. Este efecto beneficioso no fue
transitorio sino que se mantuvo en cortes sucesivos. La tolerancia vegetal al estrés (en términos de
actividades antioxidantes) es potenciada por estos tratamientos conjuntamente aplicados.
60
MYCORRHIZAS OF TEMPERATE FORESTS IN SOUTH-CENTRAL CHILE

Godoy, R.1, Etcheverría, P.2 & Boy, J.3
1
Instituto de Botánica, Universidad Austral de Chile, Valdivia, Chile. rgodoy@uach.cl
2
Departamento Ciencias Químicas, Universidad de la Frontera, Temuco, Chile
3
Institute of Soil Science, Leibniz University Hannover, Germany
Chilean Old-Growth Forests constitute an important part of the world’s temperate rainforest reserve.
They are relevant for biodiversity conservation and provision of several environmental services,
both on local and global scale. Among these services the high ability of Chilean Old-Growth Forests
to sequester carbon is increasingly gaining attention in a changing world. Growing under a low
element deposition regime scarcely altering nutrient cycling, these highly productive pristine forests
have evolved efficient mechanisms to retain nutrients even under elevated leaching due to high
precipitation (> 5000 mm year-1). Especially Nothofagus stands on volcanic soils are N and P
limited. Avoiding losses of these nutrients, either in dissolved or gaseous form, therefore represents
an essential adaptation strategy in which mycorrhiza plays a key role.
Here we revisit the first descriptions of mycorrhizal associations with vascular plants of Old-
Growth Forests along with the studies on mycorrhizal processes and functioning, putting special
emphasis on endemic conifers and local Nothofagus species. Furthermore, we present controlled
inoculation experiments in laboratory, greenhouse and nursery, review the opportunities of applied
mycorrhizal research, and finally examine the tools on offer for the restoration of degraded land. All
this knowledge is crucial for understanding, modeling, and forecasting environmental change and
should be considered in sustainable resource management, conservation programs, and the design of
future research.
61
CONSTRUCTION OF VECTORS TO IMPROVE BOTH PHOSPHATE AND PHYTATE

UTILIZATION BY RHIZOBACTERIA
Fernández, M.T.*, Gonzáles, T., Fraga, R., Rodríguez, H. and Goire, I.
Instituto Cubano de Investigación de los Derivados de la Caña de Azúcar (ICIDCA), Vía Blanca
804, AP 4026, CP 11000, Ciudad de la Habana, Cuba.
*e-mail: maritere.fernandez@icidca.edu.cu
The current environmental problematic has generated the need to develop new methods to increase
agricultural yields and the development of a sustainable agriculture around the world. To avoid the
negative effects of current fertilization methods, a global effort is needed for develop biofertilizers
that allow a more efficient use of the phosphorus (P) in agricultural soils. This technology requires
the development of systems that allow the efficient transfer of genes involved in the P solubilization
by bacterial strains, under biological safety procedures. The insertion of these genes in organisms
that lack the ability to solubilize P would eliminate the need for mixing different bacterial
populations used as inoculants. In this study, we constructed three vectors for chromosomal
integration of: 1) phoC gene, which encodes an acid phosphatase enzyme, 2) PQQ operon from
Klebsiella pneumoniae which is cofactor acting in the P solubilization, and 3) phyL gene from
Bacillus licheniformis, a known phytase-producing rhizobacteria. These genes were cloned into the
suicide delivery vector pJMT6 (a derivative vector from the pUT/mini Tn5 system). The pJMT6 was
initially used to genetically modify Pseudomonas putida KT2442 through random integration of
mini-Tn5 in its chromosome. This vector also carries the genes kilA telAB, which encode tellurite
resistance, a selection marker used for the construction of strains intended to be released to the
environment. The recombinants construction was transformed and expressed in Escherichia coli
CC118λpir. Also, it was obtained a recombinant strain, Pseudomonas putida N-14, isolated from
rhizosphere of papaya with phoC gene integrated into its chromosome. The recombinant strain
expressed the introduced gene, showed a significant acid phosphatase activity compared with the
parental strain which did not produce the enzyme. The gene integration in the chromosome of P.
putida N-14 did not affect the production of siderophores. The obtained results have allowed the
genetic improvement of a rhizobacterial strain and its promising use as biofertilizer.
62
PHYTATE-DEGRADING BACTERIA AND THEIR BIOTECHNOLOGICAL POTENTIAL

IN AGRICULTURE
Jorquera, M.A.* and Mora, M.L.
Center of Plant, Soil Interaction and Natural Resources Biotechnology, Scientifical and
Technological Bioresource Nucleus, Universidad de La Frontera, Av. Francisco Salazar 01145,
Temuco, Chile. *e-mail: mjorquera@ufro.cl
Phytate is one of the most abundant organic phosphorus (P) compounds in many soils. Chilean
volcanic soils have a great content of total P and organic P, but their availability for plants is low.
The rhizosphere of plants harbours a wide diversity of microorganisms, including bacteria able to
release phosphates from organic and inorganic P forms through organic acids production and
enzymatic activity (phosphatases and phytases). The capacity to utilize insoluble phytate, as sole P
source, on agar media by culturable bacteria has been evaluated in rhizosphere of pasture plants
grown in Chilean volcanic soils. From diverse pastures (natural and under fertilization), around 46-
97% of culturable bacteria were capable to mineralize phytate. The use of degenerate primer set,
designed for amplification of Bacillus phytase gene, showed positive DNA amplification in 1-12%
of rhizospheric isolates. Sequencing of 16S rRNA and phytase genes revealed the presence of
Paenibacillus isolates carrying 3-phytase gene in three sampled pastures. The software Cn3D, freely
available in NCBI website (http://www.ncbi.nlm.nih.gov), confirmed that the detected phytase in
Paenibacillus isolates belong to β-propeller phytase group. In addition, phytase activity (2.1 mU ml-
1
) in cell pellet was detected, suggesting the functionality of these isolates to degrade phytate as
substrate. Until now, our results have revealed the occurrence of phytate-degrading bacteria in the
rhizosphere of natural and fertilized pastures grown in Chilean volcanic soils. Also, molecular and
bioinformatic tools have allowed the accurate detection and characterization of β-propeller phytase
in Paenibacillus populations. Despite their limited genomic information, there has been a growing
interest for the ecology and biotechnological application of phytate-degrading bacteria and their
phytases in agriculture and other fields. Therefore, converting the phytate in soils and organic
residues into phosphate available for plant will reduce the dependence of agriculture on P-fertilizers.
The reduction in the use of P-fertilizers will have positive impacts on environment, because a high
intake of inorganic phosphate results in over-fertilization of soils. On the other hands, the phytate
degradation during food processing for animal and human appears to be a strategy for reducing
micronutrient malnutrition and a controlled dephosphorylation of phytate may result in individual
metabolic active components in the diet maintaining human health and preventing chronic diseases.
Acknowledgments: The authors are grateful for support from Fondecyt Initiation into Research
Project no. 11080159, Bicentenary Program in Science and Technology Grant PSD26 and Fondecyt
no.1061262.
63
THE ECOLOGY OF MICROBIAL COMMUNITIES IN THE PLANT RHIZOSPHERE

Crowley, D.E.
Department of Environmental Sciences, University of California, Riverside, CA 92521

crowley@ucr.edu
Microorganisms affect the growth and health of plants through many different processes that
include symbioses, mineralization of nutrients, production of growth hormones, competition,
parasitism, and disease. While much previous work has focused on the interactions of plants with
individual microorganisms, in nature all such interactions are set within microbial communities. One
of the goals of agricultural science is to manage the composition of these microflora to promote
plant health and nutrient acquisition while suppressing plant deleterious organisms that inhibit root
growth or that cause disease. This requires an understanding of the rhizosphere ecology and how
microbial community activities are influenced by local variations in physical and chemical
conditions. Finally, the activities that occur at microscalesmust be integrated to obtain a view of how
plant health and nutrition can be manipulated by either traditional and nontraditional management
practices.
Molecular biology and chemical analytical methods currently provide complimentary approaches
for examining the composition and function of microbial communities, but are constrained by the
challenge of working at the very small scales that delimit specific habitats. Previously, much of the
research on rhizosphere ecology has focused on processes that are involved in the initial colonization
of roots and the temporal development of microbial communities along the root axes. The root apices
are of particular interest as this zone is one of the most carbon rich environments, where root
exudates provide abundant resources that can be exploited by opportunistic microorganisms. Some of
theseopportunist cause disease, whereas others function to mobilize mineral elements that can be
accessed by both plants and microorganisms. Generally, microorganisms are more competitive for
mineral elements than their plant hosts, and can rapidly immobilize nutrients in their biomass. This
can result in temporary local depression of the bioavailability of nitrogen, phosphorus, and trace
elements, which in turn induces specific stress responses.
As an example, one of the most limiting elements under high carbon conditions is the trace metal
iron, which has extremely low solubility under aerobic conditions. Competition for iron results in the
induction of iron stress responses in both the plant and the rhizosphere microflora that can ultimately
determine the composition of the microbial community, and the suppression of disease organisms.
The microbial responses to iron deficiency largely revolve around the production of iron chelators,
called siderophores, that are secreted into the environment. Virtually all microorganisms produce
siderophores, each of which varies with respect to its chemical properties and affinity for different
ion. Some siderophores are generically used by many different microorganisms and can also be used
by plants as iron sources. Still other siderophores require the expression of highly specific membrane
transport systems that facilitate uptake of the molecule. Further contributing to the chemical milleau,
plants produce organic acids that help to solubilize iron, and in the case of Poaceae, include
phytosiderophores that are released into the rhizosphere in the zone of elongation behind the root
apices. After release into the soil solution, the chelators then attack the mineral surfaces of iron
oxides, stripping away individual iron atoms. Further competition then ensues, as iron is exchanged
between chelators having different affinity for the metal. During the interplay between grasses that
64
produce phytosiderophores and microorganisms, microbial siderophores are generally much more
effective chelators and microorganisms have first access to the iron that is solubilized. However, the
plant can overcome this advantagewith a massive influx of phytosiderophores via a diurnal pulses
behind the root apex where microbial populations are at relatively low densities. Still other
microorganisms can use phytosiderophores as iron sources, and will suppress their own production of
siderophores to the minimum extent needed to strip iron from phytosiderophores for their own
growth.Ultimately the competition for iron can result in the suppression of plant disease causing
microorganisms that are unable to establish in the rhizosphere.
While iron plays a key role in the initial colonization of roots, still other processes come into play
as the microbial community matures and undergoes changes in composition as the roots mature. the
lower abundance of labile carbon and crowded environment on the densely colonized older results in
a probiotic effect that prevents the establishment of opportunistic pathogens that require large
amounts of abundant carbon. Here it is speculated that antibiotics, produced in the stationary phase of
microbial growth, may have a role in protection of plant roots from disease. Other sites in the
rhizosphere where such competition can occur again include the sites of lateral root emergence where
the root cortex cells are ruptured by the elongation of new root apices from the meristematic tissue in
the interior of the root.
While research on the colonization of roots represents one aspect ofhow microbial communities
interact, there are many other questions that require careful consideration of rhizosphere ecology.
These include the role of microorganisms in mobilizing phosphorus, the priming of organic matter
degradation, recycling of nutrient elements in soil organic matter, degradation of chemical
pollutants, and the interactions between plant growth promoting bacteria and roots that involve the
release of plant growth hormones or their precursor molecules in the plant rhizosphere.
An important example that is now receiving considerable attention involves the plant growth
hormone ethylene. Ethylene is produced by the plant in response to stress and helps to mediate the
plant stress response to drought, salinity, disease and other environmental factors that impair plant
growth. In this case, the precursor molecule is aminocyclopropane carboxylate (ACC), which is
transformed by the plant via an oxidase enzyme into ethylene. When ethylene is produced in high
quantities this can result in an over-reaction that can result in a downward spiral in plant health. In
countering this effect, many plant growth promoting rhizosphere microorganisms carry the gene for
an enzyme, ACC deaminase, which destroys the ethylene precursor and alleviates stress ethylene
production, thereby enabling the plant roots to resume growth. The benefit to the microorganisms is
reflected in the increased availability of nitrogen that becomes available when ACC is degraded.
From a practical perspective, the understanding of the various processes that influence plant
growth provide the foundation for new biotechnology that can be used for agriculture, for example
by using biofertilizers and soil inoculants to alter the composition of the rhizosphere. In the case of
ACC degrading bacteria, inoculation of plant roots is being examined to increase plant tolerance to
drought and salinity, and to improve plant water use efficiency. Similarly, antibiotic and siderophore
producing bacteria have been shown to have the potential to reduce root disease, and hormone
producing bacteria can improve root growth and yields.As these new technologies emerge, it will be
essential to carefully consider the traits that confer rhizosphere competence, and how soils can be
managed to achieve effective population densities of plant growth promoting bacteria. This will
require the evaluation of gene expression and microbial activity in specific root zone where the
65
processes affecting root growth are directly relevant. In theera of metagenomics, microarrays and
bioinformatics, these new experimental approaches are rapidly changing the paradigm from single
organisms interactions with plants to consideration entire microbial communities and differentiation
of their functions at discrete microsites in the plant rhizosphere.
66
Simposio 3.
Microbiología y Rizosfera
67
68
DETECTION AND QUANTIFICATION OF INDOLE ACETIC ACID IN

RHIZOBACTERIA STRAINS ISOLATED FROM VOLCANIC SOILS GRASSLANDS OF
SOUTHERN CHILE.
Acuña-Sobarzo J1; Jorquera M2; Mora M.L2.
1
Doctorate in Natural Resource Sciences,Universidad de La Frontera.
Email: acuna.sobarzo@gmail.com
2
Biotechnological Bioresource Nucleus, Universidad de La Frontera.
Email: mjorquera@ufro.cl; mariluz@ufro.cl
Indole acetic acid (IAA) is a natural-occurring auxin in most plants. Auxins are phyhormones that
regulate several processes during plant development, such as: growth, cell division and the
formation and elongation of stems and roots. Compared with the current use of auxins obtained by
chemical synthesis, auxins produced by microorganisms are of great importance. The application of
bacteria inoculum capable to produce auxins, may be a suitable alternative in the context of the
sustainable agriculture. In this context, recent studies have showed that strains of the genus Bacillus
sp.(Ali et al. 2009), Pseudomonas sp.(Karnwal 2009) and Enterobacter sp. (Sergeeva et al. 2007)
isolated from the rhizosphere of different plants are capable of producing these metabolites. The aim
of this research was to detect and quantify the production of indole acetic acid (IAA) in
rhizobacterial strains isolated from pastures grown in volcanic soils of Southern Chile. The strains
evaluated were: Paenobacillus sp., Bacillus sp., Pseudomonas sp. and Enterobacter sp., previously
isolated from the rhizosphere of pastures from La Araucania and Los Rios regions. Production of
IAA was detected by colorimetric reaction using the Van Urk Salkowsky reagent (Patten and Glick,
2002). Quantification of IAA was performed by means of a calibration curve made with synthetic
IAA standards. The results showed that Paenobacillus sp. strain 60P3, 60P22 and Bacillus sp. C15
strain had productions of IAA ranging between 2.0 to 4.6 mg ml-1. This result suggests that these
strains could be adequate candidates for further studies on biofertilizer.
Keywords: Paenobacillus, Bacillus, Pseudomona, Enterobacter, Indol acetic acid.

Acknowledgements: UFRO Doctoral Scholarship; Project Fondecyt Nº 1061262; Project Fondecyt
Initiation Nº 11080159.
Ali B; Sabri AN, Ljung K, Hasnain S. (2009). Quantification of indol 3- acetic acid from plant
associated Basillus ssp. and their phytostimulatory effect on Vigna Radiata (L). World Journal of
Microbiology and Biotechnology. 25: 519-526.
Karnwal A. (2009). Production of indol acetic acid by fluorescent Pseudomonas in the presence of
L- tryptophan and rice root exudates. Journal of plant Pathology 91:61-63.
Seergeva E., Hirkala D., Nelson L. (2007). Production of indole-3-acetic acid, aromatic amino acid
aminotransferase activities and plant growth promotion by Pantoea agglomerans rhizosphere
isolates. Plant and soil. 297: 1-13.
69
EFFECT OF THE APPLICATION OF NATURAL PRODUCTS PROMOTERS OF THE

VEGETAL GROWING AS STIMULATORS OF THE ARBUSCULAR MYCORRHIZAL IN
EXTENSIVE CROPS OF THE AGRICULTURAL INTEREST IN THE CENTRAL-
SOUTHERN ZONE OF CHILE.
Castillo CG1,2
1
Universidad Católica de Temuco, Facultad de Recursos Naturales, Escuela de Agronomía, Temuco,
Chile, e-mail ccastill@uct.cl. 2Scientific and Technological Bioresource Nucleus, Bioren-UFRO.
Traditional agricultural methods currently use enormous quantities of fertilizer to increase crop
yields, resulting in the pollution of soil and water. Ecological agriculture is coming to the forefront
as a solution to this worldwide problem. This discipline avoids the use of chemicals and seeks
alternatives that avoid causing damage to agro-ecosystems. Among these tools can be found the use
of bio-fertilizers, and these include arbuscular mycorrhizal fungi (AMF), which form mutual
symbiotic associations with most crops that are of agricultural interest. They increase the amount of
nutrients acquisition by the plant, especially P, Cu and Zn. However, AMF are obligatory symbionts
and they do not reproduce if they do not have a host. This limits inoculation to the level of fruit
orchards and vegetable nurseries. Their population can only be manipulated in extensive crops
through the use of different farming systems, including crop rotation.
On the other hand, natural products (NP) are being used in agriculture to stimulate plant growth.
One of the ingredients of these NPs is formononetin, an isoflavone which is obtained from the roots
of red clover. Recent studies have shown that this NP also has a stimulating effect on AMF.
However, it is not known what effect other currently applied NPs would have on AMF populations.
Therefore, this project will attempt to investigate the effect of some NPs on the early stimulation
of mycorrhization and AMF populations in extensive crops that are of agricultural interest for the
Central-Southern zone of Chile. To carry out this research, NPs that can be applied in different ways
to soil, seeds or leaves will be used. Different tests at a greenhouse level will allow any NPs that
stimulate mycorrhization to be selected, regardless of whether this happens with native populations
or in sterile soil with re-inoculated AMF. Early mycorrhization will be assessed by quantifying
fungal colonization in the roots of the crops.
Similarly, a test will be carried out using experimental micro-plots with different cultivars of
wheat to investigate whether the stimulation provided by NPs has an effect to varietals level. In this
test, early mycorrhization will be studied, along with the effect on fungal populations.
Finally, depending on the results obtained in the greenhouse, the interaction between the NP and
AMF in terms of early mycorrhization and fungal populations will be determined in field plot tests,
using a short rotation of wheat-corn host crops compared with a rotation that includes a non-host
crop, rape. It is hoped that adding NPs to a certain farming system will increase the yield of the
cereal as a result of an improvement to early mycorrhization.
Acknowledgements: Fondecyt 11090014.
70
ARBUSCULAR MYCORRHIZAL FUNGI AND GLOMALIN AS SOIL ORGANIC

MATTER STABILIZATION AGENTS OF SOIL IN A MEDITERRANEAN
AGROECOSYSTEM AT CENTRAL CHILE
Curaqueo, G1., Acevedo, E2., Rubio, R1., Borie, F1.
1
Center of Amelioration and Sustainability of Volcanic Soils. BIOREN-UFRO. Universidad de La
Frontera, Casilla 54-D. Temuco, Chile, Email gcura@ufro.cl
2
Facultad de Ciencias Agronómicas. Universidad de Chile, Casilla 1004, Santiago, Chile.
The arbuscular mycorrhizal fungi (AMF) and its product glomalin (glomalin related soil protein:
GRSP), a glycoprotein which present a high recalcitrance in the soil have been mentioned or
involved in soil C stabilization. Hence, AMF through the protection of organic matter into soil
aggregates, where the combined action of AMF hyphae and the production of GRSP play an
important role in the soil aggregation process. The activity of AMF and soil glomalin content are
affect mainly for the tillage systems used, influencing the carbon dynamics in the agroecosystems
and therefore the aggregation process. The aims of this study were i) To evaluate the effect of no
tillage (NT) and conventional tillage (CT) on arbuscular mycorrhizal propagules, levels and stability
of GRSP ii) To determine the contribution of GRSP to soil C pool and iii) To relate some
mycorrhizal characteristic with organic matter and water stables aggregates among others. In the top
soil (0-5 cm depth) we evaluated the GRSP content, total and active AMF mycelium length, AMF
spore number; water stables aggregates and GRSP content in macro and microaggregates. In
addition, GRSP stability (recalcitrance) was determined by soil incubation for 400 days at 20ºC,
measuring the GRSP content each 50 days. The study was carried out in an Mollisol (Entic
Haploxerolls) of Central Chile managed 6 years under no-tillage and conventional tillage using a
spring wheat (Triticum turgidum L.) - corn (Zea mays L.) rotation. The results showed higher values
in the GRSP content and AMF hyphal length in NT treatment compared with CT. Thus, the GRSP
contents were 3.96 and 8.16 mg g-1 for NT y CT respectively as well GRSP showed 36.8% of C in
its structure, representing a 3.9% of total soil C under CT and 8.2% under NT. The spore numbers
was higher in CT than NT. The total mycelium obtained 3.65 m g-1 in CT and 4.98 m g-1 in NT and
its viability increased in NT compared with CT. This trend also was observed on % of WSA, since
under CT were obtained lower values of aggregation (32%) than under NT (59%), showing higher
GRSP concentration in macroaggregates than microaggregates. The GRSP content decreased 40%
after the incubation period. Results showed a positive effect of NT in relation with CT treatment on
the characteristics evaluated, suggesting a important role of AMF and GRSP on soil aggregation and
the stability of organic matter in the soil of the Mediterranean agroecosystem studied. Other
important aspect to highlight is the promotion of soil management as NT systems, which increase
the action of AMF and stimulate the GRSP production.
This research was made with support of FONDECYT Project Nº 1060372.
71
ABIOTIC STRESS AFFECTING THE MYCORRHIZAL OF FOREST ECOSYSTEMS

FROM CENTRAL SOUTHERN CHILE, AN OVERVIEW
Etcheverría, P. and Godoy, R.
Instituto de Botánica, Universidad Austral de Chile, Valdivia Chile.
Temperate forests in the central-south of Chile are in restrictive environments as low N and P
availability, summer drought and low temperature in winter. Although these limitation, forests have
developed strategies to grow there. Among them, the symbiosis with mycorrhizal fungi have a key
role in plant nutrition and in stress conditions. Some of this stress are fire, which is mostly man
made and very destructives and by other hand, the drought periods. The effects they have on the
symbiosis in these forests are poorly understood. Thus, the aim of this work is review the literature
of abiotic stress effects on mycorrhizal symbiosis in forest ecosystems from central south of Chile.
The effect of fire have been studied on arbuscular mycorrhizas (AM) and ectomycorrhizas (EC)
only in Araucaria-Nothofagus forest and Nothofagus alpina from Tolhuaca National Park. In both
kind of mycorrhizas there were a reduction in colonization intensity and species or morphotypes
involved. AM fungi showed a fire resistance, which is important in vegetal restoration. Some EC
morphotypes disappeared in post-fire conditions and other were exclusive for post-fire condition. It
was observed a dominance of Descolea antarctica in young trees of N. alpine.
The effect of drought conditions was studied on N. dombeyi young plants. The EC fungi, D.
antarctica and Pisolithus tinctorius decreased the root colonization under drought condition but they
increased foliar N and P concentration in the host plant as well as they stimulated plant growth. By
other hand, N. dombeyi had lower oxidative stress that non mycorrhizal plants under drought
condition and the response of both fungi were different upon the same environmental condition.
The effect of stress factor on the mycorrhizal fungi and host plant is plant host species and
mycorrhizal fungi species dependent. For that reason is very much important acquired knowledge
about how different stress factors affect the symbiosis and their performance to respond a changing
scenario and improved the restoration strategies.
72
NOVEL BACTERIA AND MICROBIAL COMMUNITIES INVOLVED IN SIMAZINE

DEGRADATION IN AGRICULTURAL SOILS OF CENTRAL CHILE
Hernández, M.1,2,4*, Jorquera, M.A.2, Moore, E3. and Seeger, M.4
1
Programa Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera, Temuco,
Chile. 2Center of Plant, Soil Interaction and Natural Resources Biotechnology, Scientifical and
Technological Bioresource Nucleus, Universidad de La Frontera, Av. Francisco Salazar 01145,
Temuco, Chile. 3Culture Collection University of Gothenburg (CCUG), Department of Clinical
Bacteriology, Sahlgrenska University Hospital, Gothenburg, Sweden. 4Laboratorio de
Microbiología Molecular y Biotecnología Ambiental, Departamento de Química, Universidad
Técnica Federico Santa María, Valparaíso, Chile. *e-mail: mthernan@ufro.cl
The s-triazine herbicides are worldwide applied for the control of leaf and grassy weeds.
Simazine is one of the most s-triazines herbicides used for agricultural and forestry in Chile, with an
annual application of 219 tons. The huge application of simazine in agricultural soils can potentially
lead to contamination of water bodies. Bacterial strains capable to degrade s-triazines have been
isolated from soils in diverse regions. The aims of this study were the characterization of s-triazine-
degrading bacteria and the identification of the bacterium, Pseudomonas sp. strain MHP41. In
addition, the short-term effects of simazine on Bacteria, Fungi and Archaea in agricultural soils
form central Chile were analyzed. The microbial diversity in soils was analyzed by culture-
independent methods. Strain MHP41 was allocated initially in the genus Pseudomonas by
phenotypic (Biolog) and 16S rRNA gene sequence analyses. Physiological and biochemical
characterizations of strain MHP41 (CCUG 50378), using API 20NE and API ZYM and other typing
assays suggest that it represents P. nitroreducens. Multi-locus sequence analyses (MLSA), utilizing
gyrB and rpoB housekeeping genes, as well as 16S-23S rRNA inter-genic spacer (IGS-1) regions,
further identified strain MHP41 as Pseudomonas nitroreducens. Microbial community composition
of soils was analyzed by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S
rRNA and 18S rRNA gene fragments. DGGE analysis revealed that simazine application increased
some groups of Bacteria, Fungi and Archaea in both soils. After simazine application in soils,
bacterial groups closely related to Pseudomonas and Acinetobacter genera were stimulated,
independently to the previous history of simazine application in the soils. Although, fungal
communities were only slightly affected by simazine application, this herbicide stimulated fungi
clones closely related to Paecilomyces and to Penicillium genera in soils with history of simazine
application. Simazine promoted changes on the composition of archaeal communities in both soils.
Main changes were related to members of Crenarchaeote. This report indicates that bacterial and
archaeal communities are mainly affected by simazine application in agricultural soils of central
Chile. Finally, the systematic characterization and classification presented in the present study
identified the strain MHP41 as the first Pseudomonas nitroreducens strain reported to degrade s-
triazine compounds. Strain MHP41 is an efficient simazine-degrading bacterium and represents a
biotechnologically important strain with potential for biodegradation and soil remediation.
Acknowledgments: The authors are grateful for support from the Millennium Nucleus on Microbial
Ecology and Environmental Biotechnology grant P04/007-F (MS), USM 130836 (MS), USM
130948(MS) Conicyt 24080081 (MH), Fondecyt 11080159 (MJ) and Bicentenary Program in
Science and Technology Grant PSD26 (MJ). MH acknowledges Mecesup FRO0601 and Conicyt
fellowships.
73
ISOLATION AND SCREENING OF POTENTIAL PLANT GROWTH PROMOTING

RHIZOBACTERIA FROM PASTURES IN CHILEAN VOLCANIC SOIL UNDER
NITROGEN FERTILIZATION
Martínez1 O.; Jorquera2 M.; Mora2 M.L.
1
Doctorate Program of Sciences in Natural Resources, Universidad de La Frontera, Chile. Email:
omartinez@ufro.cl. 2Center of Plant, Soil Interaction and Natural Resources Biotechnology,
Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Chile.
Agricultural production in Southern Chile is based on acid and moderately acid soils. Pastures are
often under a regime of fertilization with ammoniacal fertilizers, which contributes to the soil
acidification process. In this study, bacteria associated to rhizopheric soils, with properties related to
plant growth-promotion, were isolated from pastures established on volcanic acidic soils under long
term nitrogen (urea) fertilization (PN) trial. Diverse strains were isolated by conventional culture
methods using rich-nutrient agar media, and some their plant growth promoting mechanisms were
identified. Rhizospheric bacteria were also studied in pasture without long term N fertilization (P0).
A total of 1176 bacteria were isolated and evaluated in vitro for their plant growth-promoting traits,
such as: phosphate solubilization (PS), phytate mineralization (PM), and production of indole acetic
acid (IAA). In relation to total culturable bacteria, the percentages of phosphobacteria (including PS
and PM) were around 85% and 58% for PN and P0, respectively. Moreover, the results showed
higher percentages of indole acetic acid producing bacteria in P0 compared with PN. The results
also revealed that 133 isolates (10-13%) simultaneously contain the three mechanisms (PS-PM-
IAA) and the majority of them (130 isolates) showed also the capacity to produce siderophore
(SID). This study, show that pasture established on volcanic soils contain significant bacterial
populations simultaneously expressing multiple mechanisms (PS-PM-IAA-SID) involved in the
plant nutrition, phytostimulation and pathogen’s bioncontrol. Thus, the rhizosphere from pastures
grown in volcanic soils constitutes a reservoir of potential PGPR strains that could be effective
under conventional fertilization regimens, increasing the efficiency of the applied chemical and
organic fertilizers.
Acknowledgements: FONDECYT Initiation into research no. 11080159, PBCT PSD26,

FONDECYT Regular no.1061262.
74
DIFFERENT PATTERNS OF LMWOA EXUDATION IN METALLOPHYTE AND

AGRICULTURAL PLANTS AT INCREASING COPPER LEVELS
Sebastián Meier*, Marysol Alvear, Fernando Borie, Paula Aguilera, and Pablo Cornejo
BIOREN-Universidad de La Frontera. P.O.Box 54-D Temuco. Chile. *E mail: fmeier@ufro.cl
The effect of copper (Cu) in plant growth, Cu accumulation in shoot and root tissues and low
molecular weight organic acids (LMWOA) root exudation have been studied in two metallophytes
from Central Chile (Oenothera affinis and Imperata condensata) and two agricultural plants
(Lupinus albus and Helianthus annus) growing under hydroponics conditions at increasing Cu levels
(0, 0.125, 0.250, 0.5, 1 and 2 mg Cu L-1). The plants were grown in mineral solution for four weeks
and after that on Cu-added solution for ten days, being harvested and analyzed after this time. The
high Cu levels produced a decrease on root elongation higher than to 60%, being O. affinis the most
affected specie, with a reduction higher than to 90%. Plant growth was proportionally reduced with
the increase in Cu levels. All the plant species showed high Cu accumulation at high Cu levels,
highlighting Oenothera affinis specie with 116 and 2657 mg Cu kg-1 in shoots and roots respectively
(4 fold than the other species analyzed). Strong differences in the LMWOA exudated by the
different plant species were determined. O. affinis exudated higher amounts of succinic acid, while
I. condensata exudated mainly citric and oxalic acids. Acid root exudates from agricultural plants
were mainly composed by citric acid. This result suggests that root exudates from metallophytes
plants play an important role for overcoming high Cu levels in polluted habitats, being an important
factor for plant selection in the design and implementation of phytoremediation programs.
Acknowledgments: FONDECYT 11080131 and CONICYT scholarship.
75
TWO PHYTASE PRODUCING BACTERIA WITH BIOTECHNOLOGICAL POTENTIAL

FOR USE IN ENHANCING P BIOAVAILABILITY IN SOILS AND MANURES
Menezes-Blackburn D. A; Jorquera M. B; Mora M. L.B
A
International Doctorate in Environmental Resources Science, Universidad de La Frontera (Chile)
and Universidad Federico II de Nápoles (Italy)., Emails dmenezes@ufro.cl
B
Biotechnological Bioresource Nucleus, Universidad de La Frontera, Chile. Email:
mjorquera@ufro.cl; mariluz@ufro.cl
Chilean volcanic soils have a high phosphorus (P) retention capacity and a high organic P content.
Phytate is the most recalcitrant and abundant form of soil organic P but its availability to plants is
low. We studied the Pi release from Ca phytate and the phytase activity of Paenibacillus sp. 60P3
and Rhanella sp. D75 bacteria strains in order to evaluate their potential use in soil and manure P-
phytate hydrolysis biotechnological applications. Phytase-screening medium (PSM) (1), containing
phytate as a sole P source, was used to evaluate the phytase activity of the studied strains and their
capacity for Pi releasing. Protein extraction was used according to Hill et al. (2007) (2) and phytase
activity assay was adapted from Greiner et al. (1993) (3). A cell associated acid phytase activity was
detected for both Paenibacillus sp. 60P3 (36.81±0.67 mU mg-1 protein) and Rhanella sp. D75
(18.74±2.77 mU mg-1 protein) strains. Alkanline phytase activity was not detected and the
functional enzyme pH observed to Paenibacillus sp. 60P3 matches the environmental pHs were it is
naturally found. ß-propeller group of phytases typically present alkaline phytase activity and
Paenibacillus sp. 60P3 phytase may be an unusual representant of this group. Although both
bacteria strains grow in liquid PSM media, no significant extracellular Pi release was detected
suggesting that most of the hydrolysed Pi was quickly immobilized in cells. Biotechnological
applications using directly phytases from these microorganisms are being considered to be tested for
soil and manure amendment treatments to increase the availability of soil and manure organic P
pool. Further investigations on the immobilization of these phytases in clays will be tested for
enhancing enzyme stability during the proposed treatments.
Key Words: Phytase; Phytate, Organic phosphorus; Bacteria;
Acknowledgements: UFRO Doctoral Scholarship; CONICYT Scholarship Support for

Implementation of Doctoral Thesis Year 2009; Project FONDECYT Nº 1061262; Project
FONDECYT Initiation Nº 11080159
J. Kerovuo, M. Lauraeus, P. Nurminen, N. Kalkkinen, J. Apajalahti, Applied and Environmental

Microbiology 64, 2079 (1998).
J. E. Hill, D. Kysela, M. Elimelech, Environ. Microbiol. 9, 3100 (Dec, 2007).
R. Greiner, Protein J. 23, 567 (Nov, 2004).
76
SCREENING AND ISOLATION OF PHOSPHATE SOLUBILIZING AND

MINERALIZING FUNGI FROM SOILS OF SOUTHERN CHILE
Morales, A., Alvear, M., Borie,F.
Universidad de La Frontera, Facultad de Ingeniería, Cs. y Adm. Dpto. Cs. Químicas.

Scientific and Technological Bioresource Nucleus.
e-mail: almoral@ufro.cl
Chile has excellent conditions to become a major food producer and exporter by means of the use of
traditional or organic farming. In this way, free-living phosphate solubilizers and mineralizers fungi
(phosphofungi) can play an important role in plant nutrition by allowing use part of accumulated P
in the soil, which has low availability for plants, especially in volcanic soils. In those soils,
phosphatase activity is relevant for allowing the mineralization of P-organic, an important form of P.
This implies the potential use of biofertilizers through inoculation techniques whose success mainly
depends on the use of native phosphofungi previously well-tested. Hence, the aim of the study was:
1) To make a screening of native phosphofungi present in volcanic soils from southern Chile. 2) To
asses the P-solubilization capacity and phosphatase activity secretion of selected phosphofungi in
four liquid media.
The screening of phosphofungi was carried out from several soils of our region under
different crops and managements using Martin medium agar-rose bengal-streptomycin, with
Ca3(PO4)2 or phytate-Ca as insoluble inorganic or organic phosphate source, respectively. A halo
around the colony indicated solubilization. After 5 d incubation an index of solubilization was
calculated as the total ratio diameter / diameter of the colony. The colonies with high phytate-Ca
solubilization capacity were analyzed for phosphatase activity secretion picking up on Martin
medium supplemented with phenolphthalein phosphate; a red halo around these colonies revealed
with ammonia vapours indicated secretion of P-ase.
Four phosphofungi strains with high index of solubilization (Penicillium sp., Penicillium
albidum, Gliocladium roseum and Penicillium thomii) were evaluated in the liquid media:
Agnihotri, Asea-Wakelin, Pikovskaya and Nahas et al. with Ca3(PO4)2 as the P source. After 7 days
soluble P, phosphatase activity, pH and fungal biomass were determined. All treatments were
performed with four replicates.
At higher index of solubilization, the number of inorganic phosphate solubilizing colonies
decreased, but increased the proportion of organic phosphate solubilizing colonies. There was P-ase
secretion in 80% of phytate solubilizer colonies analyzed on Martin medium. In liquid medium, the
greater solubilization of P was in Asea-Wakelin, Nahas et al. and Pikovskaya and in these there was
also the greatest decrease in pH; however, in liquid medium Asea-Wakelin the metabolism of fungi
is higher oriented to the solubilization P and phosphatase production.
In conclusion, it is possible to isolate phosphofungi with high potential capacity of P
solubilizing and mineralizing to be used to mobilize accumulated P in volcanic soils. The Martin
medium is useful for screening of native phosphofungi, while the liquid medium Asea- Wakelin
appear to produce a better quantitative evaluation.
Acknowledgement: Grant DIUFRO DI08-0054, Universidad de La Frontera
77
MICROBIAL BIOFERTILIZERS: PREPARATION OF MYCORRHIZAL INOCULANTS

ON POROUS INERT MATRICES
Rubio R1, Castillo C1, Borie F1, Castillo CG2
1
Universidad de la Frontera, Departamento de Química y Recursos Naturales, Temuco, Chile, e-mail
rrubio@ufro.cl
2
Universidad Católica de Temuco, Facultad de Recursos Naturales, Escuela de Agronomía, Temuco,
Chile.
Scientific and Technological Bioresource Nucleus, Bioren
In the Region de La Araucanía, including fruit vegetables grown pepper and tomato stands not only
for quantities that are used for fresh consumption but also as a raw material for industry and
medicine. The production process of these vegetables stage involves a seedbed with subsequent
transplantation to soil. The inoculum of arbuscular mycorrhizal fungi (AMF) is used as a
biofertilizer, bioregulator and bioprotector successful establishment of crops, allowing production of
better quality and lower chemical inputs. At present, still suffers from methodologies that will
produce large amounts of biofertilizer production being restricted to an overall growth of the AMF
and the plant called "pot culture”.
The overall project objective is to prepare a mycorrhizal biofertilizer with a selection of AMF
morphotypes from vegetable crops in the Region de La Araucanía to play at an early stage, small fee
for "pot culture" and "hydroaeroponic culture" and later in large quantities by "production box"
using volcanic slag as a substrate.
From locality of Angol, Lumaco and Purén morphotypes were isolated and reproduced by AMF
in pot culture as a host using corn and tagetes. On the other hand, lettuce seedlings inoculated with
Glomus claroideum were used to reproduce the fungus by hydroaeroponic culture, but for this
system of reproduction, better response was achieved with the commercial inoculum.
The nursery inoculation with morphotype Glomus claroideum in Capsicum annuum L. cv.
"Cacho de Cabra" was very effective in shortening ripening 21 days and achieving a larger fruit,
compared with the use of inoculation with a commercial AMF.
We had reproduced massively Glomus claroideum in boxes (2m x 2m x 0,2m) using as substrate
fine slag originating from Llaima volcano and 200 L of biofertilizer were obtained, with which
seedlings were inoculated with speedling and pepper and lettuce flower-beds, resulting in successful
inoculation speedling where significant differences were observed in some plant parameters as
height and strength in relation to the control without inoculum.
It is concluded that the use of biofertilizer on bed of pepper and lettuce, speeds up physiological
ripeness of the vegetables.
Acknowledgements: Fondecyt 1070283.
78
CHANGES IN THE DENSITY OF MYCORRHIZAL FUNGI PROPAGULES

ASSOCIATED TO OENOTHERA AFFINIS IN COPPER POLLUTED SOILS
Seguel, A., Meier, S., García, C., Borie, F., y Cornejo, P
Center of Amelioration and Sustainability of Volcanic Soils. BIOREN-UFRO. Universidad de La

Frontera, Casilla 54-D. Temuco, Chile. Email aseguel@ufro.cl
The phytoextraction and / or phytostabilization of metals such as Cu can be enhanced by the

formation of mycorrhizal symbiosis in metallophyte plants, which is particularly relevant in soils
contaminated by mining activities as those of Puchuncaví Valley (Region de Valparaiso), near
Ventanas Smelter (ENAMI). The aim of this study was to analyze the influence of increasing Cu
levels on the density of propagules of arbuscular mycorrhizal fungi (AMF) associated with
Oenothera affinis, a pseudometallophyte present in Cu-polluted soils from Puchuncaví Valley.
Plants of O. affinis were established in: i) sterile soil without inoculation (-M), ii) native AMF
inoculated to contaminated soil(+M), and iii) inoculated with Glomus claroideum (GC) isolated
from agricultural soils in the Region de La Araucania, Chile. In the case of implementing different
levels of Cu were considered: (0) without addition of Cu to substrate, consisting of a mixture
sepiolite: sand 1:1 p:p, (1) a mixture of soil:sand:sepiolite 1:1:1 p:p:p, (2) sterilized natural soil;
natural soil sterilized and added with (3) 75 mg Cu kg-1, (4) 150 mg Cu kg-1 (5) 225 mg Cu kg -1,
and (6) 300 mg Cu kg-1. In all cases with an increase in Cu content, we used a solution of CuSO4,
which was added to the substrate installments for 4 weeks for stabilization in the soil. For each
treatment 4 replicates were used. The results showed a decrease of colonization in +M treatments at
increasing Cu levels (20% in (1), 6% (6)). A high colonization in GC treatments was observed in the
absence and at high Cu levels (34% in (0) and 23% (6)). GC High densities of AMF spores were
observed in soils at high levels of Cu (250 spores in 100 gss-1 in treatment (6)), a trend also observed
in natural soil. Additionally, there was increased production of biomass in the sand/sepiolite
substrate, reducing it to levels means of Cu, but increased to high levels of metal, in concordance
with the growth of spores in the soil. Accordingly the results suggested an important role of the
AMF, adapted and not adapted to the establishment of O. affinis in Cu-polluted soils.
Acknowledgements: This work was supported by Fondecyt 3070052 (P. Cornejo) and DI089-1003
Univ. de La Frontera (F. Borie) Grants.
79
80
Simposio 4.
Suelos, Fisiología y Nutrición Vegetal
Moderadores: Dra. María de la Luz Mora; Dra.Miren Alberdi.
1. Dra. María de la Luz Mora, Universidad de La Frontera, Chile:”Interacción

suelo-planta en suelos volcánicos”.
2. Dra. Miren Alberdi, Universidad de La Frontera, Chile: “Abiotic stress effects

on physiological and biochemical traits of Blueberry (Vaccinium corymbosum
L.) cultivars”.
3. Dr. Alexander Ivanov, University of Western Ontario, Canada: “Carboxylation

activity of Rubisco in Arabidopsis thaliana under Cd and Cu toxicity - role of
carbonic anhydrase and Rubisco activase in adaptation to stress”.
4. Dr. Nanthi Bolan. University of South Australia: “Bioaccumulation and

biotransformation of heavy metal(oid)s in relation to bioremediation - using a
case studies involving bacterial induced lead immobilization”.
5. Dr. Antonio Violante, Università Federico II, Italy: “Factors Affecting the
Mobility and Bioavailability of (Micro)Nutrients, Heavy Metals and Metalloids in
Soil Environments”.
81
82
SOIL-PLANT INTERACTIONS IN VOLCANIC SOILS

María de La Luz Mora G.
Director of Center of Plant, Soil Interaction and Natural Resources Biotechnology; Scientific and
Technological (BIOREN). La Frontera University, Avenida Francisco Salazar 01145, Temuco,
Chile. Email address: mariluz@ufro.cl
For a long time our Plant-Soil Interaction and biotechnological Center have studied the soil-plant
interaction in pastures and cereal crops growing in volcanic acidic Chilean soils because of their
economical importance for milk and meat production and human food in Southern Chile. Beef and
dairy market in Southern Chile is based on grassland production, but large portions of soil used for
this activity are acidic, so that soil acidification is one of the main limitations for cereal crops and
grassland production in the area (Mora et al., 1999a). Acidification is mainly caused by heavy
rainfall during winter period, which results in the leaching of the main cations (CEC). It is also the
result of acidifying fertilizers (urea, MAP etc) (Mora and Demanet, 1999). Acidification leads to the
increase of aluminium (Al) and manganese (Mn) in soil solution. Reduction of root growth (Mora et
al., 1999b; Mora et al., 2005) and plant toxicity may derive. About 50% of Chilean Andisols have
high soil acidity levels, and increased amounts of exchangeable aluminium (Al) that is highly toxic
to plants (Mora et al., 2002, 2004). Whereas the correlation between soil acidity and the release of
Al in Chilean Andisols is clear, the relationship between soil pH and the amount of plant available
manganese (Mn) in these soils is less understood because it also depends on redox conditions. This
reduces yield in crops and grasslands (Mora et al., 1999, 2002, 2004, 2005; Rosas et al. 2007; Mora
et al. 2009), affecting also the quality of grain and pasture. In order to ameliorate soil acidity
conditions in our soils we use lime. However, some time the rhizosphere soil response is different
from that of the bulk soil, because of the different nature of the exudates released by the different
species and cultivars.
In our soils, the high organic P content, the high inorganic P fixation and, in turn, the low P
available and the high Al and Mn soluble concentrations at low pH are the most important limiting
factors of crop production in Southern Chile. Therefore, it is very difficult to separate the effects due
to low P or high Al and Mn in plant because Al/Mn toxicity and P deficiency often coexist in acid
soils, and these factors may strongly interact to each other through chemical and biochemical
reactions. We hypothesize that phosphobacteria are an important biotechnological tool to increase
available P in the rhizosphere through the action of their phosphatases and phytases (Jorquera et al.
2008 and unpublished results). In crop production we apply heavy phosphate rate to produce high
yield, but often with a low efficiency. Also, it seems very useful to utilize organic P sources within
the soil like dairy wastes improving the P soluble in water and in bicarbonate fraction (Fuentes et
al., 2009) and P biomass (Baeza et al. un-published).
Furthermore, Al and Mn and low pH produce an oxidative stress in crop cultivated in these soils.
Mn differential tolerance in perennial ryegrass appears to be related to ascorbate peroxidase and
guaiacol peroxidase activities. (Rosas et al., 2009). We have determined that Se increases the
antioxidant response in plant. However, the Se content in our soils is very low. The behaviour of
selenite anion in Andisols follows the same pattern of phosphate (Barrow et al., 2005), while that of
selenate is very similar to sulphate (un-published results), although selenite in plant decreased with
application of S to soils by increasing the Se-organic fraction (Cartes et al., 2005). Our studies with
soils and crop plants indicated that we need to use selenite, because we were able to produce more
83
antioxidative responses in the plant (Cartes et al., 2005; 2006, 2009; Mora et al., 2008; Motomura et
al., 2008) with less Se contaminated soils.
Organic acid secreted by plant roots and micro organisms as stress response, have the capacity to
complex metals like Al and Mn. The exudates released by different species and cultivars as citric,
oxalic and malic acids can be adsorbed onto variable charge minerals by inner-sphere coordination
and can compete with phosphate or sulphate or other anions for active site on colloid surface (Jara et
al., 2006). Sulphate retention capacity of our soils is also very dependent on pH and organic matter
content (Mora et al., 2005), and normally we applied sulphur to the soils to maintain and optimize
food production. Some S applications as gypsum increase dry matter production and decrease Al
toxicity by favouring the formation of Al-sulphate complexes in the rhizosphere (Mora et al.,
1999b). Moreover, we studied Mo availability because this anion is an important factor for clover
nutrition and productivity. We determined that molybdate has great affinity for iron oxides in soils
(Vistoso et al., 2009) and Mo availability for plant growth is strongly dependent on soil pH. In
addition, nitrogenase and nodules activities were regulated by Mo soil and plant interaction (López
et al., 2007). Furthermore, we reported that Mo added to clay-phosphate complexes slight decreased
phosphatase activity (Rosas et al., 2008).
Nowadays we are very interested in improving knowledge on how to manage the rhizosphere of the
main crops produced in Southern Chile. Indeed, we believe that we can identify some
phosphobacteria able to accumulate selenium. They could be applied as biofertilizer, slowly
releasing Se and P to the rhizosphere. Also we are working in different biofertilizer using
immobilized enzymes on natural and synthetic nanoclays for their application in the rhizosphere.
Both kinds of fertilizers will increase these nutrients in plant and improve metabolic processes and
food quality.
References
Barrow, N.J., Cartes, P. and Mora, M.L. (2005). Modifications to the Freundlich equation to
describe anion sorption over a large range and to describe competition between pairs of ions.
European Journal of Soil Science, 56: 601-606.
Cartes, P., Gianfreda, L. and Mora, M.L. (2005). Uptake of selenium and its antioxidant activity
in ryegrass when applied as selenite and selenate forms. Plant and Soil, 276: 359-367.
Cartes, P., Shene, C. and Mora, M.L. (2006). Selenium distribution in ryegrass and its antioxidant
role as affected by sulfur fertilization. Plant and Soil 285: 187-195.
Cartes, P., Jara, A.A., Pinilla, L., Rosas, A. and Mora, M.L. (2009). Selenium improves the
antioxidant ability against aluminium -induced oxidative stress in ryegrass plants. Annals of Applied
Biology (In press).
Fuentes, B., Jorquera, M. and Mora, M.L. (2009). Dynamic of phosphorus and associated
phytate-mineralizing bacteria during the aerobic degradation process of dairy cattle feces.
Chemosphere 74: 325-331.
Jara A.A., Violante, A. Pigna, M. and Mora, M.L. (2006). Mutual Interactions of sulfate, oxalate,
citrate and phosphate on synthetic and natural allophanes. Soil Science Society of American Journal.
70: 337-346.
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Jorquera, M., Hernández, M., Rengel, Z., Marschner, P. and Mora, M.L. (2008). Isolation of
culturable phosphobacteria with both phytate-mineralization and phosphate-solubilization activity
from the rhizosphere of plants grown in a volcanic soil. Biology and Fertility of Soils 44: 1025-
1034.
López, R., Alvear, M., Gianfreda, L. and Mora, M.L. (2007). Molybdenum availability in
Andisols and its effect on biological parameters of soil and red clover (Trifolium pratense L.). Soil
Science 172: 913-924.
Mora, M.L. and Demanet, R. (1999a). Uso de enmiendas calcáreas en suelos acidificados.
Frontera Agrícola 5 (1 and 2): 43-58 (Technical Publication).
Mora, M.L., Schnettler, B. and Demanet, R. (1999b). Effect of liming and gypsum on soil
chemistry, yield and mineral composition of ryegrass grown in an acidic. Communications in Soil
and Plant Analysis 30 (9 and 10): 1251-1266.
Mora, M.L., P. Cartes, Demanet, R. and Cornforth, I.S. (2002). The effects of lime and gypsum
on pasture growth and composition on an acid Andisol in Chile, South America. Communications in
Soil Science and Plant Analysis 33 (13 and 14): 2069-2081.
Mora, M.L., Alfaro, M., Sterh, W., Williams, P.H. and Demanet, R. (2004). Production and
mineral composition of a Lolium multiflorum pasture growing on an acidic Andisol with two levels
of acidity. Journal of Soil Science and Plant Nutrition 4 (1): 28-40.
Mora, M.L., Shene, C. Violante, A., Demanet, R. and Bolan N.S. (2005). The effect of organic
matter and soil chemical properties on sulfate availability in chilean volcanic soils. In. Soil abiotic
and biotic interactions and the impact on the ecosystem and human welfare, Huang, P.M., Violante,
A., Bollag, J.M. and Vityakon P. (eds). ISBN 1-57808-344-3; C. 444, Science Publishers, Inc.,New
Hapshire , USA , Chapter 13. pp: 223-244.
Mora, M.L., Pinilla, L., Rosas, A. and Cartes, P. (2008). Selenium uptake and its influence on the
antioxidative system of white clover as affected by lime and phosphorus fertilization. Plant and Soil
303: 139-149.
Mora, M.L., Rosas, A., Ribera, A. and Rengel, Z. (2009). Differential tolerance to Mn toxicity in
perennial ryegrass genotypes: involvement of antioxidative enzymes and root exudation of
carboxylates. Plant and Soil 320: 79-89.
Motomura, Y., Reyes-Díaz, M. and Mora, M.L. (2008). Influence of selenite on the total
polyphenol content and antioxidative activity of aqueous and ethanolic extracts in sprouts of four
agronomic species. Journal of Soil Science and Plant Nutrition 8(1): 5-67.
Rosas, A., Rengel, Z. and Mora, M.L. (2007). Manganese supply and pH influence growth,
carboxylate exudation and peroxidase activity of ryegrass and white clover. Journal of Plant
Nutrition 30:253-270.
Rosas, A., Mora, M.L., Jara, A., Lopez, R., Rao, M. and Gianfreda, L. (2008). Catalytic
behavior of acid phosphatase immobilized on natural supports in the presence of manganese or
molybdenum. Geoderma 145: 77-83.
Vistoso, E.M., Bolan, N., Theng, B.K.G. and Mora M.L. (2009). Kinetics of molybdate and
phosphate sorption by some Chilean Andisols. Journal of Soil Science and Plant Nutrition 9(1): 55-
67.
85
ABIOTIC STRESS EFFECTS ON PHYSIOLOGICAL AND BIOCHEMICAL TRAITS IN

BLUEBERRY (Vaccinium corymbosum L.) CULTIVARS
Miren Alberdi, Marjorie Reyes-Díaz, Alejandra Ribera, María de la Luz Mora
Department of Chemical Science, Center for Plant, Soil Interaction and Natural Resources
Biotechnology, Scientific and Technological Bioresources Nucleus (BIOREN-UFRO),
malberdi@ufro.cl
The deciduous highbush Vaccinium corymbosum (Blueberry) is an important crop species in south
central Chile, where it grows on volcanic soils (Andisols) characterized by high organic matter
contents, reduced nitrogen (N) and phosphorus (P) availability, and acidity (pH ≤ 5.5). This acidity
is associated with toxic forms of aluminum (Al3+) and manganese (Mn2+), which may perturb plant
metabolism and productivity. These toxic forms exacerbate the production of reactive oxygen
species (ROS), inducing oxidative damage, which negatively affects plant physiological and
biochemical processes. Among these processes plant growth, photosynthetic performance, synthesis
of chlorophyll, carotenoids, enzymes, amino acids and proteins, among others, are affected. The
damage induced by oxidative stress depends on the antioxidant capacities of plants. It is important to
mention that manganese plays a double role in plant metabolism: as an essential micronutrient and
as a toxic element at higher concentrations and acidity, and as micronutrient Mn, it is important to
the water-splitting system in the photosynthesis process, providing electrons to PSII. The effects of
Al and Mn toxicity on plants are well documented, particularly in the roots, but less is known about
the effects of Al and Mn toxicities on the physiological and biochemical responses of highbush
blueberry grown under acidic conditions and under other abiotic stress conditions.
We report on the studies of our work group regarding the effects of Al and Mn toxicity on some
physiological and biochemical traits (root growth, Al and Mn accumulation, lipid peroxidation
(LP), antioxidant activities (AA), photosynthesis and photochemical efficiency of PSII (maximum
quantum yield: Fv/Fm, ratio of variable to maximum fluorescence and effective quantum yield:
ФPSII) in blueberry cultivars (Brigitta, Legacy and Bluegold) growing in hydroponic solution
containing increased concentrations of Al3+ as Al Cl3 (0, 25,50 75 and 100 µM) at pH 4.5 and Mn2+
(2, 10, 50,250,500 and 1000 µM Mn ) as MnCl2 x 4 H2O at pH 4.8 for different times (0 to 48 h,
and 0 to 20 days, short and long term, respectively) in a greenhouse. The Al and Mn resistance of
the cultivars were estimated based on the physiological and photochemical parameters. The effect
of UV-B radiation (280-320nm range) on the photosynthetic capacities of blueberry cultivars were
also investigated using UV-B lamps (Phillips Q-panel 313) with a maximum irradiance of 4W m-2
simulating a typical summer day in the Province of Cautín (39ºLS).. In addition, the antioxidant
activity (AA) and total phenolic (TPH) and total anthocyanins (TA) contents in blueberry fruits
(whole fruit, shell and skin) at different maturity stages (from 100% green to 100% blue) grown
under field conditions was evaluated. The differential gene expression in response to Al stress
through cDNA-AFLP technique in blueberry roots has been also studied.
The results about Al toxicity showed that in the short term (48h), Brigitta practically did not change
its Fv/Fm and ФPSII in comparison with the control by effect of the Al treatment, whereas in
Legacy and Bluegold a decrease (P<0.05) of these parameters by the Al- treatments was found.
Thus, based on these parameters, Brigitta appeared as the most Al resistant cultivar followed by
Legacy and Bluegold as the most Al sensitive. The highest leaf Al-accumulation was exhibit by the
most Al-resistant cultivar (Brigitta), which suggest an internal mechanism to ameliorate Al toxicity.
By contrast, in the long term, Legacy had the best photosynthetic performance (ФPSII) under Al-
86
treatment, appearing as the most Al resistant cultivar followed by Brigitta, while Bluegold remained
the most Al sensitive (lower ФPSII). The highest Al resistance of Legacy was also corroborated
with a lower root lipid peroxidation and a higher antioxidant activity (AA) with respect the other
cultivars, especially at 7 days, suggesting an activation of the defense mechanisms through radical
scavenging activity at this time, which afterwards favored a decrease in the lipid peroxidation.
Despite their different Al resistance both Legacy and Bluegold displayed similar pattern of Al
accumulation in leaves, although Al contents of Legacy were higher than Bluegold up 100µ M Al
treatment.
With respect to Mn toxicity, blueberry cultivars treated with increased concentrations of Mn showed
that the Fv/Fm parameters practically remained in the range of healthy leaves (around 0.8) at any
concentration and time of treatment. Major differences between the cultivars showed the values of
ФPSII at 30 days of Mn treatment. At this time, Bluegold decreased its ФPSII by more than 50%
(P<0.05) at concentrations of 50 to 1000 µ M Mn compared with the controls, whereas in Legacy
and Brigitta a lower (around 28%) but statistically significant decrease (P<0.05) was observed at the
highest Mn concentration (1000 µM). Interestingly, although Brigitta showed a similar ФPSII
decrease as Legacy at the highest Mn concentrations at 30 days, at 15 day values were significantly
lower than at 30 days at both 500 and 1000 µM Mn treatments. This suggests a slight recovery at the
end of the experiment, without reaching the control values. Values of ФPSII were higher in Legacy
than in the other cultivars under Mn treatments. Thus, according this parameter the most Al resistant
cultivar was Legacy followed by Briggita and Bluegold the most sensitive. ETR showed the same
course as ФPSII. Mn contents of leaves fluctuated between 300-400 mg kg-1 in the controls and 645-
866 mg kg-1 at the highest level of Al treatment. From these values the Mn leaf accumulation was
calculated, with the highest Mn leaf accumulation being obtained in cv Legacy (248%) followed by
Brigitta (189%) and Bluegold (60%). In general, the cultivars maintained a similar behavior of Mn
and Al resistance under these stresses.
The effect of UV-B on the photosynthetic performance of young and mature leaves of blueberry
cultivars growing in hydroponics Hoagland solution under greenhouse conditions for 15 days was
studied. Young Brigitta and Legacy leaves decreased (around 40%) their CO2 assimilation when
exposed to UV-B radiation in relation to non-irradiated plants. However, the adult leaves of these
cultivars did not show any differences in CO2 assimilation. Adult leaves of Bluegold significantly
decreased its photosynthetic rate (P<0.05). On the other hand, UV-B treated adult Legacy leaves and
young Bluegold leaves showed differences (P<0.05) in the Fv/Fm values with respect to the non-
treated leaves. No effect of UV-B radiation on the Fv/Fm was observed in the Brigitta leaves. In
relation to the ФPSII, no differences between the leaf age of each cultivar was observed with the
UV-B treatment, but differences between the cultivars were found, with lower values for Brigitta
(P<0.05). Thus, according to this parameter cultivar Brigitta was the most susceptible to UV-B
radiation.
The results with respect to fruit quality indicated that AA of ripe whole fruits (100% blue) varied
among the cultivars in the order Legacy > Brigitta > Bluegold. Skin extracts, in all maturity stages
and for all cultivars, showing the high AA compared with those of whole fruit and shell. In general,
AA of whole fruits decreased at increasing maturity from 100% green to 75% red stages, whereas
from 75% red to 100% blue stages AA increased steadily. On the other hand, in shell and skin
extracts, at increasing maturity stages AA decreased and increased lineally, respectively. The
contents of total phenolic (TPH) content and total anthocyanins (TA) varied among fruit tissues in
the order skin > whole fruit > shell. In whole fruits, TPH decreased from 100% green to 75% red
and increased from 75% to 100% blue maturity stages. In shell and skin extracts, TPH at increasing
87
maturity decreased and increased, respectively. In conclusion, the AA of 100% green fruits and
100% blue fruit was similar. The high content of TPH and TA could explain the higher levels of AA
in 100% green and 100% blue fruits than those observed at immature stages (from 25% red to 100
red).
We have identified genes associated with resistance to Al and oxidative stress and have now begun
the molecular characterization of the genes as glutathione s-transferable, ATP-citrate lyase,
calmodulin and H+ vacuolar pyrophosphatase. Some of the projections for the future are to
characterize the functional level of these genes using transgenic Arabidopsis plants or blueberry.
It is concluded that in view of the different Al response obtained in the genotypes investigated in the
short and long term experiments, care must be take with the time-duration of stress in the assessment
of the Al resistance or susceptibility. We think that the short term excluded the acclimation
possibility in some plants, which is variable and depends on the genotype and environmental factors.
It can be also concluded that genotypes, which have the capacity to respond efficiently to one stress,
can also show an efficient response to a different stress. This can be relayed in the similarity of some
stress responses mechanisms.
The present exposition is inserted in the projects with the financial support of Fondecyt 1080372 to
M. Alberdi and L.M Mora, Fondecyt 11080231 to M.Reyes, Bicentenario PSD-26 to M. Reyes and
LM Mora, Fondecyt (Postdoctoral) 3090074 to A. Ribera.
88
CARBOXYLATION ACTIVITY OF RUBISCO IN Arabidopsis thaliana UNDER CD AND

CU TOXICITY - ROLE OF CARBONIC ANHYDRASE AND RUBISCO ACTIVASE IN
ADAPTATION TO STRESS CONDITIONS
A. Siedlecka1, O. Moskvin2,3, G. Samuelsson2, Z. Krupa1, A.G. Ivanov2,4
1
Department of Plant Physiology, Maria Curie-Sklodowska University, Akademicka 19, 20-033
Lublin, Poland, 2Umeå Plant Science Center, Department of Plant Physiology, Umeå University, S-
90183 Umea, Sweden, 3Institute of Basic Biological Problems, Russian Academy of Sciences,
Pushchino Moscow Region 142290, Russia, 4Department of Biology, University of Western Ontario,
London, Ontario, N6A 5B7 Canada.
Wild type (WT) and Rubisco activase (RA)-antisense mutant (RLDI00) of Arabidopsis thaliana
plants were exposed to different concentrations of Cd and Cu. Accumulation of toxic metals in plant
leaves was strongly dependent on the concentration of both toxic metals in the nutrient medium and
was much higher for Cd. Higher toxicity of Cd has been related to the higher accumulation of Cd in
comparison with Cu. Despite this, primary photosynthetic capacity of Photosystem II (PSII) in both
WT and RLD100 mutant measured as Fv/Fm remained unaffected by Cd and Cu. Rubisco activase
(RLD100) mutant exhibited lower capacity for photochemical quenching (qP) and lower yield of
PSII-dependent electron transport and was more sensitive to both Cu and Cd stress. These changes
were accompanied by differential responses of both protein abundance and expression levels of
Rubisco and carbonic anhydrase (CA) in WT and RLD100 mutant. Not only Rubisco and CA
abundance and their genes expression, but also Rubisco and CA activities seemed to be regulated in
two different ways upon Cd and Cu stress, as well as upon low and high concentrations of both toxic
metals. At moderate Cd stress conditions carboxylation activity maintenance seems to be CA-
dependent and energy independent, while adaptation to severe Cd stress became Rubisco activase
(RA)-dependent, thus energy-dependent. During adaptation to Cu stress CA has been immediately
inhibited and could not participate in carboxylation maintenance. Acclimation to moderate Cu stress
has been RA-dependent, while at strong toxicity of this metal additional support came from
increased Rubisco synthesis. Both mechanisms are energy-dependent and, in general, severe Cu
stress imposes harsher metabolic restrictions which could be thought as more difficult to overcome.
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BIOACCUMULATION AND BIOTRANSFORMATION OF HEAVY METAL(LOID)S IN

RELATION TO BIOREMEDIATION: USING A CASE STUDY INVOLVING
BACTERIAL-INDUCED LEAD IMMOBILIZATION.
Jinhee ParkA, B, Nanthi BolanA,B and Ravi NaiduA,B
A
CRC-Contamination Assessment and Remediation of Environment (CRC CARE);
Nanthi.Bolan@unisa.edu.au.
B
CERAR, University of South Australia, Mawson Lakes, SA-5095
Introduction
Soil represents the major sink for metal(loid)s released into the biosphere through both geogenic
(i.e., weathering or pedogenic) and anthropogenic (i.e., human activities) processes. The mobility
and bioavailability of metal(loid)s in soils are affected by adsorption onto mineral surfaces,
precipitation, formation of stable complexes and bioaccumulation and biotransformation by
microorganisms. Soil is a biologically-active integral component of the terrestrial ecosystem in
which higher plants, soil constituents and soil organisms interact, and the available energy in the
form of organic and inorganic compounds promotes microbial activity. The dynamics of
metal(loid)s in soils depends not only on their physico-chemical interactions with inorganic and
organic soil constituents but also on biological interaction associated, to a large part, with the
microbial activities of the soil-plant system.
Bioaccumulation and biotransformation processes can influence the solubility and subsequent
mobility of these metal(loid)s in soils by altering their speciation and oxidation/reduction state.
These processes play a major role in the ecotoxicology and environmental health of metal(loid)s.
Since it is unlikely that the natural phenomenon is optimal in metal(loid)s removal from
contaminated sites, the bioaccumulation and biotransformation processes can be readily managed
for efficient removal. The aim of the paper is to examine the influence of microbial processes in the
accumulation and transformation of metal(loid)s using a case study involving bacterial-induced lead
(Pb) immobilization.
Bacterial-induced immobilization of lead

Both soluble and insoluble phosphate compounds are also used to immobilize heavy metal
contaminated environments through phosphate-heavy metal precipitation. While soluble P compounds
are effective in the immobilization of heavy metals such as Pb, they can lead to groundwater pollution
through leaching of P. However, insoluble P compounds need to be solubilized for these compounds to
become effective in the immobilization of heavy metals. The objectives of this study are (i) to isolate
phosphate solubilizing bacteria (PSB) from P amended and Pb contaminated soils, (ii) to characterize
PSB in relation to P solubilization mechanism and (iii) to examine the effect of PSB on P-induced
immobilization of lead.
90
Materials and Methods

Ten soil samples used for PSB isolation were locally collected from P amended and Pb
contaminated sites. The soil samples were analyzed for pH, electrical conductivity (EC), total Pb,
NH4NO3 extractable Pb, and Olsen P.
To extract bacteria from soil, 0.5 g of soil was mixed with 50 mL of autoclaved 0.2 % NaCl solution
(soil: solution = 1: 100) and shaken for 16 hours. The isolation of PSB was carried out in NBRIP
medium containing insoluble tricalcium phosphate [Ca3(PO4)2] as the source of P. The colonies with
clear halo were considered as PSB. The PSB strains that are most effective in the solubilization of P
were identified using crude DNA extractions followed by PCR analysis.
The solubilization of P by PSB was measured using insoluble tricalcium phosphate [Ca3(PO4)2] in
NBRIP broth medium. The acid phosphatase activity in the culture media was measured by following
the ρ-nitrophenol reaction method. The organic acid content of the culture media was measured using
an ion chromatography.
The immobilization of Pb was tested with different levels of rock phosphate and soluble P
compound (KH2PO4). Lead spiked sterile soil samples were treated with various levels of P sources in
the presence and absence of PSB for 2 weeks and the amount of NH4NO3 extractable Pb was
monitored.
Results and discussion

19 morphologically different PSB strains were isolated from 10 different control, P fertilizer
amended and Pb contaminated soils. The number of PSB was relatively higher in P amended soils than
Pb contaminated soils. Lead is toxic to the microorganism which may be attributed to low level of PSB
in Pb contaminated soils. The two bacterial strains which showed the highest P solubilization capacity
were putatively identified as Pantoea sp. and Enterobacter cloacae.
Among 19 different bacterial strains isolated from these souls, 6 strains solubilised high levels of
(300mg/L) from insoluble tricalcium phosphate. pH, phosphatase enzyme activity and organic acid
concentration in bacterial culture were analyzed and their correlation with solubilized P was evaluated
to describe the mechanism of P solubilization by PSB. Acid phosphatase activity produced by the
isolated bacteria ranged from 0.0034 mM to 0.1420 mM as measured by p-nitrophenol production. The
PSB strains isolated from P amended soils showed relatively higher acid phosphatase activity compared
to the PSB from non amended soils and Pb contaminated soils. The amount of organic acid produced by
the isolated bacteria ranged from 20 to 112 mg/L. The PSB strains isolated from P amended soils
produced more organic acid than PSB from Pb contaminated soils. The most commonly produced
organic acids by the 19 isolated bacteria were acetic, pyruvic, fumaric and citric acids.
While there was no significant correlation between acid phosphatase activity and amount of
solubilized P, organic acid showed a positive correlation and pH showed a negative correlation with
solubilized P. It is known that production of organic acids and acid phosphatase play a major role in the
solubilisation of P in soils. Although the mechanism of P solubilization by organic acid was not well
91
understood, the pH decrease resulting from the release of organic acids could be related to P
solubilization (Eq. 1).
Ca10(PO4)6F2 + 12H+ 10Ca2+ + 6H2PO4- + 2F- (1)
The concentration of NH4NO3 extractable Pb decreased with increasing level of both soluble P and rock
phosphate and in the case of rock phosphate the effect was more pronounced in the presence than
absence of PSB. While the soluble P sources are likely to immobilize Pb rapidly, the rock phosphate
addition supplies a constant level P slowly to soil solution, thereby achieving long term immobilization
of Pb in the environment. The solubility of P and Pb sources determines the kinetics of pyromorphite
precipitation process which is likely to be rapid once both P and Pb become available in solution. The
dissolution of rock phosphate and precipitation of Pb as a carbonated fluoropyromorphite like mineral is
the primary mechanism for Pb immobilization by rock phosphate.
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FACTORS AFFECTING THE MOBILITY AND BIOAVAILABILITY OF (MICRO)

NUTRIENTS, HEAVY METALS AND METALLOIDS IN SOIL ENVIRONMENTS
A. Violante
Dipartimento di Scienze del Suolo, della Pianta e dell’Ambiente, Università di Napoli Federico II,
Portici (Napoli), Italy. violante@unina.it
Many factors, such as pH, nature of the sorbents, presence and concentration of organic and
inorganic ligands, including humic and fulvic acid, root exudates and nutrients, affect the mobility,
bioavailability, leaching and toxicity of trace elements. Furthermore, redox reactions, both biotic
and abiotic, are of great importance in controlling the oxidation state and thus, the mobility and the
toxicity of many elements, such as Cr, Se, Co, Pb, As, Ni and Cu.
The soil components responsible for trace element sorption include, soil humic substances,
phyllosilicates, carbonates and variable charge minerals (constituents such as Fe, Al, Mn and Ti
oxides, short-range ordered aluminosilicates as well as phyllosilicates coated by OH-Al or OH-Fe
species whose charge varies with the pH of the soil solution). Recently, evidence on the sorption of
heavy metals on microorganisms have been reported. Soil components differ greatly in their
sorption capacities, their cation and anion exchange capacities, and the binding energies of their
sorption sites.
A body of evidence has demonstrated that humic matter and metal oxides are much more
effective scavengers of trace elements in cationic form, than even the most efficient sorbent among
phyllosilicates, indicating that specific sorption and other complexation processes are the dominant
binding mechanisms. Trace elements in cationic form are probably not dominantly sorbed on 001
faces of phyllosilicates because they are always vastly outnumbered by other cations with which
they compete. They may be strongly sorbed only on the edges of the phyllosilicates. However, clay
minerals have also an important role as carriers of associated oxides and humic substances forming
organo-mineral complexes, which present peculiar sorption capacities different from those of each
single soil constituent.
Variable charge minerals selectively sorb polyvalent cations even when their surfaces are
positively charged (solution pH values lower than the point of zero charge [PZC] of the sorbent).
Most transition cations (Pb, Cu, Cr, Ni, Co, Zn, Al, Fe, Mn) are often sorbed more strongly than
alkaline earth cations. Spectroscopic techniques such as electron spin resonance (ESR) and
Extended X-ray Absorption Fine Structure Spectroscopy (EXAFS) have been used for the
identification of metal complexes at the surfaces of Al, Fe or Mn oxides, silicate clays and soil
organic matter. In the last decade extensive research has been carried out on the heterogeneous
precipitation of trace elements on the surfaces of minerals, using modern spectroscopy techniques
such as synchrotron-based X-ray absorption spectroscopy.
Many factors such as pH, surface properties of the sorbents, the number of sites available for
sorption, the nature and charge of Me-L species in solution influence trace element sorption onto
soil inorganic components (phyllosilicates and variable charge minerals) in the presence of
inorganic and biological ligands. Inorganic and organic ligands (e.g., organic acids) which form
strong complexes with trace element cation usually prevent or reverse their association with
negatively charged sorbents, as clay minerals, by forming stable dissolved or dispersed negatively
charged complexes with the cations. In contrast, the presence of certain foreign ligands, occurring
naturally in the rhizosphere, such as siderophores produced by microorganisms and
93
phytosiderophores exuded by plants, may promote the formation of positive complexes and,
consequently, the sorption of trace elements onto phyllosilicates.
The processes, which affect the sorption of trace element cations onto variable charge
minerals in the presence of complexing agents, are particularly complex and are different from those
onto phyllosilicates. Usually, high concentrations of low molecular weight organic acids may
promote the formation of negative complexes with metal ions and favour their sorption onto variable
charge minerals, whereas siderophores may promote the formation of positive complexes,
preventing sorption. In conclusion, biological ligands play a very important role on the mobility of
trace elements at soil-root interface.
Trace elements which exist in anionic form are sorbed primarily by chemisorption at reactive
sites of metal oxides and allophanes and at the edges of phyllosilicates. Sorption of anions onto
variable charge minerals and soils varies with pH. With increasing pH, within a certain range,
sorption decreases (due to a decrease of positive charge of minerals) or else increases to a maximum
close to the pKa for anions of monoprotic conjugate acids and then decreases. Anions may be
specifically or nonspecifically sorbed. Ligands which are specifically sorbed replace OH- or OH2
groups from the surfaces of variable charge minerals. Specific sorption is also termed inner-sphere
sorption because it involves direct coordination to the surface metal atom. Nonspecific sorption is
also termed outer-sphere sorption and is influenced by the ionic strength of the system.
The presence of organic and inorganic ligands also affects the sorption of trace elements in
anionic form by competing for available sorption sites and/or reducing the surface charge of the
variable charge minerals and soils. The competition depends on the affinity of the anions for the
surfaces of the sorbents as well as the nature and surface properties of the minerals and soils.
Competition may be easily observed at sufficient high surface coverage. Time of reaction and the
surface coverage have also a great influence on the competitive sorption between trace elements and
organic and inorganic ligands. Most competitive sorption studies have been carried out adding the
ions contemporaneously. In natural environments, however, it is more likely that the ions will come
in contact with a sorbent sequentially, i.e., the solid is exposed to one ion first, with the second ion
coming in contact with a solid at a later time. The sorption of metals and metalloids is strongly
affected by the order of addition of organic and inorganic ligands and trace elements on the sorbents.
It has been demonstrated that larger amounts of selected heavy metals (Pb, Cu) were sorbed when
chelating organic anions (oxalate, tartrate) were added before or after the metals as referred to the
systems where the heavy metals were added as a mixture with the organic ligands or alone.
In contrast to sorption studies relatively little information is available on the desorption of
metals and metalloids from soils or soil components. Presence of inorganic and organic ligands,
such as phosphate, sulfate and root exudates, has a significant impact on the desorption of trace
elements. A number of factors, such as the type, mineralogy and crystallinity of the sorbents, the pH,
the pe, the surface coverage, the residence time of metals and metalloids on the surfaces of soil
components may affect the desorption of trace elements. Some studies on the effect of residence
time on the desorption of some heavy metals and metalloids have been carried out. Usually, the
longer the residence time, the greater the decrease in trace element desorption, because of a
rearrangement of surface complexes and/or a conversion of surface complexes into surface
precipitates. In soil environments trace elements may be not only sorbed on but also coprecipitated
in metal oxides. Their partial removal by nutrients or organic ligands from coprecipitates has
received some attention only in the last years.
The mobility and bioavailability, and hence potential toxicity of trace elements depend on
their concentration in soil solution, the nature of its association with other soluble species, and the
94
soil’s ability to release the metal from the solid phase to replenish that removed from soil solution
by the plants. Fractionation of trace metal cations into operationally defined forms under the
sequential action of given leachants with increasing aggressiveness is a common approach for
distinguishing the operationally defined various species of heavy metals according to their physico-
chemical mobility and potential bioavailability. Finally, soil microorganisms are affected by the
presence of high metal concentrations in soil, but the organisms in turn influence the availability of
metals in soil either directly, through alterations of pH, Eh, biosorption and uptake, or indirectly in
the rhizosphere through their effect on plant growth, root exudation, and resulting rhizosphere
chemistry.
95
96
Simposio 4.
Suelos, Fisiología y Nutrición Vegetal
97
98
MULTICOMPONENT ADSORPTION OF CU AND ZN ON ALLOPHANIC CLAY

Alejandra A. Jara1,2, Claudia Pezo2 and María de la Luz Mora1,2
1
Departamento de Ciencias Químicas, 2Scientific and Technological Bioresource Nucleus,
Universidad de La Frontera, Temuco, Chile. Corresponding author: aljara@ufro.cl
Adsorption of divalent metal ions on mineral surfaces is one of the important sorption process
regulating mobility, bioavailability and toxicity in natural systems like soils (Hodgson, 1963; Jenne,
1977). The mechanisms that influence the solubility and speciation of the metals in soils require
higher attention to understand the process that governs the metal chemical form and their behavior
to be uptaken by plants. The solubility and, consequently the availability of metals in soils depend of
different factors, like soil pH, organic matter content, oxidant conditions and type of clay in soil.
The aim of this study was study the competitive adsorption of Cu and Zn on clay fraction extracted
from an Andisol of Southern Chile. The experiments were carried out in batch system for 24 h at
pH, ionic strength (KCl 0.1 M) and temperature (25 ºC) constants, with clay fraction/solution ratio
0.1 g/10 mL. The metals in solution were determined by atomic absorption spectrophotometer
(AAS). Zn and Cu isotherms were conducted from pH 4.5 to 6.0, showing an increase in the
adsorption with the pH. These results are corresponding with the negative charge of the surface (IEP
= 2.56) and surfaces with variable charge. The isotherm of Cu and Zn evaluated by Freundlich and
Langmuir models at pH constant (5.5) suggest that Cu exhibit a higher affinity than Zn for the
allophanic clay surface. This observation is in agreement with the later desorption studies. Zn
desorption percent (10.3 %) at pH 5.0 was slightly higher than Cu (6.8 %) in the first hour.
However, at 10 h the desorption capacity was similar for both metals (2 %). In allophanic clay the
competitive adsorption is expect that the sites density is not a limit factor (high surface area, and low
metal concentration should not have effect on the adsorption.The competitive adsorption
experiments showed that Zn do not have effect on Cu adsorption, but Zn adsorption in presence of
Cu increased to initial molar ratio Zn/Cu > 2.5. Similar results were reported by Violante et al.
(2005) in experiments about Zn and Cu adsorption on organo-mineral complexes. It is possible that
the adsorption mechanisms of Zn and Cu are similar, there are studies that may to form monodentate
and bidetante complexes on the goethite surface (Xu et al., 2006). Nevertheless, the types of
complexes do not explain both why the adsorption increase and their specific interaction with the
surface.
Acknowledgments
Financial support from Fondo Nacional de Desarrollo Científico y Tecnológico (11070241) is
acknowledged.
References
Hodgson, J.F., 1963. Chemistry of micronutrient elements in soils. Adv. Agron. 15, 119-159.
Jenne, E.A., Trace element sorption by sediments and soils-sites and processes. In: W. Chappell and
K. Peterson, Editors, Symposium on molybdenum in the environment, Marcell Dekker, New York,
1977, pp. 425–453.
Violante A., Ricciardella, M., Pigna, M. y Papasso, R. 2005. Effects of organic ligands on the
sorption of trace elements onto metal oxides and organo-mineral complexes. In Biogeochemistry of
Trace Elements in the Rhizosphere, ed. Huang, P.M. and Gobran G.R. Elsevier B.V, 157-182.
Xu, Y., Axe, L., Yee, N. and Dyer, J.A. 2006. Bidentate complexation modeling of heavy metal
adsorption and competition on goethite. Environ. Sci. Technol., 40, 2213-2218.
99
NITROGEN AND PHOSPHOROUS DYNAMICS IN A PERENNIAL RYEGRASS PASTURE

OF SOUTHERN CHILE
A. A. Jara1, R., Demanet2, E. Stohmann1, M. González1, E. Cantero2 and M. L. Mora1*
1
Center of Plant, Soil Interaction and Natural Resources Biotechnology; Scientific and
Technological (BIOREN). 2Instituto de Agroindustria. La Frontera University, Avenida Francisco
Salazar 01145, Temuco, Chile. *Corresponding author: mariluz@ufro.cl
In intensive pastures in Southern Chile, farmers apply large amounts of phosphorus and nitrogen
to achieve the optimum dry matter yield. But, in general the effect of N fertilizer on P and N losses
has not received major attention. In Chile, grassland systems are commonly fertilized with urea at N
supply ranging from 45 to 300 kg N ha-1 (Mora et al., 2004; 2007; Cartes et al., 2009). The nutrient
dynamics in soil-plant system depends on the fertilization rate and the environmental condition. In
this context, the aim of this work was to obtain complementary studies about the nutrient dynamics
in the rhizosphere in acidic conditions under nitrogen (N) and phosphorus (P) fertilization in an
Andisol of Southern Chile. Ryegrass pastures were established in an Andisol belonging to Freire
Serie on March, 2009 in the Maquehue Experimental Station of the Universidad de La Frontera.
Four N doses (0, 40, 80 and 120 kg N ha-1 as urea) were applied in combination with four P doses (0,
80, 160 and 240 kg P2O5 ha-1 as triple superphosphate) in triplicate plots. In order to quantify the N
leaching we put a lysimeter in each plot. Also, the soil was fertilized with sulphur, potassium and
magnesium as Sulpomag (200 kg ha-1). The seed doses used were equivalent to 22 kg seed ha-1. The
seeds were previously pelletized with sodium selenite in doses equivalent to 20 g ha-1. In the soil
samples obtained between April and September, 2009 were evaluated soil pH, Olsen-P, N-NH4+ and
N-NO3- (in soil and in leaching) and soil urease activity. The soil pH increased after N fertilization
(April, 21) from pH 5.3 to 5.7 and thereafter it returned to original values (5.4). Later, pH values
were maintained constants or slightly increased according to natural variation. Olsen-P increased as
P fertilizes rates increased, but also it appeared that it returned to its original values (around 18 mg P
kg-1) and showed no significantly differences with the treatment because of high P fixation and P
uptake by plants. On the other hand, urease activities ranged from 250 to 500 µ mol g-1 2h-1 in the
time sampled. After urea application it was observed that urease activity increased with the N doses
in all P treatments and also with supplied P. We are observing an important effect of P fertilizer on
urease activity and consequently an increment in N-NO3- in the soils. Nitrification process increased
the N-NO3- concentration about 110 mg N kg-1, meanwhile N-NH4+ was kept around 18 mg N kg-1.
In general, N-NH4+ losses decreased as P rate fertilizer increased while N-NO3- losses increased,
because nitrification increased by P supplied. The dry matter production increased with both P and
N rates, from 3.3 to 6.6 ton ha-1 at 120 kg N ha-1 and 240 kg P2O5 ha-1 in the sample period
(approximately 7 months).
Acknowledgements. This work was supported by the FONDECYT N° 1061262.
Mora, M.L., Ordoñez, C., Cartes, P., Vistoso, E., Pino, J., Jara, A., Demanet, R. 2004. Reciclaje de nitrógeno
proveniente de purines en una pastura de Lolium perenne L. In: M.L. Mora (editors), Simposio de las Ciencias del
Suelo: Residuos orgánicos y su uso en sistemas agroforestales. Sociedad Chilena de la Ciencia del Suelo. Temuco,
Chile. Universidad de La Frontera. p. 243-256.
Mora, M.L., Cartes, P., Núñez, P., Salazar, M., Demanet, R. 2007. Movement of NO3--N and NH4+-N in an Andisol and
its influence on ryegrass production in a short term study. J. Soil Sci. Plant Nutr., 7, 46-64.
Cartes, P., Jara, A.A., Demanet, R. and Mora, M.L. 2009. Urease activity and nitrogen mineralization kinetics as
affected by temperature and urea input rate in southern Chilean Andisols. J. Soil. Sci. Plant Nutr., 9, 69-82.
100
ANTIOXIDANT ACTIVITY AND TOTAL PHENOLIC AND ANTHOCYANINS

CONTENTS IN HIGHBUSH BLUEBERRY FRUITS AT DIFFERENT MATURITY
STAGES
Ribera, A.E.1*; Reyes-Díaz, M.(1); Alberdi, M.(1); Verdugo, M.(1,2); Mora, M.L. (1)
1
Technological (BIOREN); 2 Post-degree Program of Natural Resources Sciences. La Frontera
University, Avenida Francisco Salazar 01145, Temuco, Chile. *aribera@ufro.cl
The antioxidant activity (AA) and total phenolic (TPH) and anthocyanin (TA) contents in highbush
blueberry (Vaccinium corymbosum L.) fruits (whole fruit, shell and skin) of different cultivars
cultivated in Chile (Legacy, Brigitta and Bluegold) at different maturity stages (from 100% green to
100% blue) were evaluated. The results indicated that AA of ripe whole fruits (100% blue) varied
among the cultivars in the order Legacy > Brigitta > Bluegold. Skin extracts, in all maturity stages
and for all cultivares, showed the high AA compared with those of whole fruit and shell. In general,
AA of whole fruits decreased at increasing maturity from 100% green to 75% red stages, whereas
from 75% red to 100% blue stage, AA increased steadily. On the other hand, in shell and skin
extracts, at increasing maturity stages AA decreased and increased lineally, respectively. TPH and
TA content varied among fruit tissues in the order Skin > whole fruit > shell. In whole fruits, TPH
decreased from 100% green to 75% red and increased from 75% to 100% blue maturity stages. In
shell and skin extracts, TPH at increasing maturity decreased and increased, respectively. In
conclusion, AA of 100% green fruits and 100% blue fruit was similar. The high content of TPH and
TA could explain the higher levels of AA in 100% green and 100% blue fruits, respectively, than
those observed at immature stages (from 25% red to 100% red). In addition, the results (HPLC
analyses) showed especially high contents of chlorogenic acid and rutin in blueberry fruits, which
varied significantly through the different maturity levels and among fruit tissues.
Acknowledgements: Postdoctoral FONDECYT N° 3090074 (Responsible researcher: Dra.

Alejandra Ribera) and Regular FONDECYT N° 1080372 (Responsible researcher: Dra. Miren
Alberdi) Grants.
101
EFFECT OF NITROGEN FERTILIZATION ON SOIL PHOSPHOROUS DYNAMICS AND

LOLIUM PERENNE L. PRODUCTION AND NUTRITION UNDER GREENHOUSE
CONDITIONS
A.E. Ribera, A.A. Jara, E. Stohmann, M. González, M. Reyes-Díaz, M. Alberdi and M.L.
Mora*.
Technological (BIOREN). La Frontera University, Avenida Francisco Salazar 01145, Temuco,
Chile. *Corresponding author: mariluz@ufro.cl
Nitrogen (N) fertilizers can modify soil pH in the rhizosphere. Actually, urea is the N source mostly
used in Southern Chilean soils, although, it has been reported that the acid reaction fertilizer causes
rhizospheric phosphorous (P) deficiency, limiting the P uptake by crops. Hence, urea can reduce the
growth and shoot P concentration in plants. The aim of this study was to evaluate the effect of urea
applications on soil nutrient distribution and forms, and on perennial ryegrass (Lolium perenne L.)
production and mineral nutrition.
The assay was carried under greenhouse conditions in pots using two Chilean Andisol
(Piedras Negras and Freire Series) treated with four doses of N (0, 50, 100 or 200 mg N kg-1, as
urea) and P (0 or 300 mg P kg-1, using triple superphosphate), all combinations with or without
limed treatment (2000 mg kg-1 of Magnecal). In addition, plants were fertilized with sulphur (S) in
doses of 50 mg kg-1 and with boron (B) at 0.5 mg kg-1. In every pot, 100 seeds, previously
palletized with sodium selenite in doses equivalent to 20 g ha-1per pot, were sown. After 60 days,
plants were harvested for determination of dry weight (DW), and N and P shoot concentrations. In
the soil from each pot was determined the follow parameters: available P, exchange bases,
exchangeable Al, and soil pH. Moreover, we determined the rhizospheric soil solution pH, which
was extracted using solution micro-suctions at 5 days intervals. Plant photosynthetic capacity was
analyzed, through the determination of net photosynthesis (using a portable CO2 infrared analyzer;
Liquor LR6400) and fluorescence emission of PSII [maximum photochemical efficiency (Fv/Fm)]
in pre-darkened leaves using a modulated fluorometer FMSII (Hansatech).
The results indicated that DW production increased in response to both, P and N
applications. Shoot P concentrations, which ranged from 1.34 to 4.17 g kg-1, proportionally
increased at increasing P doses. In both soils, shoot P concentration in ryegrass decreased in a doses
dependent manner with N application. As expected, the percent of shoot N increased at increasing
N doses. Soil available P decreased in response to lime application. The addition of N significantly
increased the concentration of exchangeable Al, Ca, Mg and K in both soils, although the higher
levels were founded in the non-limed soils. N fertilization decreased soil pH values, which ranged
from 4.9 to 5.6 in Freire soil and from 4.3 to 5.7 in Piedras Negras soil. Theses pH decreases were
also detected in the rhizospheric soil solution. Ryegrass plants grown in Piedras Negras soil without
P applications (1 month of growth) showed low values of maximum photochemical efficiency of
PSII (Fv/Fm) and net photosynthesis (µmol CO2 m-2s-1), while plants grown under all other
fertilization treatments showed normal levels for both parameters.
Acknowledgements. This work was supported by the FONDECYT N° 1061262.
102
IDENTIFICACIÓN DE ARCILLAS EN SUELOS DE LA REGIÓN DE ANTOFAGASTA

Y SU USO COMO ADSORBENTES DE METALES PESADOS.
Fariña Ca. , Miranda Na., Agüero Pa., Alarcón N bc., Fuentes B bc*.
a
Ingeniería Civil Ambiental, Departamento de Ingeniería Química, Universidad Católica del Norte,
Antofagasta. Chile. b Departamento de Ingeniería Química, Universidad Católica del Norte.
Antofagasta, * e-mail bfuentes@ucn.cl. c Centro para el Desarrollo de Tecnologías de Explotación
Sustentable de Recursos Hídricos en Zonas Áridas, (CEITSAZA). Antofagasta. Chile.
Los metales pesados son contaminantes no biodegradables y tóxicos. En la región de

Antofagasta son frecuentes en suelos y aguas, y su presencia se debe a origen natural y
antropogénico relacionado con procesos extractivos mineros. Los suelos de la zona están
constituidos por diferentes arcillas que pueden ser utilizadas en el tratamiento de efluentes mineros
para la remoción de metales pesados. El objetivo de este trabajo fue identificar los minerales
presentes en suelos de la Región de Antofagasta y probar su efectividad como adsorbentes de
metales pesados. Se tomaron muestras de suelo (perfil 0-20 cm) desde distintas localidades
(Antofagasta, Calama, Mejillones, Aguas Blancas I y Aguas Blancas II). Las muestras fueron
secadas a 105ºC y tamizadas por malla de 0,5 mm. Luego se analizaron por difracción de rayos X
(XRD). El suelo Aguas Blancas II se utilizó para realizar pruebas de adsorción de Zn y Cu a
distintos pH (entre 5 y 8). Los ensayos de adsorción se realizaron a 25ºC, se utilizaron 0,25 g de
suelo y 25mL de solución de metal (40 mg l-1) con fuerza iónica 0,01 en KCl. La cinética de
adsorción se realizó hasta 48 h utilizando solución de metales de 100 mg l-1 la suspensión se ajustó a
pH 7. Los análisis de XRD mostraron la presencia de diferentes arcillas en los suelos estudiados:
zeolita (4-9%), caolinita (4-5%), illita (20-13%), clorita (2-10%). Sólo en el suelo Aguas Blancas II
se identificó montmorillonita (9%) y vermiculita (13%), y se cuantificó el mayor contenido de
zeolita (9%). Las pruebas de adsorción indicaron que a pH 7 se logró remover un 96% de Cu y un
62% de Zn. Los estudios cinéticos indicaron que el tiempo de equilibrio para la adsorción de Zn fue
de 24 h. No fue posible determinar el tiempo de equilibrio para la adsorción de Cu, ya que con la
concentración utilizada se produjo 100% de adsorción de Cu en todos los tiempos probados.
Estudios posteriores de isotermas de adsorción permitirán determinar la capacidad de adsorción del
suelo. El suelo Aguas Blancas II presentó una elevada capacidad de adsorción de Cu y Zn, lo cual
fue atribuido al alto contenido de arcillas presentes, por lo tanto se proyecta su uso en el tratamiento
de efluentes mineros.
Agradecimientos:
Proyecto DGIP, UCN, 2009. Desarrollo de un adsorbente órgano-mineral para la adsorción de
metales pesados desde efluentes líquidos.
103
IRRIGACIÓN DE UN SUELO SALINO CON AGUA RESIDUAL PROVENIENTE DE

LA INDUSTRIA CERVECERA: EFECTO SOBRE LA CONDUCTIVIDAD Y EL PH.
Miranda Na., Fariña Ca., Agüero Pa., Alarcón N bc., Fuentes Bbc*.
a
Ingeniería Civil Ambiental, Departamento de Ingeniería Química, Universidad Católica del Norte,
Antofagasta. Chile. b Departamento de Ingeniería Química, Universidad Católica del Norte.
Antofagasta, * e-mail bfuentes@ucn.cl. c Centro para el Desarrollo de Tecnologías de Explotación
Sustentable de Recursos Hídricos en Zonas Áridas, (CEITSAZA). Antofagasta. Chile.
En la región de Antofagasta, la salinidad de los suelos, la baja disponibilidad y calidad del recurso
hídrico son un problema común que limita la actividad agrícola, el uso de técnicas de
fitoremediación de suelos contaminados y el desarrollo de cultivos energéticos. El objetivo de este
estudio fue evaluar los efectos que produce la irrigación de agua residual de la industria cervecera
sobre la conductividad eléctrica (CE), el pH y el contenido de materia orgánica (MO) del suelo. Se
realizaron ensayos de irrigación en columnas utilizando un suelo de la comuna de Antofagasta (pH:
7,0; CE: 5 mScm-1; MO: 0,3%), agua residual de la industria cervecera (pH: 6,7; CE: 5 mScm-1) y
agua destilada como control. El volumen total de irrigación fue de 1000 mL y la medición de
parámetros (CE y pH) se realizó cada 100 mL de lixiviado. Finalizado el experimento, se dividió la
columna de suelo en 5 partes (cada una de 3,5 cm) y se determinó CE, pH y MO. Los resultados
mostraron que la CE de los lixiviados del suelo irrigado con agua y con agua residual variaron entre
18 y 3 mScm-1 y entre 16 y 8 mScm-1, respectivamente. En tanto que el pH de los lixiviados
incrementó de 7,5 a 8,5 y de 7,0 a 7,5 cuando se utilizó agua destilada y agua residual,
respectivamente. El suelo irrigado con agua destilada alcanzó valores de CE de 0,7 mScm-1,
mientras que el suelo irrigado con agua residual presentó valores de 2,8 mScm-1, en los primeros 3,5
cm. La irrigación con agua destilada provocó un incrementó de pH hasta 8,5, en cambio la
irrigación con agua residual incrementó el pH del suelo hasta 7,8. La incorporación de agua residual
incrementó el contenido de MO en el perfil del suelo alcanzando valores de 0,7%. La irrigación con
agua residual, mejoró las características físico-químicas del suelo de Antofagasta, lo cual se vio
reflejado por el decrecimiento de la conductividad y por la menor variación del pH del suelo, debido
probablemente a la incorporación de materia orgánica.
Agradecimientos: Proyecto DGIP, UCN, 2009. Desarrollo de un adsorbente órgano-mineral para la

adsorción de metales pesados desde efluentes líquidos.
104
DISPONIBILIDAD Y MOVILIDAD DE NITRÓGENO EN UN ANDISOL Y SU

INFLUENCIA SOBRE LA PRODUCCIÓN DE Lolium perenne.
Paredes C.1 *, y Mora M.L. 1, 2
1
Programa de Doctorado en Ciencias de Recursos Naturales. Universidad de La Frontera. Av.
Francisco Salazar 01145, Temuco, Chile. 2Scientific and Technological Bioresource Nucleus
(BIOREN), Center of Plant, Soil Interaction and Natural Resources Biotechnology, Universidad de
La Frontera, Temuco, Chile, Email mariluz@ufro.cl.* e-mail autor: cparedes@ufro.cl.
El Nitrógeno (N) ocupa una posición única entre los elementos esenciales derivados del suelo para
el crecimiento de la plantas, debido a su compleja biogeoquímica y las grandes cantidades
requeridas por las plantas, en comparación con otros elementos. El N es uno de los elementos con el
cual se obtienen los mayores efectos en rendimiento, sin embargo se ha reportado que altas dosis
provocan alteraciones en el crecimiento de raíces y brotes. Además se ha demostrado que las plantas
muestran un crecimiento diferencial en respuesta a las fuentes de suministro de N, ya sea NH4+ o
NO-3. El objetivo de este estudio fue evaluar la aplicación de dos fuentes de N (N-N03 y N-NH4+)
en condiciones de invernadero, sobre la producción de L. perenne cv. Nui, la concentración de N en
el suelo, en la planta y en el lixiviado. El ensayo de invernadero se llevo a cabo utilizando tubos de
PVC (simil a lisímetros), con un diámetro interno de 11.3 cm y 24 cm de largo. Para evitar el paso
de las raíces, se coloco una malla de nylon de 35 µm de diámetro de poro, en el extremo inferior del
lisímetro y para permitir la recolección de los lixiviados se utilizo una tapa del mismo material con
una salida que permitía la colección del lixiviado por goteo. Cada lísimetro fue llenado con 1.100 g
de suelo previamente incubado con 300 mg P kg-1, aplicado al suelo como KH2PO4. Se aplicaron
dosis de N como KNO3 y (NH4)2SO4 equivalente a 0, 100 y 300 mg N kg-1 s. Se consideraron 3
repeticiones por niveles de N. Para la determinación de N foliar, parte aérea y raíces, se utilizo la
metodología descrita por Sadzawka et al. (2007). La determinación de N disponible y pH en el suelo
se realizó mediante la metodología descrita por Sadzawka et al. (2006). El N lixiviado desde el
suelo como N-NO3- fue determinado por cromatografía iónica. La concentración de N-NH4+ fue
determinado mediante electrodo selectivo de amoniaco con medición directa.Los resultados de
materia seca, indicaron que la producción aumentó con la dosis aplicada de 300 N-NO3- y 100 N-
NH4+, sin embargo con la dosis de 300 N-NH4+ la producción disminuyó significativamente. La
concentración de N varió entre 3 a 5 mg kg-1 en la parte aérea siendo mayor con la concentración de
300 N-NH4+. En el caso de las raíces, la mayor concentración fue de 3.3 mg kg-1 con la mayor dosis
de NH4+.Como era de esperar se produjo una variación en el pH, dependiendo de la fuente de N
aplicado, mostrando valores de 5,2 + 0,2 cuando la fuente era N-NO3- y valores de 4,3 + 0,2 cuando
la fuente era N-NH4+. Las concentraciones de N disponible ya sea como NO3- o NH4+, siempre
fueron mayores significativamente con las dosis más altas y a mayor profundidad 10-20 cm. Esto
también se aprecia en las concentraciones de NO3- y NH4+ en el lixiviado. Se observa además, la
movilidad que presenta el ion NO3- en el suelo quedando de manifiesto las pérdidas producidas por
el fenómeno de lixiviación.
REFERENCIAS
Sadzawka, A., Carrasco, M. A., Demanet, R., Flores, H., Grez, Z.R., Mora, M.L., Neaman, A.
(2007). Métodos de análisis de tejidos vegetales. Segunda edición. CNA, Sociedad Chilena
Ciencia del Suelo.
Sadzawka, A., Carrasco, M. A., Flores, H., Grez, Z.R., Mora, M.L., Neaman, A. (2006). Métodos
de análisis recomendados para los suelos de Chile. CNA, Sociedad Chilena Ciencia del Suelo.
105
DIFFERENTIAL ANALYSIS OF GENE EXPRESSION BY cDNA-AFLP IN HIGHBUSH

BLUEBERRY (Vaccinium corymbosum L.) UNDER ALUMINUM TOXICITY
Inostroza-Blancheteau, C.1,2, Aquea, F.2, Reyes-Díaz, M.3, Alberdi, M.4, Mora, M.L.4, and
Arce-Johnson P*2.
1
Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera, Casilla 54-
D, Temuco, Chile, e-mail: cinostroza@ufro.cl. 2Laboratorio de Bioquímica, Departamento de
Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia, Universidad
Católica de Chile, Casilla 114-D, Santiago, Chile, e-mail: parce@bio.puc.cl. 3Instituto de
Agroindustria, Center of Plant, Soil Interaction and Natural Resources Biotechnology, Scientific and
Technological Bioresource Nucleus (BIOREN-UFRO), Universidad de La Frontera, Casilla 54-D,
Temuco, Chile. 4Departamento de Ciencias Químicas, Center of Plant, Soil Interaction and Natural
Resources Biotechnology, Scientific and Technological Bioresource Nucleus (BIOREN-UFRO),
Universidad de La Frontera, Casilla 54-D, Temuco, Chile.
Highbush blueberry (Vaccinium corymbosum L.) is cultivated in Chile mainly in acid soils. In these
soils aluminum (Al) toxicity is the major limiting factor for crop productivity. The first symptom of
Al toxicity in plant is a rapid inhibition of root growth. Several Al-regulated genes have been
identified in the roots of different plant species. To investigate the molecular bases of Al toxicity
and Al resistance of blueberry, cDNA-amplified fragment length polymorphism (cDNA-AFLP) was
used for identify Al-regulated genes in roots of an Al-tolerant genotype, Brigitta, and an Al-sensitive
ones, Bluegold. One year old seedlings of both genotypes were transferred to hydroponic medium
(Hoagland's solution) for 7 days and subsequently transferred to a solution of calcium chloride
(CaCl2) supplemented with 0 and 100 µM Al3+ nominal as aluminum chloride (AlCl3). Root samples
were taken at 0, 2, 6, 12 and 48 hours of treatment and frozen at -80ºC until RNA extraction.
Selective amplifications with 28 primer combinations allowed the visualization of about 3,800
transcript-derived fragments (TDFs), 168 of which (4.4%) were differentially expressed between
cultivars or Al treatments. These TDFs were designed as VCAL (Vaccinium corymbosum
Aluminum), were selected, sequenced and their homologies sought in the databases. Several of these
genes perform functions in cellular metabolism, transport, signal transduction, photosynthesis and
energy, as well as response to abiotic stress. Among TDFs identified in tolerant cultivar, three of
them codified for genes associated with oxidative stress, a glutation-S-transferase (GST),
calmodulin and vacuolar H+-pyrophosphatase. In the sensitive cultivar we have identified a ATP-
citrate lyase, gene related with resistance to Al in Lupinus sp. The molecular characterization of
these genes is a step important in its use as potential candidates for Al resistance in highbush
blueberry.
This research was supported by Millennium Nucleus for Plant Functional Genomics, Fondecyt-
1080372, Fondecyt-11080231, PSD74-2006 and CONICYT Ph.D. Fellowship
106
EFFECT OF Al/Ca RELATION ON PHOTOSYNTHETIC CAPACITY OF HIGHBUSH

BLUEBERRY CULTIVARS (Vaccinium corymbosum L.).
Meriño C1*, Reyes-Díaz M2, Alberdi M2 and Mora ML2
1
Programa de Doctorado en Ciencias de Recursos Naturales. 2Center of Plant, Soil Interaction and
Natural Resources Biotechnology, Scientific and Technological Bioresource Nucleus (BIOREN-
UFRO), Universidad de La Frontera, Casilla 54-D, Temuco, Chile. *Corresponding author:
cmerino@ufro.cl
Keywords: Al/Ca relation, gypsum, photosynthesis, Vaccinium corymbosum L.
In Chile, the most agronomic activities are developed in Andisols (pH range 4.5 to 5.5), such as,
highbush blueberry (Vaccinium corymbosum L.) crop. Although this species is good adapted to acid
soils, is very sensitive to aluminium phytotoxic (Al3+), which constitutes a problem for its
productivity. On the other hand, different calcium (Ca) sources are used to improve blueberry fruit
quality and yield. Ca is an essential nutrient for many physiological and biochemical processes. The
interactions between Ca2+ and Al3+ are probably the more important factors affecting Ca2+ uptake,
and transport in plants grown in acid soils (pH≤5.5). It has been recognized that Al toxicity is
ameliorated by basic cations especially Ca.
Two blueberry cultivars (Legacy and Brigitta) growing in nutrient solution (Hoagland solution pH
4.5) for 15 days, under greenhouse (temperature controlled 20-25 ºC, 16/ 8h light/dark regimen,
80% relative air humidity and photosynthetic photon flux density (PPFD; photometer Licor LI-
250A) average of 300 µmol photons m-2 s-1) were treated with increased aluminium concentrations:
25, 50, 100 and 200 µM as AlCl3 and calcium concentrations: 2.5, 5.0 and 10 mM applied as
gypsum (CaSO4) and their interactions. It was determinated that the most adequate and inadequate
Al/Ca ratios to induce changes in physiological parameters such as photosynthesis (CO2
assimilation; Licor-6400), fluorescence parameters (Fv/Fm: maximal photochemical efficiency,
ΦPSII: Effective photochemical efficiency, NPQ: Non-photochemical quenching and ETR: electron
transport rate; FMSII Hansatech) and biomass as root and shoot growth. The results indicated that
Brigitta showed a significant increase of fresh weight (FW), shoot and roots length for 2.5, 5 and
10mM CaSO4 treatments, while Legacy increase these parameters in Al and Ca treatments.
Photochemical efficiency (Fv/FM and ΦPSII) was decreased with 100 and 200 µM Al in two
cultivars. On the other hand, Brigitta was the most affected cultivar in its CO2 assimilation by Al
treatments. In addition, Ca with Al treatment stimulate the growth of blueberry plants, by contrast
Al treatment (100 and 200 µM) were negative effects on photochemical parameters, indicating that
Ca ameliorate this Al toxicity condition.
Acknowledgments: FONDECYT Nº 11080231, Berries Sn. Luis.
107
IMPROVING PROTEIN EXTRACTION METHODS FROM DIFFERENT PLANT

ORGANS OF AGRICULTURAL CROPS FOR ENHANCED PROTEOMIC ANALYSIS
C. WULFF-ZOTTELE1*, I. CARUSO2, B. GRECO2, A. E. RIBERA FONSECA1 AND M. L.

MORA GIL1
1
Center of Plant, Soil Interaction and Natural Resources Biotechnology. Scientific and
Technological Nucleus, La Frontera University, Temuco, Chile. 2Department of Soil, Plant,
Environmental and Animal Production Sciences, University of Naples Federico II, Portici (Naples),
Italy. * mail of corresponding authors: cwullff@ufro.cl; immacolata.caruso@unina.it
protein extraction, polyphenols, potato, tomato, blueberry
The proteome allows a deep understanding of changes in the gene expression of proteins and their
post-translational modifications in organisms exposed to different environmental conditions. During
the last four decades, the development of mass spectrometry protein sequencing, electrophoresis,
and bioinformatics tools, permitted the improvement of proteomics. Currently, the techniques most
often employed for analyzing large protein populations are 2-DE and HPLC. Both techniques can be
coupled, either with ESI MS or MALDI-TOF MS, to identify proteins. A critical aspect in protein
analysis is the amount of interfering compounds. They did not allow a good resolution in protein
separation through the utilization of 2D-polyacrilamide gel electrophoresis. Compared to other
organisms, plants are generally more problematic for proteomic analysis, because their tissues are
rich in interfering compounds, such as cell wall components, pigments, polyphenols, organic acids
and proteases. In this work, we describe an efficient method for total protein extraction from leaves
harvested from potato and tomato plants and blueberry fruits (peel, flesh and complete fruit). Based
on the protocols of protein extraction described by Damerval et al, 1986 and Wang et al., 2003, we
developed a new protein extraction method consisting in the utilization of a first step of protein
precipitation using 10%TCA, 0.07% 2-mercaptoethanol in acetone solution, and in a second step of
a phenol extraction from acetone powder extracts. Total amount of protein were quantified using
Bradford assay. Good protein yields were obtained, ranging from 1.0 to 2.5 mg of total protein per
300 mg of fresh tissue. 10 to 25 g of total protein was separated through the utilization of SDS-
PAGE, and stained with Coomasie Blue. Furthermore, 300 g of proteins extracted from developed
potato leaves were used for 2D gel protein separation.
Results from SDS-PAGE and 2D-gel allowed concluding that the protocol of protein extraction
is effective to decrease the amount of interfering compounds and to obtain well-resolved protein
bands detected using SDS-PAGE and 2-DE of potato and tomato leaves, and different tissues of
blueberry fruits.
Acknowledgments: This work was funded by the grants MECE-FRO601 and Scientifical
research projects of Universidad de la Frontera (DIUFRO Nº 4023.206), and the Colaboration
Program between the Ph.D. programs of Natural Resources of Universidad de la Frontera, and
University of Naples Federico II.
108
IDENTIFICATION OF PROTEINS INVOLVED IN THE ADAPTATION RESPONSES TO

ALUMINUM STRESS IN ROOT TISSUES OF RYEGRASS GROWN IN DIFFERENT
CONDITIONS OF SULFUR FERTILISATION
Wulff-Zottele C.1,3,Stohmann E.1, Mora M.L.1,2.
(1) Instituto de Agroindustria, Universidad de la Frontera, Francisco Salazar 01145, Temuco, Chile.
(2) Departamento de Ciencias Químicas, Universidad de la Frontera, Temuco, Chile. E-.mail:
cwulff@ufro.cl. (3) Center of Plant, Soil Interaction and Natural Resources Biotechnology.
Scientific and Technological Nucleus, La Frontera University, Temuco, Chile. E-mail of
corresponding author: cwulff@ufro.cl
Soil acidity is one of the yield-limiting factors in soils used for agricultural purposes in the world.
Acid injury of root tissues is mainly caused by aluminum toxicity, and it is the basis of physiological
alterations associated to the uptake of macronutrients. In addition, volcanic acid soils (Andisols)
show high sorption capacities that are finally reflected in low availability of sulfate and phosphorus
for plant nutrition. To overcome the problems of sulfate and phosphate nutrient availability and root
tissue injury by aluminum toxicity, gypsum (Calcium sulfate) and lime (Calcium carbonate) are
mechanically mixed into the volcanic soils in the field. Studies at chemical level suggests that the
addition of gypsum increase the uptake of macronutrients (e.g. sulfate), and it could be associated to
the activation of physiological mechanisms against aluminum toxicity in plant tissues. Considering
the aforementioned facts, we decided to study the modifications in the protein profile of root tissues
of plants treated with combinatorial conditions of aluminum stress and increased conditions of
ammonium sulfate fertilization, aimed to establish the role of sulfur in the enhancement of the
response of plant against Al-stress. Perennial ryegrass (L. perenne cv. Jumbo) was tested in a
factorial design with four ammonium sulfate fertilization conditions (120 µM, 240 µM, 360 µM, and
480 µM) and Al-stress (200 µM) in Taylor Foy´s nutrient solution. At day 28 of treatment, root
tissues were collected and used for protein isolation using 10% TCA in acetone solution. 7.5 g of
total amount of protein of each root extract was separated through the application of one
dimensional gel electrophoresis under denaturing conditions (SDS-PAGE). Proteins bands that have
shown significant modifications in their expression in Al-treated plants, as compared to controls,
were isolated from the gels, trypsin digested, and sequenced using MALDI-TOF mass spectrometry.
The mass spectra results obtained were submitted to Mascot 2.2 and Internet databases in order to
search homologies in other plant species. The results of protein separation by SDS-PAGE permitted
the identification of an enhancement in the protein expression profile in ryegrass roots treated with
combined conditions of Al-stress and fertilization doses between 120 to 360 µM of ammonium
sulfate. Interestingly, 13 proteins displayed a significant induction in root tissues of plants grown in
conditions of Al-stress (200 µM) and 360 µM of ammonium sulfate fertilization. In addition,
MALDI-TOF allowed the identification four protein bands, corresponding respectively to a small 17
KDa heat shock protein, class III peroxidase, calcium bindig protein, and a putative nucleotide
dispnhosphatase.
General conclusion: Adequate sulfur fertilization induces the expression of proteins that enhance
the physiological response against aluminum toxicity in ryegrass roots.
Acknowledgments: This work was funded by the grants MECE-FRO601 and Scientifical
research projects of Universidad de la Frontera (DIUFRO Nº 4023.206)
109
GENETIC AND PHYSIOLOCICAL CHARACTERIZATION OF THE DROUGHT

RESPONSE IN TOMATO
GRECO B. , DE STEFANO R. , REYES-DIAZ M.2, WULFF-ZOTTELE C.2, DI MATTEO
1* 1
A.1, MORA GIL M. L.2 AND BARONE A.1
1
Department of Soil, Plant, Environmental and Animal Production Sciences, University of Naples
Federico II, Portici (Naples), Italy. 2Center of Plant, Soil Interaction and Natural Resources
Biotechnology. Scientific and Technological Nucleus (BIOREN-UFRO), La Frontera University,
Temuco, Chile. *Corresponding author: barbara.greco@unina.it
Drought stress in plants is one of the resulting effects of climatic change in the world due to CO2
emissions, and its consequences cause major yield losses. Most crop plants, including tomato
(Solanum lycopersicum), are sensitive to drought stress. Substantial genetic variation for Drought
Tolerance exists within the cultivated tomato, as well as in other related wild species. However, the
genetic variability and differences in the response to drought stress in tomato species is not well
understood to warrant its use for developing drought-tolerant cultivars. The aim of this work is to
identify polymorphisms of genes involved in drought tolerance in different tomato cultivars and
wild species through the utilization of the PCR technique. Primers were designed to detect Single
Nucleotide Polymorphisms (SNPs) in putative drought stress-related genes, such as MKP1 (MAP
kinase phosphatase), Asr2 (named after abscicic acid [ABA], stress, ripening), TSW12 (a lipid
transfer protein gene). By analyzing the sequences obtained from cultivars and wild species of
tomato a total of 62 point mutations were identified in MKP1 gene, 17 mutations in Asr2 and 10
mutations in TSW12. In addition, the effect of the water deficit was assessed on the photosynthetic
performance in young leaves of tomato plants grown in Andisols collected from the Maquehue
station of Universidad de la Frontera (Temuco series). Tomato plants were pre-grown in volcanic
soil, and fertilized, during two weeks in the greenhouse. The experiment was started when in
drought-treated plants the water potential in soil was 3.0 Atm, and in control plants was 0.3 Atm.
Photosynthetic performance of PSII and CO2 exchange in young tomato leaf tissues were measured
at the starting day (time 0) of the experiment, the first day in which the water potential was 0.3 Atm
in treated plants, and after 15 days of drought conditions. In the treated plants, the maximum
quantum yield (Fv/Fm) was 5% lower than the control at 15 days. The effective quantum yield
(ФPSII) was 22% lower in the treated plants than the control ones, while the treated plants showed
12% higher non-photochemical quenching (NPQ). In CO2 assimilation the values from treated
plants were around 50% lower than the control ones. At the moment, the general conclusion of the
work is that the combination of genetic and physiological studies could help to identify
polymorphisms in genes that can be associated to the tolerant or susceptible phenotype, leading to
the design of useful molecular markers for assisted selection programs.
110
GREENHOUSE GASES EMISSIONS FROM AGRICULTURAL SOILS AND

MITIGATION STRATEGY
Muñoz, C.1, Paulino, L.1, Stolpe, N.1, Ovalle, C.2, Suescún, F.3 and Zagal, E.1
1
Department of Soil and Natural Resources, Universidad de Concepción, Campus Chillán, Chile;
2
Departamento de Producción Animal, Instituto Nacional de Investigaciones Agropecuarias (INIA)
Quilamapu, Chillán, Chile; 3Master Program in Sciences, University of Concepcion, Chillán, Chile.
Author of correspondence: cristinamunoz@udec.cl; ezagal@udec.cl
The anthropogenic activities are potential sources of Greenhouse Gases (GHG) emissions where
CO2 and N2O are of relevance in agricultural systems. In South America few information is
available about in situ quantification of GHG from agricultural and agroforestry systems. In Chile, a
national inventory was done based on factors proposed by the Intergovernmental Panel of Climate
Change (IPCC) for the most representative systems, but reveled the lack of information for their
particular characteristics of soil (meanly volcanic ones) and climate considering the most relevant
productive activities at the country or region scale. Our research group has the main aims to assess
biogeochemical processes related to GHG emissions (meanly CO2 and N2O) from soil under
different degrees of anthropogenic influence. We study in situ and ex situ GHG emissions from
different agricultural systems including agriculture uses and management of the Central-South of
Chile, using field and laboratory experiments. In addition, we analyze the microbiological process
and abiotic conditions that influence the GHG emissions from soils. The preliminary results to show
that in volcanic soils the N2O emission have a conservative patron with minimum emissions rate
under no-till system, and the CO2 emission is strongly influenced by water content and temperature
of soil. In the study of mitigation strategy we study the effect of some vegetal metabolites over the
nitrification and denitrification processes, founded that some vegetal species can reduce the losses of
nitrogen with the production of metabolites with capacity to inhibit biological process like soil
respiration and microbial biomass in the soil.
Acknowledgments: Fondecyt Project (N° 1080076), Programa Bicentenario de Ciencia y

Tecnología, “Inserción de Investigadores a la Academia 2005 (PSD-12)”, CONICYT-World Bank-
Universidad de Concepción and DIUC Project (N°208.123.010-1.0).
111
THE EFFECT OF AL/CA RATIO ON NITROGEN, MEAN RELATIVE GROWTH RATE

AND RUBISCO CONTENT IN VACCINIUM CORYMBOSUM L.
Alarcón E1*, Reyes-Díaz M2 and Alberdi M3
1
Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera, Temuco,
Chile, *email: edith.alarcon@gmail.com. 2Instituto de Agroindustria, Center of Plant, Soil
Interaction and Natural Resources Biotechnology, Scientific and Technological Bioresource
Nucleus (BIOREN-UFRO), Universidad de La Frontera, Casilla 54-D, Temuco, Chile.
3
Departamento de Ciencias Químicas, Center of Plant, Soil Interaction and Natural Resources
Biotechnology, Scientific and Technological Bioresource Nucleus (BIOREN-UFRO), Universidad
de La Frontera, Casilla 54-D. Temuco, Chile.
Acid soils are limiting factor of agriculture productivity in around the world. In South of Chile the
soils derived from ash volcanic are acid (Andisols). The acidity of Andisol permit the Al
solubilization and its transformation in aluminium phytotoxic (Al3+). The amendments application
(as gypsum for example) are frequently used in blueberry cultivation (Vaccinium corymbosum L.) to
ameliorate the acidity and aluminium phytotoxicity. However, the few reports about the gypsum
application in Andisols and its influence on the physiological performance of plants are been
reported. Based on these antecedents the objective of this work was to determine the optimal
relationship between aluminium and calcium in V. corymbosum. Legacy cultivar growing in
hydroponic condition (Hoagland solution) under greenhouse with different treatments during 15
days was investigated. The treatments were: Control (Hoagland solution), increased aluminium
concentrations 25, 50, 100 and 200 µM as AlCl3 and calcium concentrations 2.5, 5.0 and 10 mM
applied as gypsum (CaSO4) and their interactions. After 15 days mean relative growth rate (MRGR),
nitrogen (N) of leaves and Rubisco contents were determined.
The preliminary results indicated that the MRGR was lower in 10mM CaSO4 treatment than the
control and others treatments. Also, N concentration of leaves were lower in the mayor
concentration of CaSO4 (10 mM) and 200 µ M Al. About this we can speculate that a mayor CaSO4
concentration can induce other type of toxicity, affecting the growth and N uptake in this species,
but more studies in this area are necessary. On the other hand, a higher Al concentration affected the
N uptake, observing lower N concentration in leaves. With respect to Rubisco content, the extraction
of total protein was standardized for blueberry and also Rubisco was visualized by Western Blot.
With these results we can concluded preliminarily that the low CaSO4 and Al concentration no
affected the growth and N uptake.
Acknowledgments: Fondecyt Nº 11080231 project, technical assistance and San Luis Farm, Lautaro.
112
SYNTHETIC NANOPARTICLES LIKE ALLOPHANE: POTENTIAL USE AS

CATALYST HETEROGENEOUS IN THE FENTON LIKE REACTION FOR ORGANIC
POLLUTANT REMOVAL
Garrido, E.G1* and Mora, M.L2.
1Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera, Temuco

Chile. 2Scientific and Technological Bioresource Nucleus (BIOREN-UFRO), Universidad de la
Frontera, Temuco Chile. *eggarrido@ufro.cl
Nanoparticles can be used in many technological applications such as electronics, medicine,

catalysis, environmental and other. The soils are natural source of nanoparticles 1. Allophane is the
most important component of the clays fraction of Andisols of Southern Chile. This aluminosilicate
occur as hollow spherules in the nanoscale range, with an external diameter between 3.5 and 5.5 nm
and wall thickness of 0.7–1.0 nm. In addition, iron oxides of short-range order, notably ferrihydrite,
are also widespread in Andisols and occur as coating of clays mineral particles. Due to allophane
nanoclays properties and natural association with iron oxide, we have proposed the use of allophane
nanoclays as catalyst in the Fenton like reaction for organic pollutant degradation 2. Recently we
were able to extract allophane nanoclays from an Andisol of Southern Chile but for industrial
application, the synthesis of nanoparticles like allophane is necessary. The Aim of this work was
synthesize nanoparticles like allophane coated with iron oxide and evaluate their catalytic activity in
the Fenton like reaction. Nanoparticles like allophane were synthesized by coprecipitation of
potassium silicate and aluminium chloride3 and coated with iron oxide by wet impregnation method
4
. The nanoparticles were characterized by TEM, FTIR and elemental analysis. The Surface charge
properties were evaluated by the Isoelectric Point (IEP), the Point of Zero Salt Effect (PZSE) and
the Zero Point Charge (ZPC). The catalytic activities of nanoparticles like allophane were studied by
discoloration of methylene blue (MB) at a concentration of 50 mg/L with H2O2 (0.3M) and 10 mg of
the catalyst. The MB discoloration was monitored by UV/Vis spectroscopy at 663 nm. All reactions
were carried out in a recirculating temperature-controlled bath at 25±1 ºC and pH 7.0. The
SiO2/Al2O3 ratio was 2.2 ± 0.27. TEM showed aggregates of individual hollow spherical
nanoparticles with similar morphology to natural and synthetic allophane. The FTIR spectres
presented the typical bands reported for allophane. The zero point charges were 6.2 (ZPC), 4.47
(IEP) and 4.1 (PZSE). The MB concentration showed a rapid decay in the first 2 min. (90% of
discoloration) and then followed by a much slower decay rate (95% and 99% of discoloration for
adsorption and Fenton reaction respectively in 72 h). This behavior was represented for a
mathematical model, in which both of them α (inverse proportional to the first-order reaction rate
coefficient k) and β (inverse proportional to the logarithm of the maximum MB discoloration)
constants were determined. The calculated values of constants α and β were 0.1185 h and 0.3124 for
adsorption and 0.326 h and 0.2108 for Fenton like reaction. The FTIR spectra for nanoparticles like
allophane after to be used as catalyst in the Fenton like reaction didn’t change. We can concluded
that synthetic nanoparticles like allophane is a promissory material that could be used as catalyst in
the Fenton like reaction for the organic pollutant removal.
[1] Calabi, F.M., Theng, B.K.G., Reyes, P., Mora, M.L. 2009. Clay Miner. 44, 161-176.
[2] Garrido-Ramírez, E.G., Theng, B.K.G., Mora, M.L. 2009. Appl. Clay Sci. In press
[3] Díaz, P., Galindo, G., Escudey, M., 1990. Bol. Soc. Chil. Quim. 35, 385–389.
[4] Mora, M.L., Escudey, M., Galindo, G., 1994. Bol. Soc. Chil. Quim. 39, 237–243.
113
COSTS OF CONSTRUCTION AND CONVERSION EFFICIENCY OF DRY MATTER OF

BLUEBERRY (VACCINIUM CORYMBOSUM L.) ORGANS
Jorquera-Fontena, E1.; Franck N2.
1
Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera. 2 Centro
de Estudios de Zonas Áridas y Departamento de Producción Agrícola, Universidad de Chile.
Plant species show wide variation in their potencial relative growth rate when grown under optimal
conditions. The construction cost (CC) represents the amount of photoassimilates needed to provide
the carbon skeletons, the reducing power and the chemical energy involved in the synthesis of a unit
weight of biomass. In crop models, CC is the coefficient used to convert the carbohydrates produced
during photosynthesis into dry matter. Another parameter for analyzing CC is the conversion
efficiency coefficient (Yg) which indicates the fraction of all carbon (C) fixed through
photosynthesis for growth process that must be rereleased in the process of synthesizing new
biomass. For the time being, only a limited set of standard values of CC and Yg are used in crop
models. In the most recent CC studies, three methods have been privileged 1) the heat of
combustion (Williams et al., 1987); 2) the elemental composition (Mcdermitt and Loomis, 1981)
and 3) the carbon, nitrogen and ash content (Vertregt and Penning de Vries, 1987), a simplification
of the elemental composition method.
This study presents preliminary results on the estimation of CC and Yg of dry matter in different
blueberry organs using the method of Vertregt and Penning de Vries, which was chosen based on its
simplicity and high goodness of fit as compared other, more complex, existing methods. Samples of
different organs (buds, stems, fruits and leaves) were collected from blueberries cv. ‘brigitta’ grown
in a commercial orchard located in the Araucania region (38º30’S, 72º25’W) of Chile under optimal
watering and fertilizing conditions. The samples were oven-dried at 70 ºC in the laboratory. Then
the carbon and ash content was measured by pirolisys and nitrogen content was measured by
Kjedhal method.
The preliminary results show estimations of CC of 1.95; 1.93; 1.91; 1.93 (g glucose g dry mass -
1
) for buds, stems, fruits and leaves, respectly indicating that CC are higher for buds than for others
organs in blueberry. These general results are slightly higher as compared to other studies, in anual
and fruits crops. The conversion efficiency coefficient varied beetwen 0.55 and 0.56 (mol C
mol C-1) being lower for buds. Therefore, 55-56% of the carbon available for growth will be
retained as new dry mass of the organs and the remaining will be respired as growth respiration.
Given Yg typically has a value of between 0.25 and 0.5.
The results presented here are the first estimations of the CC and Yg of blueberry organs (no
values are presented in the literature to the author’s knowledge), we therefore envision to persue this
study in order to increase the precision of the estimated parameter values which will then be
incorporated into a blueberry carbon balance model.
114
METABOLIC AND MORPHO-ANATOMICAL ANALYSIS IN SYNTHETIC

AUTOTETRAPLOIDS OF WILD POTATO SPECIES
CARUSO I.1, ARONNE G.2, AVERSANO R1, DAL PIAZ F3., DE MICCO V2, FASANO
C.1, GARRAMONE R.1, LEPORE L.3, SCOGNAMIGLIO N.1 AND CARPUTO D.1
1
Federico II, Via Università 100, 80055 Portici. 2 Department of Arboriculture, Botany and Plant
Pathology, University of Naples Federico II, Via Università 100, 80055 Portici. 3Department of
Pharmaceutical Sciences, University of Salerno, Via Ponte don Melillo, 84084 Fisciano (SA).* mail
of corresponding author: immacolata.caruso@unina.it
polyploids, Solanum, leaf area, stomata parameters, secondary metabolites
The common occurrence of polyploidy suggests an evolutionary advantage of having multiple sets
of genetic material for adaptive evolution. Polyploidy produces anatomical, biochemical and genetic
changes that affect physiological processes in plants. Morpho-anatomical effects include changes in
organ size and number of cells and organelles. Biochemical effects include changes in amounts of
gene products (gene dosage effects of polyploidy) and changes in biochemical diversity. Potato
provides an excellent model system for studies on polyploidy, since the genus Solanum shows an
exceptional tolerance of ploidy manipulation. The aims of this study were to produce 4x genotypes
from two 2x wild species (S. commersonii and S. bulbocastanum) and to characterize obtained
polyploids from the morpho-anatomical and biochemical standpoint. Nine 4x genotypes (five from
S. commersonii and four from S. bulbocastanum,) were produced by oryzalin treatment and
analyzed. While in S. commersonii the leaf area reduced with increased ploidy level, in S.
bulbocastanum two 4x genotypes had a leaf area larger than the 2x progenitor and two did not.
Results from analysis of stomata frequency (per mm2) and length (guard-cell length, pole to pole)
showed significant differences between 2x progenitors and 4x genotypes. However, we did not
observe a clear relationship between stomata parameters and polyploidization. As for metabolite
profiles, the analysis of 4x genotypes in S. commersonii showed that leaf content of total alkaloids
was significantly lower in the 4x genotypes than in the 2x progenitor. By contrast, 4x genotypes
showed an increased amount of some phenol compounds, as caffeic and ferulic acids and rutine. To
elucidate the molecular events associated to polyploidization, gene expression and epigenetic studies
are being carried out. Using the whole transcriptome approach screening of microarray, we will
have a complete eyeshot of how gene expression profile is modulated after polyploidization. In
addition, epigenetic regulation of gene expression will be studied using MSAP (Methylation
Sensitive Amplified Polymorphism) analyses.
115
SOIL WETTABILITY: A COMPARISON OF METHODS FOR ITS EVALUATION

Ramírez, J.
Departamento de Ciencias Químicas, Universidad de la Frontera, Temuco. Av. Francisco Salazar

01145, Casilla 54-D. Temuco, Chile. ramirezsuelos@googlemail.com
The wettability is defined as the ability of a fluid to wet a solid. This process depends on the
interaction between adhesion and cohesion forces, resulting in phenomena like liquid spreading on
solid surfaces and liquid rise in capillaries. In soil science, the wettability is a major factor affecting
the erosion rate and occurrence of surface runoffs. Wettability acts also at different scales into the
soil profile, influencing the dynamics of aggregation and movement of nutrients. The wettability is
characterized by the contact angle (CA). A higher CA indicates higher hydrophobicity. The present
research compares the results of different methods to calculate the CA in porous media for future
application in real soils. Four non-consolidated porous materials were used: Silt, sand and glass
beads of two different fractions (40-70 µm and 150-250 µm). Samples with different levels of
hydrophobicity were obtained by mixing hydrophilic (untreated) and hydrophobic particles (treated
with Dichlorodimethylsilane) at different rates. The CA was measured with the Sessile Drop
Method (θSDM), the Capillary Rise Method (θCRM) and a method consisting in the measurement of
the maximal capillary height of water and ethanol of the same materials, previously packed into
plastic columns (diameter: 1.8 cm; height: 150 cm) (θEQ). All methods and materials showed a direct
relation between the CA and the hydrophobicity level (higher amount of hydrophobic particles)
(R2>0.90). Assuming that the big-scale θEQ method can be related to the wetting behavior of a real
soil system, both small-scale methods (θSDM and θCRM) showed deviations of the CA, especially for
the most hydrophilic samples as also for silty particles. θSDM showed a subestimation of the CA up
to 30º approximately, while θCRM showed an overestimation up to 20º - 30º. It is concluded that the
differences between the methods regarding the CA are explained due to their different theoretical
backgrounds: while θSDM measures the CA directly on a graduate scale, θCRM calculates the CA
based on the wetting kinetics into the porous system. Further research is needed in order to correct
both small-scale methods in relation to the wetting behavior of real soils.
116
NATURAL AND NANOPARTICLES FROM AN ANDISOL AND ITS APPLICATION IN

ATRAZINE CONTROLLED RELEASE FORMULATIONS
Cea, M.*, Mora, M.L.*
*Scientific and Technological bioresources Nucleus, Universidad de La Frontera, Temuco, Chile
Chemical surface fuctionalization and surface structuring particularly on the nanometer scale play
an increasing role in many fields of technological application. However, the production of
nanoparticle with small diameters and a narrow size distribution is challenging and expensive.
Therefore, natural nanoparticles are a cheap alternative if the size distribution is narrow and the
extraction of the particles from soil material is economically efficient. Our goal was to provide a
nanostructured surface based on natural nanoparticles to develop atrazine controlled release
formulations. To obtain the nanometric fraction of soils (nanoclays covered with organic matter) we
used the methodology described by Li and Hu (2003). The nanoparticles were characterized by
transmission electron microscopy (TEM), scanning electron microscopy (SEM), infrared
spectroscopy (FTIR), isoelectric point (PIE) and specific surface area Brunauer-Emmett-Teller
(SSABET). Microspheres containing nanoparticles and ethylcellulose were prepared by the solvent
evaporation technique according to the techniques described by Fernandez-Urzuno et al. (2000) and
Dong and Feng (2005). The surface morphology of the microspheres was examined by SEM. The
microspheres size was measured by image analysis. In order to determine the efficiency of the
microencapsulation process, and atrazine content of the microspheres, samples were dissolved in
methanol (1mg mL-1) and analyzed by High Performance Liquid Chromatography (HPLC). The
encapsulation efficiency was measured according to the parameters: LH (encapsulated
herbicide/final weight of the microspheres), SR (percentage of microspheres to the total weight of
polymer and atrazine used) and EE (percentage of herbicide encapsulated with the herbicide used in
formulating). The Transmission electron microscopy micrographs revealed more or less condensed
aggregates of nanoparticle and the obtaining material corresponds to nanoclays with a particle
average size less than 100 nm. The FTIR spectra showed characteristic bands of the soil organic
matter (fulvic and humic acids). Low values of PIE (3.2) are attributed to the organic matter
covering of nanoclays. The SSABET was 8 m2g-1. With regard to the controlled release formulations,
the size ranged from 200 m to 500 m and, the results indicated a HL range of 7.5 % to 9 %. SR
values fluctuated between 61-72 %, finally EE values fluctuated between 27-39 %. A next stage of
the research is to evaluate the water release kinetics of atrazine from obtained formulations.
Acknowledgments: We wish to thank FONDECYT proyect Nº 3080039.
References
Dong, Y. and Feng, Si-Shen. 2005. Poly(D,L-lactide-co-glycolide)/montmorillonite nanoparticles
for oral delivery of anticancer drugs. Biomaterials,26: 6068-6076.
Fernandez-Urrusuño, R., Gines, J.M. and Morillo, E. 2000. Development of controlled release
formulations of alachlor in ethylcellulose. J. Microencapsulation. 17(3): 331-342.
Li Z., Hu N., 2003. Direct electrochemistry of heme proteins in their layer-by-layer films with clay
nanoparticles. Journal of Electroanalytical Chemistry. 558: 155 – 165.
117
STRUCTURAL CHARACTERIZATION OF NATURAL NANOMATERIALS:

POTENTIAL USE TO INCREASE THE PHOSPHORUS MINERALIZATION.
Marcela Calabia, Alejandra Jarab, James Bendallc, Mark Wellandc and María de la Luz
Morad
a
Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera. b Departamento de Ciencias
Químicas, Universidad de La Frontera. c Nanoscience Centre of The University of Cambridge, England. d
Scientific and Technological Bioresource Nucleus (BIOREN-UFRO), Universidad de La Frontera. Av.
Francisco Salazar 01145, P.O. Box 54-D, Temuco, Chile. mcalabi@ufro.cl
Nanoparticles occur widely in the natural environment. Particles within nanoscale range have been studied in
soil sciences few decades ago to understand the behavior of the soil environment (Wada 1987; Parffit et al.
1983). However, these particles have not been studied in the nanoscience point of view. In nanoscience,
material design and surface feature of nanomaterials play an increasing role in many fields of technological
application such as: electronics, medicine, biocatalysis, material science and others. Although, synthetic
nanomaterials are relevant in nanoscience for a wide possibility of applications (Garrido et al. 2010), the
production of nanoparticle with small diameters and a narrow size distribution is expensive and difficult.
Nanoclays is a real alternative to get a material with nanometric size. In soils derived from volcanic ash as
Andisols (Calabi et al. 2009), is possible to find different structures of nanoparticles like aluminosilicate with
nano-ball (allophane) and nano-tube (imogolite) morphology within clay fraction. The more important
constituent of Andisols is allophane a non-crystalline aluminosilicate with morphology of hollow spherule
with an outer diameter of 3.5–5.0 nm. These nanoparticles form stable microaggregates, with pores within the
nanoscale range with similar physical characteristics to silica nanomaterials which are very important in
biocatalysis (Kim et al. 2006). Previous results from our investigation group shown that acid phosphatase
immobilized on allophanic clays from Andisols of Southern Chile increase its catalytic efficiency (Rosas et
al. 2009). The aims of this study were: i) to characterize natural allophanic clay and nanoclay fraction with
and without organic matter from an Andisol and ii) to evaluate allophanic and montmorillonite nanoclays as
support to immobilize acid phosphatase. An Andisol, from Piedras Negras Series, taken from Southern Chile
was sampled within 0–20 cm of depth, sieved to 2 mm mesh and air-dried. One part of the soil was treated
with 30% hydrogen peroxide to remove the soil organic matter. The separation of particle-size < 2 µm
fractions was performed by sedimentation procedures based on Stoke's law. The nanoclays was extracted
from allophanic clays and montmorillonite clay (AppliChem A6918, LOT 7W007719), those were extracted
using clays pre-treated in the similar way as described by Li and Hu (2003). Allophanic clays and nanoclays
were characterized throughout energy dispersive X ray (EDX), transmission electron (TEM) and scanning
electron (SEM) microscopy, electronic diffraction (ED) and atomic force microscopy (AFM). The complexes
were formed by interaction between acid phosphatase and allophanic and montmorillonite nanoclays, and
used as model systems to simulate enzymatic reactions in heterogeneous environment. The enzymatic
activities of free and immobilised phosphatase were assayed with 6 mM p-nitrophenylphosphate (p-NPP) and
the concentration of p-nitrophenol was determined by measuring of the absorbance at 405 nm with a
spectrophotometer (Rao et al. 2000, Rosas et al. 2009). The kinetics parameters (Vmax and Km values) were
calculated according to Michaelis–Menten equation. The microscopy analysis showed that the methodology
of the nanoclays extraction allowed obtain aggregates with the high proportions of mesoporous, which are
suitable to enzymatic immobilization. The nanoclay fractions showed that organic matter governs the feature
behavior and is very recalcitrant, suggesting that this nano-fraction play an important role in carbon
sequestration. Finally, we demonstrated that the phosphatase immobilized have a significant increase both,
the catalytic efficiency (Vmax/Km) and the substrate affinity (1/Km) when allophanic and montmorillonite
nanoclays were used like material support.
Key Words: Natural nanoclays; allophane; aluminosilicate, enzyme immobilization, acid phosphatase
118
EFFECT OF GYPSUM APPLICATION ON PHOTOSYNTHETIC PERFORMANCE OF

VACCINIUM CORYMBOSUM L.
Reyes-Díaz M1*, Meriño C2, Mora ML1 and Alberdi M1
1
Center of Plant, Soil Interaction and Natural Resources Biotechnology, Scientific and
Technological Bioresource Nucleus (BIOREN-UFRO), Universidad de La Frontera, Casilla 54-D,
Temuco, Chile, *reyesm@ufro.cl. 2Programa de Doctorado en Ciencias de Recursos Naturales,
Universidad de La Frontera, Temuco, Chile.
In southern Chile, soils derived from volcanic ashes (Andisols) with an acid pH are frequent. The
acidity of this soil type may permit the Al solubilization from silicates and oxides and its
transformation in phytotoxic aluminium (Al3+) as occurred in other allophanic Andisols. Al toxicity,
which induce ROS in several plants, seems to be important in limiting the plant growth and
production in agricultural volcanic ash soils in Southern Chile. It is known, that gypsum treatment of
acid Andisols reduced exchangeable Al and its toxicity, increasing the yield of some crops. On the
other hand, the gypsum application could also supply water-soluble calcium (Ca2+) without
significantly pH changes. Ca2+ appears to be indispensable for normal functioning of the
photosynthetic apparatus and it is an essential key constituent for root hair formation. The
interactions between Al3+ and Ca2+ are probably the most important factors affecting Ca2+ uptake
and transport in plants grown in acid soils (pH < 5.5). These conditions seems to be very important
for blueberry which need soil acidity for growth. On the other hand, the few reports about the
utilization of gypsum in Andisols deal to its influence on the soil but not with respect to the effects
on the physiological performance of plants.
Currently, in different areas of Southern Chile the most preponderant agronomic activity is
represented by the cultivation of highbush blueberry (Vaccinium corymbosum L.). Although
blueberry is adapted to acidity, the response to the interaction between acid substrate and aluminium
and gypsum applications and their implications in its photosynthetic performance remains unknown.
In this work Brigitta, Legacy and Bluegold cultivars growing in hydroponic solution (Hoagland)
were subjected to following treatments: control (Hoagland solution); 2.5 mM CaSO4; 5 mM
CaSO4; 100 µ M Al; 100 µM Al + 2.5 mM CaSO4; 100 µM Al + 5 mM CaSO4 during 15 days.
After this time the growth rate, photosynthesis (CO2 assimilation), fluorescence parameters of PSII
(Fv/Fm, ФPSII and ETR) and antioxidant activity (AA) were performed.
A negative effect (about 50%) in the mean relative growth rate (MRGR) was found in Brigitta
and Bluegold under Al treatment and with a 5mM CaSO4 with respect to the controls, whereas in
Legacy practically no changes were found. The addition of 2.5 and 5 mM CaSO4 in presence of Al-
stress ameliorated the negative effect on the MRGR in Bluegold, while in Brigitta that occurred only
at the high CaSO4 concentration. Markedly differences in CO2 assimilation were observed only in
Bluegold under Al toxicity, showing a slight recovery with gypsum application without to reach the
control values. Similar results were obtained for the photochemical parameters. In roots and leaves
AA increased with the Al and gypsum treatments, specially in Brigitta. In conclusion, our results
showed that the gypsum amendments could be a promissory tool in the improvement of Al toxicity
of blueberry cultivars growing in Andisols.
Acknowledgments: Fondecyt Nº 11080231 and 1080372 projects, technical assistance and Berries
San Luis, Lautaro.
119
AN ASSESSMENT OF THE SE BENEFITS FOR PASTURES SPECIES GROWING IN

SOUTHERN CHILE
Cartes, P.1,*; Gianfreda, L.2; Jara, A.1; Shene, C.1; Pinilla, L.1; Rosas, A.3; Mora, M.L.1
1
Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Temuco, Chile.
2
Dipartimento di Scienze del Suolo, della Pianta, dell’ Ambiente e delle Produzioni Animali,
Università di Napoli Federico II, Portici, Italy. 3Departamento de Recursos Naturales, Universidad
de Concepción, Chillán, Chile. *Corresponding author: pcartes@ufro.cl
Selenium (Se) is regarded as an essential trace element for animal and human health because its
biological role as a component of a number of specific enzymes, but the current molecular evidences
does not confirm the presence of essential Se-proteins in vascular plants. Nevertheless, since 1990
decade several reports have shown the beneficial effect of Se in terms of plant growth, delay of
senescence and defense against abiotic stress such us UV and cadmium through the activation of the
plant antioxidative system (e.g. Harttikainen, 2005; Filek et al., 2008; Pedrero et al., 2008).
We are very interested in the management of Se as a fertilizer because Se deficiency has been
reported in soils and pastures of Southern Chile (Soil and Plant Analysis Laboratory database) and
low Se intake has been associated with nutritional disorders in grazing cattle (e.g. Wittwer et al.,
2002). In addition, most of pastures of Southern Chile are developed on acidic Andisols, which are
characterized by both the high phytotoxic Al3+ in soil solution and the low phosphorus (P)
availability for the plant growth. In this way, widespread agricultural practices to improve the yield
on these soils include the application of lime, gypsum and large amounts of P fertilizers, leading to
changes in the Se availability in the soil-plant system. One way to increase the Se level in grasslands
systems (and therefore to increase the Se intake in the food chain) is through the application of Se as
a fertilizer to the soil. However, in the Chilean market there are not Se-fertilizers, which support the
need to develop technologies for Se application for deficient pastures areas. During several years,
we have conducted a series of experiments to assess the benefits of Se application on the
antioxidative system of pasture species commonly sown in Southern Chile in order to improve the
forage quality.
In our first approach to the study of the biological role of Se (Cartes et al., 2005), we
investigated the effects of Se chemical form (selenite and selenate) and their rates of application on
the antioxidative system of ryegrass (Lolium perenne). We evaluated the activity of the antioxidant
enzyme glutathione peroxidase (GSH-Px) and lipid peroxidation (TBARS, an index of oxidative
stress) in the shoots of plants grown in an Andisol at increasing rates of Se (selenite or selenate)
application under greenhouse conditions. Our results indicated that selenite and selenate increased
progressively the shoot Se concentration in ryegrass, and the greatest Se concentration was achieved
in selenate-treated plants. We observed that Se lowered the level of lipid peroxidation at shoot Se
concentration up to 20 mg kg-1, irrespective of the chemical form of applied Se. However, the
activity of GSH-Px was related closely with the Se chemical form rather than the shoot Se
concentration. Thus, although both Se chemical forms activated GSH-Px, selenite was more
efficient than selenate to induce the enzyme activity, and for this reason we selected selenite as Se
source for further studies.
120
Later on, we decided to study the effect of sulfur (S) fertilization on Se distribution into organic
and inorganic fractions in ryegrass (Cartes et al., 2006) due to Se is metabolized in plants by the S
assimilation pathway. We hypothesized that increasing rates of S application to Andisols affects the
dynamics of Se assimilation into organic forms as well as the role of Se as an antioxidant in selenite-
treated plants. Ryegrass plants were cultivated in a pot experiment at a rate of selenite application
of 2 mg Se kg-1 soil and increasing S supply levels (0 to100 mg S kg-1 soil). Sulfur application
decreased by at least 33% the shoot Se concentration, and the organic-Se residual fraction seemed to
increase at expense of the organic-Se soluble fraction (aminoacids and soluble protein fraction). The
addition of S at increasing supply levels also diminished the selenite antioxidant ability: the GSH-Px
activity was reduced and TBARS accumulation was increased as a consequence of the competitive
interaction between selenite and sulfate in plant tissues.
In another greenhouse experiment, we employed successfully the technology of pelletization of

seeds with selenite to increase the Se content in white clover (Trifolium repens) and to evaluate the
impact of the level of soil acidity and P fertilization on the plant antioxidant responses (Mora et al.,
2008). We found that the addition of lime and P to the soil improved the plant antioxidant capacity
by increasing peroxidase (POD) and ascorbate peroxidase (APX) activities and by decreasing the
level of lipid peroxidation at shoot Se concentration up to 0.2 mg Se kg-1. Under different
combinations of soil acidity and P fertilization plant taken up Se differentially, which affected the
antioxidative properties of Se in terms of protection of biological membranes and the activation of
antioxidant enzymes.
Since aluminum (Al) induce oxidative damage in vascular plants, more recently we have studied
the ability of Se to counteract Al-induced oxidative stress in hydroponically cultured ryegrass plants
supplied with selenite (Cartes et al., 2009). Our results showed that Al triggered oxidative stress in
ryegrass roots, but Se applied up to 2 µM diminished the oxidative damage of membranes.
Significant changes in the activities of the antioxidant enzymes superoxide dismutase (SOD), APX
and POD occurred by effect of selenite application. Our results provided evidence that at low
concentration Se alleviated Al-stress in the roots of ryegrass plants by means of two mechanisms: (i)
the enhancement of the spontaneous dismutation of superoxide radicals and (ii) the subsequent
activation of the POD antioxidant enzyme.
Therefore, from the results of our studies, it can be concluded that the benefits of Se for plant
nutrition appear to be related not only with the Se chemical form and Se supply level, but also with
the availability of essential and toxic elements in the soil-plant system.
Acknowledgements:
These works were supported by the FONDECYT Projects 11020934, 1061262 and 11080215
and by the Bicentenary Project PSD 26 CONICYT-UFRO.
120-B
References:
Cartes, P., Gianfreda, L. and Mora, M.L. 2005. Uptake of selenium and its antioxidant activity in
ryegrass when applied as selenate and selenite forms. Plant Soil. 276, 359–367.
Cartes P., Shene C. and Mora M.L. 2006. Selenium distribution in ryegrass and its antioxidant
role as affected by sulfur fertilization. Plant Soil. 285, 187–195.
Cartes, P., Jara, A., Pinilla, L., Rosas, A. and Mora, M.L. 2009. Selenium improves the
antioxidant ability against aluminium-induced oxidative stress in ryegrass roots. Ann. Appl. Biol. (in
press).
Filek, M., Keskinen, R., Hartikainen, H., Szarejko, I., Janiak, A., Miszalski, Z. and Golda A.
2008. The protective role of selenium in rape seedlings subjected to cadmium stress. J. Plant
Physiol. 165, 833–844.
Hartikainen, H. 2005. Biogeochemistry of selenium and its impact on food chain quality and
human health. J. Trace Elem. Med. Bio. 18, 309–318.
Mora, M.L., Pinilla L., Rosas A. and Cartes, P. 2008. Selenium uptake and its influence on the
antioxidative system of white clover as affected by lime and phosphorus fertilization. Plant Soil.
303, 139–149.
Pedrero, Z.; Madrid, Y., Hartikainen, H. and Cámara, C. 2008. Protective effect of selenium in
broccoli (Brassica oleracea) plants subjected to cadmium exposure. J. Agric. Food Chem. 56, 266–
271.
Wittwer, F., Araneda, P., Ceballos, A., Contreras, P. A., Andaur, M. y Böhmwald, H. 2002.
Actividad glutatión peroxidasa (GSH-Px) en sangre de bovinos a pastoreo de la IX Región, Chile y
su relación con la concentración de selenio en el forraje. Arch. Med. Vet. 34: 49–57.
120-C
EFECT OF CLIMATE VARIABILITY ON CROP SYSTEM

Orrego Raúl1,3 and Ávila Andres2
1
Programa de Doctorado en Ciencias de Recursos Naturales. 2 Departamento de Ingeniería
Matemática. 3Centro Agronómico de Investigación y Desarrollo (CAID). Universidad de la
Frontera, Temuco, Chile. R.Orrego@ufromail.cl
One of the main problems of agriculture is related with meteorological variables. Many
researches have shown examples where climate variables restrict crop production (Meza y Silva,
2009; Hansen et al., 2006). Nowadays, this issue is more important because of climate change
evidence (IPCC, 2007), which has been used by several researchers to evaluate the effect of climate
change on crop systems (Meza y Silva, 2009; Parry et al., 2005: Thomson et al., 2005).
Although climate is an uncontrolled phenomenon, there are management practices for
mitigate their impacts (Meza y Silva, 2009; Scheneider et al., 2000). For implementing and
evaluating them, it is necessary to measure the effect of climate variability on crop systems.
Simulation is the most common approach to estimate the effect of weather condition on agriculture
(Challinor et al, 2009; Meza et al., 2008; Thomson et al., 2005). The goal of this work is to quantify
the impacts of climate variability on crop systems.
We used a barley crop models running on several scenarios of climate condition. The
scenarios considerate were: temperature changes (±1, and ±5 °C), precipitations changes (±10 and
±40 mm month-1), and both change effects (+1°C with -10 mm month-1, -1°C with + 10mm month-
1
, +1°C with +10 mm month-1 and -1°C with -10 mm month-1). One hundred simulations were
performed for each scenario by a stochastic climate generator. Total drought matter productivity
(kg), yield (kg of grain drought matter), size of productive cycle (days), and agronomic efficiency
(Ton N kg-1 N) were evaluated. For testing the differences, one-way ANOVA (isolated variables)
and two-ways ANOVA (combination) were used. Homogeneous clusters are identified by Tukey's
HSD test (Honestly Significant Difference). All analyses were evaluated with 95% of significant
level.
Results shown that crops are more sensitive to temperature than precipitation. The effect of
an increase in one degree (C°) are: 10.37 % yield decrease, 5.43% cycle size increase, and no
significant effect in the agronomic efficiency and total dry matter .The effects of a decrease in one
degree (°C) are: 7.79% yield increase, 5.73% cycle size decrease, 6.59% agronomy efficiency
decrease , and no significant effect in dry matter. Precipitation effect is not significant if it is
considered 10 mm monh-1 variations, but it is significant when 40 mm variation is considered. There
are not effects of interaction when 1°C and 10 mm are considered together.
Because the high impact of small changes in precipitation and temperature, we concluded
that climate is a significant variable on crop system.
121
MANGANESE TOXICITY IN BLUEBERRY (Vaccinium corymbosum L.) CULTIVARS : ITS

EFFECTS ON SOME PHOTOCHEMICAL AND BIOCHEMICAL PARAMETERS OF
LEAVES.
Millaleo R 1*, Reyes-Díaz M2, Mora ML2, Alberdi M. 2
1
Doctorate in Science of Natural Resources, La Frontera University. 2 Departament of Chemical
Science, Center for Plant, Soil Interaction and Natural Resource Biotechnology, Scientific and
Technological Bioresources Nucleus (BIOREN-UFRO). *email: rmillaleo@ufro.cl
Manganese (Mn) is micronutrient essential for the plant metabolism, being available for plants
as Mn2+. However, an excess of this element is present in acid soils, which can be toxic for plants as
occurs in volcanic acid soils (Andisols and Ultisols) from Southern of Chile. In these soil types,
highbush blueberry (Vaccinium corymbosum L.) is a very important crop, because of economical
and nutritional significance of its fruits, due to its high antioxidant contents. Due the high acidity of
Andisols (pH between 4,5 to 5,5) it is expected that a greater amount of Mn can be available causing
phytotoxicity, which may affect negatively the productivity and metabolism in this crop. Mn
phytotoxicity as well phytotoxicity of aluminium (Al3+) increased the normal amounts of reactive
oxygen species (ROS), inducing oxidative damage. This feature may results in morphological,
physiological and biochemical alterations in plant species. The objective of this research is to study
the effect of Mn excess on some photochemical parameters of photosystem II (PSII) (maximum
photochemical efficiency, Fv/Fm and effective photochemical efficiency, ФPSII) and biochemical
parameters (antioxidant capacity, AA) in blueberry cultivars.
One year old plants of three cultivars of blueberry (Brigitta, Legacy y Bluegold) were
hydroponically cultivated in Hoagland solution (pH 4.8), under continuous aeration and greenhouse
conditions. Five increasing concentrations of Mn (MnCl2): 10, 50, 250, 500 y 1000 µM plus a
control (2 µM) were applied. Environmental conditions of greenhouse were: 25/20 ºC (day/night),
photoperiod 16/8 h (light/dark), with 70% of air relative humidity and a mean photosynthetic foton
flux of 300 µmol m–2 s–1. The experiment was run twice for different times (15 and 30 days). After
each time photochemical parameters (throughout fluorescence emission using a FMS II modulated
fluorimeter, Hansatech, UK) and antioxidant capacity measurements were determined.
Additionally, the Mn contents of leaves were evaluated. With the obtained results we have
evaluated the Mn tolerance of the investigated cultivars.
The maximal photochemical efficiency (Fv/Fm) of cultivars showed slight variations under Mn
treatments with respect to the control, with exception of Brigitta where a decrease of this parameter
was shown at each time. Major changes were found in the ФPSII, which was generally higher in
Legacy than in the other cultivars. At the end of experiment Bluegold presented the greater decrease
(> 50%) under the highest Mn treatment, whereas in Legacy and Brigitta the decrease was lower.
Mn contents of control leaves fluctuated between 300 to 400 mg kg-1 augmenting to 645-866 mg kg-
1
at the highest Mn treatment. The AA of Bluegold practically did not varied throught the treatment.
In Legacy the major variation were found at 15 days at 10 and 50 µ M Mn, whereas in Brigitta that
ocurrs at 10 and 25 µ M Mn.
It is concluded that the most Mn sensitive cultivars was Bluegold and the most Mn tolerant were
Legacy and Brigitta, where Legacy accumulate higher Mn contents at the higher Mn treatments.
Acknowlegdments: Fondecyt Project1080372.
122
POTENTIAL CARBON MINERALIZATION IN VOLCANIC SUBSOIL OF A PRISTINE

TEMPERATE RAIN FOREST OF SOUTHERN CHILE
Rodrigo Neculman a, Marcelo Panichini a, Francisco Matus a, b and Roberto Godoy c
a
Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de la Frontera, Temuco-
Chile, b Departamento de Ciencias Químicas, Universidad de La Frontera, Temuco-Chile c Instituto
de Botánica, Universidad Austral de Chile, Valdivia-Chile. Correspondence: R. Neculman. E-mail:
rneculman@ufro.cl
Carbon (C) in the subsoil has gained importance due to its high storage capacity and means
residence time of hundreds to thousands of years. However, studies of stabilization of C in the
subsoil are still scarce. The process leading to the reduction in the potential loss of soil organic
matter (SOM) is defined as C stabilization and depends primarily on the organic input rate and
chemical composition of the substrate and external factors such as soil texture, microbial activity
and soil pH. The precipitation and temperature are two key environmental conditions that also affect
the stability of soil C. Studies of the subsoil indicate that the stabilization of SOM is mainly due to
its chemical adsorption with aluminum (Al) and Iron (Fe) oxides. Chilean allophanic soils are
characterized by its high content of SOM, low pH and presence of amorphous clay minerals
(allophane). The temperate rainforest of southern Chile have been developed in these soils under a
temperate climate, with low temperatures (4.5°C) and high precipitation (5,000-7,000 mm), where
the woody vegetation produced a litter with a slow decomposition rates resulting in an accumulation
of SOM in the surface soil. Therefore the study of C at depth soil is essential for understanding the
dynamics of C. The aim of this study was to compare the C respiration in the surface and depth in a
pristine temperate rainforest of one Andisol under optimal laboratory conditions. These soils are
well developed (Silty loam in surface and Sandy loam in depth) and are located in San Pablo de
Tregua, Panguipulli (39 ° 38 'S, 72 ° 05' W). Soil samples were collected at different depths (0-20
cm and >60 cm) and incubated at 50% of water holding capacity for 60 days at 20 ± 2°C. The rate of
C mineralization was assessed at intervals of 5, 10, 20 and 60 days. The results showed an increase
in cumulative C mineralization at the surface with respect to the mineralization in depth soil. The
rate of C mineralization showed significant differences in surface and depth for each time interval
evaluated. The interpretation of the results suggests a lower potential for C mineralization in the
subsoil compared to the surface where the texture of the soil profiles significantly affected, resulting
in a less efficient use of C by microbial communities in depth. Therefore, we assume that the
reduced C respiration in the subsoil generates a greater stability of native SOM in these forests.
Key Words: SOM stabilization, C-respiration, Allophanic soils, Rate of C mineralization
123
EFECTO DE TOXICIDAD POR MANGANESO Y RADIACIÓN UV-B SOBRE LA

EFICIENCIA FOTOQUÍMICA DEL FOTOSISTEMA II (PSII) DEL ARÁNDANO ALTO
(VACCINIUM CORYMBOSUM L.)
Rojas-Lillo Y1, Reyes-Díaz M2, Alberdi M3 y Mora ML3
.
1
Programa de Magíster en Ciencias de Recursos Naturales, Universidad de La Frontera, e-mail:
yrojaslillo@gmail.com. 2Instituto de Agroindustria, Universidad de La Frontera, Casilla 54-D, e-
mail: reyesm@ufro.cl. 3Departamento de Ciencias Químicas, Universidad de La Frontera, Casilla
54-D, Temuco, Chile, e-mails: malberdi@ufro.cl, mariluz@ufro.cl.
La toxicidad por Manganeso (Mn+2) puede tener consecuencias en el desarrollo vegetal,

limitando el crecimiento y rendimiento de cultivos, principalmente, en suelos ácidos. En el sur de
Chile, suelos derivados de cenizas volcánicas (Andisoles), con un pH ácido son frecuentes.
Asimismo, factores ambientales adversos como la radiación UV-B, pueden potenciar en forma
sinérgica el estrés que produce la toxicidad por Mn. En esta región el cultivo de arándano
(Vaccinium corymbosum L.) ha cobrado gran importancia. Por lo tanto, el objetivo de este trabajo
fue evaluar la respuesta fotoquímica de esta especie en base a parámetros de fluorescencia
(eficiencia fotoquímica máxima (Fv/Fm), eficiencia fotoquímica efectiva (ФPSII), tasa de transporte
de electrones (ETR) y apagamiento no fotoquímico (NPQ)), frente a la acción de niveles tóxicos de
Mn y radiación UV-B. Se consideraron 2 cultivares con tolerancia diferencial al Mn, Brigitta
(tolerante) y Bluegold (sensible), los cuales fueron instalados en cultivo hidropónico con niveles de
Mn tóxicos (500 µM) + radiación UV-B (4 W-2S-1) (CR) y tratamientos con Mn sin RUV-B (SR),
simulándose un ciclo diario de radiación UV-B típico de verano de la Provincia de Cautín (39º LS)
durante 30 días. Ambos tratamientos tuvieron sus respectivos controles. Se utilizó un ANOVA de 2
vías y un test a posteriori de Tukey para el análisis estadístico.
Los resultados muestran que existen diferencias significativas entre los 7 y 30 días de
tratamiento en los parámetros fotoquímicos (P< 0.01) en ambos cultivares. Aunque Fv/Fm no
mostró diferencias entre cultivares, si se observaron entre los tratamientos CR y SR en Bluegold y
Brigitta. ETR y ΦPSII no variaron entre cultivares, ni entre tratamientos, pero si durante los días de
tratamiento en ambos parámetros. NPQ, que es un indicador de la disipación de energía no
fotoquímica en forma de calor, mostró variaciones significativas entre Bluegold y Brigitta,
presentando Bluegold valores más bajos que Brigitta. Además, en este mismo parámetro se
registraron diferencias significativas entre los tratamientos CR y SR.
Se concluye que la RUV-B junto con las concentraciones toxicas de Mn ejercerían un efecto
sinérgico de estrés en ambos cultivares, considerándose, además, a la RUV-B como el principal
factor de estrés que determina las diferencias entre tratamientos durante el tiempo de exposición.
Agradecimientos: Fondecyt 1080372, Bicentenario PSD-26.
124
EFFECTS OF DIFFERENT AGRICULTURAL MANAGEMENT ON SOIL QUALITY IN

AN IMPORTANT AGRICULTURAL AREA OF SOUTHERN ITALY
Scotti R.a, Sultana S.b, Scelza R.a, Marzaioli R.b, D’Ascoli R.b, Rao M.A.a
a
Federico II, via Università 100, 80055 Portici, Italy. E-mail: riccardo.scotti@unina.it
b
Department of Environmental Sciences, Second University of Naples, via Vivaldi 43, 81100
Caserta, Italy
Intensive agricultural management can have negative effects on soil quality, as progressive
reduction of organic matter (SOM), alterations in physical and chemical properties, and changes in
edaphic microflora. In this context, demand of suitable indicators able to assess the impact of
different agricultural managements on soil quality has increased. It has shown that soil characters,
based on biological and biochemical properties, are able to respond to small changes in soil
conditions, thus providing information on subtle alterations in soil quality.
Aim of this study was to evaluate changes in soil quality, by using biological and biochemical
parameters as fertility indicators, in an agricultural soil under different agricultural management,
sited in Campania Region (Italy). The work was scheduled in a preliminary monitoring phase on soil
fertility in 5 areas with different geo-pedological characteristics. We selected in each area 4 farms
(AZ), characterized by intensive agriculture and greenhouses, and 1 control farm (C), in which the
soil were sampled in orchards or uncultivated fields.. After this phase, among all previously
characterized soils, we selected soils from two farms to study the effect on soil quality of different
organic amendments. In particular, a compost from municipal solid waste and wood (scraps of
poplars pruning) were used in different doses and ratios. Soil samplings were carried out after one
month from the addition of organic matter and then after 4 months. Samplings is continuing every 4
months until a year.
The results of the monitoring study showed a great variability among the farms. A clear
evidence was that the control samples, not subjected to intensive farming, had always better values
of chemical properties and higher enzymatic activities and microbial biomass to indicate a negative
effect on soil fertility of using no organic amendment in intensive agriculture practices. In the
second phase, the use of different organic amendments showed a positive effect on soil fertility
under intensive farming. In general, all enzymatic activities and organic carbon content increased
after 1 month, and after 4 months from amendments application were still higher. Similarly, but with
some exceptions, microbial biomass and soil potential activity showed significantly higher values in
soils added with organic amendments, for both farms and samplings.
The project was founded by CCIIAA of Salerno
125
CHARACTERIZATION OF GARDERMOEN AIRPORT SOIL CONTAMINATED BY DE-

ICING COMPOUNDS AND POSSIBLE BIOREMEDIATION STRATEGY
Scotti R.a*, Rao M.A.a, Colarieti M.L.c, Toscano G.c, Cavalca L.b, Andreoni V.b, Gianfreda L.a,
Greco G. Jr.c
a
Federico II, via Università 100, 80055 Portici, Italy.
b
DiSTAM-MAAE Università degli Studi di Milano, via Celoria 2, 20133 Milano, Italy
c
DIC, Università di Napoli Federico II, Piazzale Tecchio 80, 80125 Napoli, Italy. *corresponding
author: e-mail: riccardo.scotti@unina.it
The new main airport of Norway, Gardermoen, Oslo was inaugurated in 1998. The area is a
glacial contact formation with sand and gravel dominating near the ground surface. Large amounts
of de-icing chemicals are commonly used for the removal of snow and ice from airplanes
(propyleneglycole) and runways (potassium formate). These contaminants are easily degradable by
biotic or abiotic factors, but they may still threaten groundwater due to the overloading of the
system. The present study is being performed in the framework of the European research project:
“SoilCAM - Soil Contamination: Advanced integrated characterisation and time-lapse Monitoring”.
Its overall objective is to determine necessary parameters for designing an optimal combination of
invasive and noninvasive methods for monitoring degradable contaminants in spatially variable soil
systems. Our research group is involved in soil physical, chemical and biochemical characterization
of soil profiles sampled in Gardermoen airport, in monitoring their contaminant contents, and in
applying bioremediation strategy to degrade contaminants. During the first part of study, soil
sampling was carried out at the airport near the runways. Samples of soil profiles (about one meter
deep) were collected and stored in aerobic and anaerobic conditions. In the second part of the study,
collected soils were characterized and the fate of de-icing compounds in the polluted soils was
evaluated. Physical and chemical properties, such as texture, pH, organic and total carbon, Fe, Mn
and pollutant levels, main biochemical and microbiological properties were determined.
The results showed very leached soils, generally with low biological and biochemical activities,
that do not hinder the quick transfer of pollutants, to the groundwater. Bacterial cultures, mainly
composed by different species of Pseudomonas, were isolated from soil collected at Gardermoen
Airport and some of them carried genes for putative propylene/diol dehydratase (pdu) genes. They
were capable of degrading propylene glycol and potassium formate and were used in lab-scale
degradation test.
126
RESPUESTA FOTOPROTECTORA DEL APARATO FOTOSINTÉTICO DE ALGAS

PARDAS FRENTE AL INCREMENTO DE LA RADIACIÓN UV Y TEMPERATURA.
Edgardo Cruces1,2, Iván Gomez2
1
Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera, e-mail:
ecruces@ufro.cl. 2Instituto de Biología Marina, UACh, e-mail: igomezo@uach.cl
Las macroalgas pardas son un importante componente dentro de los ecosistemas costeros,
constituyendo un importante refugio, hábitat, alimento y sustrato para animales móviles y sésiles.
Siendo, una importante fuente de productos naturales para la industria de la acuicultura y
bioquímicas. En general, las algas, han desarrollado diferentes estrategias fisiológicas para
fotoprotegerse frente a los distintos estreses a los que se encuentran sometidos (desecación, alta
irradiancia, radiación UV, temperaturas, etc.), observándose una relación especie-específicos en la
capacidad de recuperación de los daños inducidos. Por lo tanto, el incremento de radiación UV
(RUV), junto con el aumento de las temperaturas producto del cambio climático pondrán a prueba
las distintas estrategias fotoprotectoras de las algas pardas. El objetivo de de este trabajo fue evaluar
la respuesta fotoquímica de tres especie de algas pardas en base a la eficiencia fotoquímica máxima
(Fv/Fm), y composición de pigmentos frente a la acción de RUV y temperatura. Para ello, se
determinó la temperatura umbral de las algas (Lessonia nigrescens, Durvillaea antarctica,
Macocystis pyrifera) basado en la medición de Fv/Fm, indicador de la eficiencia cuántica,
considerando 20°, 28°, 30 ° y 35 ° C. Además, se utilizaron tres temperaturas en combinación con
RUV (10 ° C, 20 ° C y 28 ° C). Con un tiempo de exposición de 2-3 horas y recuperación de 4
horas. El análisis de clorofilas (a y c) y carotenoides totales se hizo extrayendo mediante N, N-
dimetilformamida. Los datos fueron comparados usando análisis de varianza (ANOVA) de dos vías,
seguidos de un test a posteriori de Tukey cuando se detectaron diferencias.
En general, D. antarctida es la que presentó la mejor aclimatación a diferentes rangos de
temperatura, seguida por M. pyrifera. Las tres especies de macroalgas mostraron a los 28° C un
Fv/Fm de entre 75 a 88% del valor inicial. A 30°C la recuperación en el Fv/Fm en las tres algas
alcanzo entre un 40 y un 70% de recuperación y a los 35°C ninguna alga se recuperó. La medición
del rendimiento cuántico óptimo de fluorescencia mostró una disminución de este parámetro en
relación con el aumento de temperatura y radiación UV. A los 28°C se observó una disminución en
la recuperación, especialmente en L. nigrescens, alcanzando solamente el 56% del valor original, a
diferencia de D. antarctica que se recuperó casi el 90% del valor inicial. A los 20°C la recuperación
alcanzó solo entre un 76 y 102%. Los contenidos de pigmentos mostraron a M. pyrifera con la
mayores concentraciones (mg g–1 por peso seco) de cl a, c y carotenoides totales. Los valores de los
pigmentos, en general, aumentan con el aumento de la temperatura. Por el contrario, la acción de la
radiación ultravioleta provoco reducción de los pigmentos fotosintéticos y accesorios. Finalmente,
como conclusión se puede señalar que D. antarctica es la especie que mostró la mejor aclimatación
a las altas temperaturas y RUV, lo que se expresa en mayores valores de Fv/Fm porcentuales, tanto
durante el tratamiento, como después de la recuperación. Y aunque la luz y la temperatura son dos
factores de estrés que co-actuan en la naturaleza, el aumento de la temperatura contribuiría a mitigar
los efectos de la radiación UV.
Agradecimientos: Fondecyt 1090494.
127
128
Simposio 5.
Biotecnología Ambiental y Manejo de Contaminantes
Moderadores Dra. María Cristina Diez; Dr. Cristian Bornhardt
1. Dra. María Cristina Diez, Universidad de La Frontera, Chile:“Hongos de

pudrición blanca en la biorremediación de ambientes contaminados con
compuestos orgánicos recalcitrantes”.
2. Dr. Cristian Bornahrdt, Universidad de La Frontera, Chile:”Eliminación de

nitrógeno en aguas residuales de alta carga”.
3. Dra. Graciela Palma, Universidad de La Frontera, Chile: “Movilidad y

transformación de pesticidas en suelo: Implicancias agronómicas y
ambientales”.
4. Dr. César Arriagada, Universidad de La Frontera, Chile: “Contribuciòn de

hongos ligninoliticos al crecimiento de especies de interés
silvoagropecuario”.
5. Mg. Felipe Gallardo, Universidad de La Frontera, Chile: “Uso de residuos

industriales provenientes de plantas de celulosa en la agricultura”.
6. Dra. Liliana Gianfreda, Università Federico II, Italy: “El rol de las enzimas en la
remediación de la contaminación ambiental”.
129
130
WHITE-ROT FUNGI FOR THE BIOREMEDIATION OF CONTAMINETD

ENVIRONMENTS WITH RECALCITRANT ORGANIC COMPOUNDS
M. C. Diez
Environmental Biotechnology Center, Scientifical and Technological Bioresource Nucleus.

Department of Chemical Engineering, Universidad de La Frontera, PO Box 54-D, Temuco, Chile.
mcdiez@ufro.cl.
Biological decomposition of organic compounds is the most important and effective way to remove
these compounds from the environment. The microorganisms have the ability to interact, both
chemically and physically, with substances, leading to structural change or the complete degradation
of the target molecule. Fungi generally biotransform pesticides and other xenobiotic by introducing
minor structural changes to the pesticide, rendering it nontoxic; the biotransformed pesticide is
released into the soil, where it is susceptible to further metabolism by bacteria. Among white-rot
fungi, P. chrysosporium and Trametes versicolor have become the most commonly used model
organisms in lignin biodegradation studies due to their good ligninolytic properties, fast growth, and
easy handling in culture. The peroxidase enzyme systems employed are non-specific, and have been
implicated in the degradation by white-rot fungi of a variety of contrasting aromatic xenobióticos,
including pesticides, although the mechanisms involved are not clearly related to ligninolytic
potential. As a way of not altering natural ecosystems, the trend in the last years has been the search
and utilization of native fungi with ligninolytic potential as well as the evaluation of technologies
that allow to optimize the conditions of degradation of organic recalcitrant compounds. In this way,
the white-rot fungus Anthracophyllum discolor, a Chilean native fungus presents a great ligninolytic
activity mainly of manganese peroxidase (MnP) enzymatic activity and therefore potential for
xenobiotic degradation.
In our research group, the main activities have been focused on the search of native fungi in
forests of the south of Chile with high ligninolytic capacity, the optimization of the growth
conditions and enzymes production, the characterization of the MnP enzyme, the evaluation of
diverse supports (lignocellulosic wastes, nanoclays) for the immobilization both the fungi and the
MnP, for the degradation of organic compounds such as chlorophenols, polycyclical aromatic
hydrocarbons (PAHs) and some pesticides. The degradation of contaminants by A. discolor using
different technologies, has been studied, as well as, the comparison with other white-rot fungi
(Phanerochaete chrysosporium and Bjerkandera adusta). The degradation of lignin by A. discolor
using airlift reactor, the degradation of chlorophenols using columns packed with allophanic soil
inoculated with the fungus, and fixed-bed reactor (biobeds) for pesticides degradation has been
studied also.
Of the mentioned studies, the principal results show that some of native white-rot fungi of
forests of the south of Chile, they present high ligninolytic potential and therefore aptitude to be
evaluated for the bioremediation of contaminated environments. The selected fungi some of them
showed at least two of the three ligninolytic enzymes (LiP, MnP and laccase) and degradation
capacity for indicators dyes.
The initial screening of the white-rot fungi showed that eleven selected strains presented high
concentrations of lignin peroxidase (Lip) and manganese peroxidase (MnP). Anthracophyllum
discolor (Sp4), produced high LiP and MnP activity compared to the control fungus Phanerochaete
chrysosporium CECT-2798. Tolerance test showed that native Chilean fungi did not present high
131
tolerance to 2,4,6-TCF and PCP but were quite tolerant to 25 and 50 mg L-1 of 2,4-DCF. However,
preacclimatization in 2,4-DCP notably improved the growth in medium with 2,4,6-TCP and PCP.
PCP in liquid medium was efficiently degraded by the fungi Anthracophyllum discolor, Lenzites
betulina (Ru-30) and Galerina patagónica (Sp3). Of the selected fungi, Anthracopyllum discolor
presented major activity and enzymatical diversity by which it was selected to realize the later
studies.
The major growth of A. discolor and enzymatical production was in medium containing glucose,
low concentration of nitrogen, in static conditions and immobilized on wheat grains. The best
conditions for ligninolytic activity were pH 5.5, temperature of 26ºC, and C/N ratio of 250. The
MnP enzyme from A. discolor was partially purified and characterized showed thermal stability
between 40 and 50 ºC, the Km values for 2,6-DMP and H2O2 were 24.83 µM y 36.98 µ M,
respectively.
The degradation of pentachlorophenol (PCP) in soil slurry cultures by Bjerkandera adusta and
Anthracophyllum discolor was evaluated. In this case, the high degradation rates obtained during the
fungal cultures are due to the agitation imposed to maintain the slurry phase, which, in turn,
increased the bioavailability of the pollutant to the fungi. From the two fungal strains evaluated, A.
discolor presented the best results of ligninolytic activity and PCP degradation (> 90%). Similar
result is observed in the degradation of lignin in reactor airlift using pelletized A. discolor as
inoculum.
The degradation of chlorophenols (2,4-DCF, 2,4,6-TCF and PCF) in columns packed with
allophanic soil inoculated with the A. discolor immobilized in wheat grains, in continuous process
was evaluated. The chlorophenols were added alone and in mixtures concentrations between 100
and 300 mg/L. The inoculated columns increased significantly the useful life and the efficiency of
degradation of the chlorophenols.
In fixed-bed reactor contaminated with PCF (100 to 300 mg/L) and inoculated with A. discolor
immobilized on wheat grains, the major removal of the chlorophenol was observed in comparison
with the not inoculated beds.
In conclusion, the white-rot fungus A. discolor has high degradative and ligninolytic capacity to
eliminate organic recalcitrant compounds as chlorophenols, PAHs and other organic recalcitrant
molecules, for which its utilization in biorremediación of contaminated environments is promissory.
In addition, bioestimulation (nutrient addition) and bioaugmentation (fungi and actinomycetes)
investigations are in development related to the degradation of pesticides in bixed-beds bioreactor.
Acknowledgements: Financed by FONDECYT, 1050614, 1090678, 3090049, 3080013,
3100118 projects.
132
NITROGEN REMOVAL IN HIGH LOADED INDUSTRIAL WASTEWATERS

Dr.-Ing. C. Bornhardt, Dr.-Ing. C. Antileo
Chemical Engineering Department, Center of Environmental Biotechnology

Scientific and Technological Bioresource Nucleus BIOREN
1. Introduction
A wide scale of industrial processes generates liquid effluents with high nitrogen concentrations
(> 200 mg N-NH4/L), which must be treated using non conventional methods, such as advanced
biological treatment for nitrogen removal. These methods are based on the utilization of intensive
technology, mainly immobilized biomass, in order to transform the ammonia to molecular nitrogen,
through an integrated nitrogen removal process: nitrification and denitrification.
The biological nitrification-denitrification process makes it possible to remove high nitrogen
loads from industrial effluents generated by e. g. the fishing, chemical and food processing
industries. During the nitrification process, ammonia-oxidizing bacteria (AOB) oxidate ammonium
(NH4+) to nitrite (NO2-), and, subsequently, nitrite-oxidizing bacteria (NOB) oxidate nitrite to nitrate
(NO3-). Then, the nitrate is sequentially reduced by heterotrophic denitrifying bacteria (Fig. 1, step
1) to molecular nitrogen (N2) as the final product. Since the nitrite is consumed during nitrification
and built up again during denitrification, the oxidation of nitrite (nitratation) becomes an
unnecessary reaction step, therefore it is feasible to denitrify the nitrite directly to molecular
nitrogen, as shown in Fig 1, step 2. Partial nitrification to nitrite (step 2) has three practical
advantages: 25% lower oxygen consumption, 40-60% lower need of organics for denitrification, and
lower sludge production.
This project proposes the optimization of the efficiency of a nitrogen removal treatment through
Nitrification-denitrification via nitrite (Step 2) and/or Simultaneous nitrification-denitrification
process (SND).
Nitrification O2
Organic-C
Step 2 Denitrification
3
NH + O → NO + 2 H + H O
+ NS − +
4 2 2 2
2
1 Step 1
NO 2 + O 2  → NO 3 NO → NO → NO → N O → N
− NB − − −
3 2 2 2
2
Figure 1: Nitrification – denitrification process. Step 1: conventional nitrogen removal; Step 2:
nitrogen removal through partial nitrification via nitrite
Previous studies have shown that it is possible to achieve partial nitrification to nitrite by
manipulating certain operating conditions in the reactor. Using a process control approach, this
133
study proposes the development of a supervisory control system that generates dynamic pH and DO
set points in order to establish environmental conditions favorable to AOB growth and unfavorable
to NOB growth over the long term.
The SND process is characterized by the fact that the nitrification and denitrification can occur
simultaneously in aerobic conditions at limiting dissolved oxygen concentrations using only one
single reactor. Taking into account that the oxygen penetrates the biofilm only the first 50 to 100
µ m, it is of great interest to explore the use of mixed autotrophic / heterotrophic biofilms, that offer
the possibility of a common space for the partial nitrification up to nitrite and the denitritation, in the
superficial and deeper zones of the biofilm, respectively.
The present project will contribute to the study of the integrated nitrogen removal process by
SND via partial nitrification up to nitrite, and to the search of optimal pH, dissolved oxygen,
ammonia nitrogen and organic matter conditions, to treat effluents with high nitrogenous load and
low organic carbon concentration in reactors operated in SBR mode, called Sequencing Batch
Biofilm Reactors (SBBRs). The objectives of this project are: to find optimal conditions for partial
nitrification, to study the SBR operation that favors the SND process via nitrite, to propose real time
monitoring strategies to increase the global nitrogen removal efficiency, and to model the SND
process via nitrite.
2. Materials and Methods

As basic infrastructure to sustain this proposal, the working group owns 2 RDBRs and 1 SBR
(suspended biomass) instrumented with sensors to transmit temperature, pH, and dissolved oxygen
on-line to a PID control system commanded by a PLC (Programmable Logic Controller) (see Fig.
2).
Figure 2. Lab-scale reactors: Activated sludge and Rotating Disk Biofilm Reactors.
The reactor with its instrumentation and control system is schematized in Figure 3. It consisted
in a single rotating disk (diameter = 35.4 cm) which was totally submerged in a 8 L reaction volume.
The disk was moved by an external motor at 3 r.p.m. Temperature was controlled at 20 ºC using a
134
thermostatic bath (Julabo, Model EC, Germany). The monitoring and control system consisted of
oxygen and pH/temperature electrodes (WTW Oxi 701, Germany, and HACH EC 310, USA).
The 0.5 M sodium carbonate solution used for H+ neutralization during nitrification, was dosed
by a membrane pump (LANG, type ELADOS EMP II, 41 L/h, Germany), and the aeration was
carried out by using pulse width modulation (PWM) for electromagnetic valve opening (Festo 457,
MSG-24DC, Germany). The air was supplied by an aeration pump (COSMOS double type 1000,
China). The data for pH, temperature, DO concentration, and the pumped carbonate solution
volume, were acquired, processed and stored every five minutes by means of a PLC (Siemens,
Simatic S7-200) connected to an IBM compatible PC, as can be seen in Figure 3.
Figure 3. Instrumentation and control for a RDBR
The aeration (length of the aerobic phase) was interrupted by the detection of bending points on
the carbonate consumption and air opening valve curves. The length of the anoxic phase (end of the
cycle) was determined by detection of bending points on the pH and/or the ORP curves.
3. Results
3.1 Nitrification-denitrification via nitrite
Figure 4 summarizes 271 days of operation for a continuous RDR, showing the influence of DO
concentration on TAN (Total Ammoniacal Nitrogen) oxidation rate and nitrite accumulation. Figure
4A shows a linear dependence of the TAN oxidation rate up to 2 mg O2/L at pH 7.5, as well as at
8.5. Therefore, the oxygen transport into the biofilm was also the limiting step for continuous
operations. As shown in Figure 4B, the nitrite accumulation was not stable and depended strongly
on DO concentration, achieving values from 60 to 95%. Additionally, the separation of curves in
Figures 4 shows a clear effect of pH, which reveals a significant influence of micro-kinetics on the
partial nitrification even at DO concentrations as low as 0.6 mg O2/L.
Fig.5 illustrates the textile material used as support for immobilization and mainly short and fat
rods absorbed in the expolysaccharide biofilm. Molecular analysis revealed the presence of
Nitrosomonas sp and Nitrobacter sp.
135
14 A Y = 11.915x + 59.29
100 B 2
Y = 5.5431X -0.2481 r = 0.9959
12 2
r =0.9867
80
TAN oxidation rate [mg N/Lh]
10 Figure 4. Partial
nitrite accumulation [%]

nitrification in continuous
8 60
operation (271d): A) TAN
Y = 6.3293x + 78.535
6
2
r = 0.9707 oxidation rate. B) nitrite
40 accumulation percentages.
4 Y = 2.9654 X -0.4285
2
r =0.999 20
2 pH 8.5
pH 7.5
0 0
0 2 4 6 0 2 4 6
DO concentration [mg O 2/L]
Figure 5. SEM of nitrifying

biofilm (A) Polyethylenstyrol as
support (50x) (B) Completely
immobilized within the matrix of
exopolysaccharide (2500x).
3.2 Simultaneous nitrification-denitrification via nitrite
Aerobic phase Anoxic phase

350
Automatic addition of Figure 6 shows the simultaneous oxidation of
external carbon source
300 acetate and ammonia nitrogen during a typical
SND cycle. For the whole cycles the total nitrogen
Concentration [mg/L]
250 removal was 94% ± 3% with 99% ± 1% of

ammonia oxidation during the aerobic phase,
200
nitrite accumulation of 80% ± 19% and the
nitrogen loss due to SND in the aerobic phase
150 Acetate
NO2--N
was 27% as mean value (60% as maximal).
100 NO3--N
TAN Figure 6. Nitrification-denitrification
Total N
50 via nitrite during a typical SBR
operation. Cycle 166 at DO=1.5 mgO2/L
0 and C-Acetate/N= 0.8.
0 2 4 6 8 10 12
time[h]
136
Figure 7 shows typical sensor profiles observed systematically in all cycles. An inflection point
appeared at 7.8 h on the carbonate profile before the plateau, indicating that no more alkalinity is required
to neutralize the protons released during nitrification. This bending point replaces the “ammonia valley”
usually observed in the pH profile. The aerobic phase was just interrupted and shifted to anoxic phase
after detection of the bending point in the % AVO profile (Fig. 7).
Aerobic phase Anoxic phase

9,0 350 80 -100 9,0 350
-2 pH
CO3 -C ORP
8,8 300 8,8 300
Consumption of carbonate [mg ]
% AVO CH3COOH-C
Consumption of acetate [mg ]

AVO, Air valve opening [%]
pH
8,6 60 -150
250 8,6 250
8,4 Nitratate apex
ORP [mv]
200 200
Double "bending 8,4
pH
-200
pH
8,2 point" just after 40

of ammonia depletion 150
150
8,0 8,2
100 100
7,8 20 -250 8,0 Nitratate knee
50 50
7,6
7,8
7,4 0 0 -300 0
4 5 6 7 8 9,0 9,5 10,0 10,5 11,0 11,5
time [h] time [h]
+
Figure 7. Cycle 166 at initial NH4 -N =280 mg/L, pH=7.5, and 23°C.
This second bending point reflects a decrease in the oxygen requirement due to ammonia depletion.
During the anoxic phase the characteristic bending points “nitrate apex and knee” can be seen at 10.8 h in
Fig. 7, indicating the end of denitrification. Afterwards, the cycle was immediately finished stopping the
dosage of external carbon.
Acknowledgements: Financial support by research grants FONDECYT 1030317 and 1070574.
4. Related papers
• Muñoz C., Rojas D., Candia O., Bornhardt C., Antileo C. (2009). Supervisory control system to
optimize partial nitrification to nitrite in an activated sludge reactor with an on-line estimate of
efficiency indices. Chemical Engineering Journal. 145 (3), 453-460
• Ciudad G., González R., Bornhardt C., Antileo C. (2007). Modes of operation and pH control
as enhancement factors for partial nitrification with oxygen transport limitation. Water Research:
20 (41) 4621-9.
• Antileo C., Roeckel M., Lindemann J., Wiesmann U. (2007). Operating parameters for high
nitrite accumulation during nitrification in a rotating biological nitrifying contactor. Water
Environment Research, 79 (9):1006-1014.
• Ciudad G., Werner A., Bornhardt C., Muñoz C., Antileo C. (2006). Differential kinetics of
ammonia- and nitrite-oxidizing bacteria: A simple kinetic study based on oxygen affinity and
proton release during nitrification. Process Biochemistry, 41(8). 1764-1772.
• Antileo C., Werner A., Ciudad G., Muñoz C., Bornhardt C., Jeison D., Urrutia H. (2006)
.Novel Operational Strategy for Partial Nitrification to Nitrite in a Sequencing Batch Rotating
Disk Reactor. Biochemical Engineering Journal, 32 (2), 69-78.
• Antileo C., Roeckel M., and Wiesmann U. (2003). “High nitrite build-up during nitrification in
a rotating disc reactor, comparison with a CSTR“. Water Environment Research. 75, 151-162.
137
MOBILITY AND TRANSFORMATION OF PESTICIDES IN SOIL: AGRONOMICAL

AND ENVIRONMENTAL IMPLICATIONS
Palma, G., Briceño, G., Aguilera, P. Candia, O. Mora, M.L. Demanet. R.
Center of Plant, Soil Interaction and Natural Resources Biotechnology. Scientific and Technological
Bioresource Nucleus. Universidad de La Frontera. Casilla 54-D Temuco. gpalma@ufro.cl
Chemical pesticides, the most effective means of pest and weed control, allow the maintenance of
current yields responding to continued increase in food demand. However, concern about the
environmental impact of pesticides and its repeated pesticide use has prompted research into the
environmental fate of these products. Specifically, soil-active and foliar application herbicides, as
well as fulfilling their purpose of weed control, are subjected after application to a series of
processes which determine their behaviour in ecosystems and which may have an impact both on
agricultural activities and on the environment. The evolution of the transfer and transport processes
which control the bio-availability of herbicides and their mobility in the environment, and the
processes of transformation or degradation which involve both chemical and biological degradation,
are influenced by many factors including climate, farming practices, soil type etc., as well as the
physical and chemical properties of the herbicides. Among all the processes which regulate the
persistence of herbicides in the soil, it is the adsorption process which conditions all others and
determines the availability of the herbicide in the soil solution. In view of the above it is
fundamental for us to obtain an understanding of how herbicides interacts with the different soil
components and how its dynamic is affected by the physico-chemical and biological properties of
the soils in the farming systems used in Chile. In addition, another important factor which should be
considered in these studies is its repeated use on the same soil and how this modifies the dynamic of
the herbicide and its effects on soil microorganisms.
Regarding the effect of the herbicides in surface water contamination we have carried out some
studies conducted in our region (Palma et al., 2004) in the Traiguén river basin. It is characterized
by agricultural and forestry land use. The agricultural area is characterized by a wide variety of
crops (wheat, raps, barley, and oats) and the forestry area was characterized by pine and eucalyptus
plantations. Residues of five pesticides in surface water were surveyed during 2001 and 2003.
Simazine, hexazinone, 2,4-D, picloram herbicides and carbendazim fungicide were selected through
a pesticide risk classification index. Six sampling stations along the river were set up based on
agricultural and forestry land use. The water sampling was carried out before and after the pesticide
application periods and in correspondence to some rain events. During sampling campaign, the
highest concentrations of pesticides were 3.0 µgL-1 for simazine and hexazinone, 4.5 µ gL-1 for
carbendazim, 9.7 µ gL-1 for 2,4-D, and 0.30 µgL-1 for picloram In sampling carried out at the
beginning of each year, no pesticides were detected. Data are discussed in function of land use and
application periods of the products, showing clear seasonal pattern pollution in the Traiguén River.
By the other hand we have studied as agronomic practices can modify the behavior of herbicides
associated with the application of amendments of animal origin and its effect on the effectiveness of
herbicides. We specifically addressed our study to maize crop and atrazine and dimethenamide
herbicides and their behavior when animal cow manure is applied as fertilization amendment. In the
south of Chile, there are edaphoclimatics conditions suitable for the production of milk and meat,
with 75% of the cattle located in this region (Salazar et al., 2003). The milk production systems have
been intensified in the last few years, increasing the amount of liquid cow manure (LCM), effluents
that are stored in open air lagoons for their application on grasslands or fodder corn crops. The large
138
volume of LCM accumulated in a reduced storage capacity provokes frequent and higher application
rates to the field per year reaching an amount up to 300,000 L ha_1. The fodder maize (Zea mayz L.)
is cropped on Andisol, characterized with high organic carbon content and acidic pH. The maize
management includes the application of LCM and atrazine as pre-plant herbicide. Specifically, for
the corn crop, the LCM is applied from August to October, before planting the corn, and its end total
amount of application is highly variable. Another associated problem with LCM application is there
is no clear indication on the amount of time needed between the last applications of LCM and
herbicide application. As a consequence, inefficient weed control can usually be observed in the
field.
The first studies were evaluated the influence of LCM application on soil properties, atrazine
adsorption, and the physicochemical controlling mechanisms in an Andisol. The LCM was applied
at rates equivalent to 0, 100,000, 200,000, and 300,000 L ha -1 resulting in treatments S-0, S-100, S-
200, and S-300, respectively. The LCM application increased dissolved organic carbon (DOC) and
pH of the soils immediately on addition, but pH returned to S-0 values 30 d after application. The
LCM application did not modify atrazine adsorption with the two lowest application rates (S-100
and S-200), but atrazine adsorption was decreased in S-300 (Kf = 0.96) compared with the control
(S-0) (Kf = 1.19), possibly due to the competitive adsorption of DOC with the pesticide. The
Fourier-transformed infrared analysis showed that LCM increased aliphaticity and presence of N-
containing groups and polysaccharide-like groups in amended soils; however, these properties did
not modify the atrazine interaction in the studied amended soils. Interestingly the addition of DOC
to soil at the high application rate (S-300) reduced atrazine adsorption in this rich OM Andisol
despite the LCM not raising the concentration of stable organic matter. The application of high rates
of liquid manure containing DOC could incurs an increased risk of pesticide leaching (Briceño et al.,
2008) and we would recommend that farmers exercise caution since applying too high a rate of
LCM could increase atrazine leaching.
To rank herbicides in relation to their leaching potential, the ground-water ubiquity score (GUS),
(Gustafson, 1989) has been used frequently (log t1/2 (4-log Koc), where t1/2 is the herbicide half-life
in soil and Koc is the sorption coeficient normalized to soil organic carbon content. Herbicides with
GUS < 1.8 are ranked as non-leachers, GUS > 2.8 represent leachers and those with GUS between
1.8 and 2.8 are considered transitional. The evaluation of leaching potential of atrazine in amended
soils with different rate of liquid cow manure application using Koc values obtained from adsorption
studies and atrazine dissipation study were calculated. The half-life of atrazine ranged between 15-
17 days and the Koc values obtained previously ranged 9-11. Gus index of atrazine in S-0, S-100, S-
200 and S-300 was 3.5 3.7, 3.7 and 3.9, respectively, suggesting that atrazine is a herbicide with a
high leaching potential (GUS> 2.8) and that this increases with LCM application. These results may
be related with a large amount content of dissolved organic matter of LCM.
A field study was carried out during 2008 year and the aim of this work has been to evaluate the
influence of dairy manure rate in dimethenamid and atrazine leaching. The rates of slurry manure
were 0, 100,000, 200,000 and 300,000 L ha-1, corresponding to treatments S-0, S-100, S-200 and S-
300, respectively. The application of herbicides was performed one day after sowing at the 1.5 kg
ha-1 and 1.3 kg ha-1 rate of the commercial formulations (atranzine 90 GW and frontier EC). The
trial was conducted in a randomized block design with three replications. Before the application of
herbicide suction lysimeters were installed at a depth of 60 cm and these were sampled at regular
intervals, associated with rain events, during 6 months. After 40 days of the herbicides application
2.4, 1.3, 2.5 and 4.4 µg of atrazine were found for plot S-0, S-100, S-200 and S-300 respectively.
These results can be correlated with the results obtained in our laboratory under controlled
139
conditions, which indicate that atrazine is adsorbed less when 300,000 L ha-1 of liquid manure was
applied, showing a high leaching potential compared to soil (GUS index of 3.5 and 3.9 respectively).
For dimethenamid the values were 3.3, 2.1, 1.2 and 0.7 µg for plots S-0, S-100, S-200 and S-300
respectively. These results can be discussed based on a relative high adsorption of dimethenamid in
soil (Koc 140). These results could be correlated with the degradation studies performed in our
laboratory where we determined the half-life of dimethenamid for these treatments, calculating the
rate of GUS, which resulted from 3.0-2.9, 2.8 and 2.6 for S-0, S -100, S-200 and S-300,
respectively.
Important results were obtained for these studies when the soil has a history of atrazine
application, specifically the half-life of atrazine decreased substantially, ranged from 5 to 8 days.
About degradation product, a little amount of metabolites were detected regarding the
extractable atrazine. The main metabolites were DEA, DIA and HA and the relative percentage of
atrazine degradation products in the soil is distinct from that of the amended soils. The soil is
characterized by the formation of a higher amount of DIA near 80%. On the other hand, in the
amended soils the relative distribution of DIA, DEA and HA is highly variable and there is no clear
pattern about their formation, although the relative distribution of degradation products seem to be
affected by the application of LCM. Modification of adsorption process, pesticide metabolisms by
the additional C and N from amendment, and enhanced co-metabolic biotransformation can be
related with these complex changes. More work about the active microorganisms would be
necessary to clearly explain these differences and the isolation and characterization of the degrading
atrazine microorganisms in some of these soils is currently undertaken.
References
Aguilera, P., Briceño, G., Candia, M., Mora, ML., Demanet, R., and Palma G. 2009. Effect of
dairy manure rate and the stabilization time of amended soils on atrazine degradation. Chemosphere
77: 785-790.
Briceño, G., Demanet, R., Mora, ML., Palma, G. 2008. Effect of liquid cow manure on andisol
properties and atrazine adsorption. J. Environ. Qual. 37:1519-1526.
Briceño, G., Palma, G., Durán, N., 2007. Influence of organic amendment on the biodegradation
and movement of pesticides. Crit. Rev. Environ. Sci. Technol. 37, 233–271.
Gustafson, D. 1989. Ground ubiquity score: A simple method for assessing pesticide
leachability. Environ. Toxicol. Chem. 8: 339-357.
Palma G., Sanchez, A., Olave, Y., Encina, F., Palma, R., Barra, R.2004. Pesticide levels in
surface waters in an agricultural–forestry basin in Southern Chile. Chemosphere 57: 763–770.
Salazar, F., Dumont, J., Santana, M., Pain, B., Chadwick, D., Owen, E., 2003. A survey of dairy
effluent management in the south of Chile. Arch. Med. Vet. 34, 215–225.
Acknowledgements: project FONDECYT 1070568.
140
CONTRIBUTION OF SAPROPHYTIC AND MYCORRHIZAL FUNGI TO THE

FORESTRY AND AGRICULTURE PLANTS SPECIES BY AMENDMENT WITH
SEWAGE SLUDGE IN GREENHOUSE TRIALS.
Arriagada, C 1, Lagos, C 1, Cornejo, P 2, Pacheco.P 1.
(1) Laboratorio Biorremediación. Departamento de Ciencias Forestales, Universidad de La Frontera.

Casilla 54-D. Temuco - Chile.
(2) Departamento de Ciencias Químicas, Universidad de La Frontera. Casilla 54-D. Temuco - Chile.
The quality and productivity of soils could be improved by the addition of organic matter but
available material is scarce (Caravaca et al., 2003). The land application of sewage sludge is
considered beneficial, as the mineral and organic constituents of the sludge can be utilized to
improve soil fertility and biological parameters, as well as plant nutrition (Arriagada et al., 2009a).
Sewage sludge (SS) is a product of wastewater treatment that has the potential to enhance soil
productivity, as it contains high levels of organic matter and nutrients. It can be used agriculturally
as a fertiliser and soil conditioner. However, sludge application on agricultural land may introduce
potential contaminants, such as heavy metals (Arriagada et al., 2009b). Uncontrolled use of these
residues may cause serious environmental problems with unforeseen effects on the soil-plant
system.
Some ligninolytic and hydrolytic enzymes have been shown to be involved in the detoxification
of SS from a municipal wastewater (Arriagada et al., 2009a). Therefore, there is a need for
guidelines in the management and use of these wastes. Biological, chemical, physical and physico-
chemical treatments have been proposed; physical pre-treatments considerably decrease its
phytotoxicity, but to date none of these approaches offers an adequate solution to the toxicity
problems. Saprophytic fungi, by breaking down cellulosic materials to simple sugars, can provide
energy sources for microorganisms including arbuscular mycorrhizal (AM) fungi. These fungi can
mobilize nutrients, degrade phytotoxic substances, enable plants to optimise the use of available
nutrients when living roots release organic and inorganic compounds with sloughed cells (Dix and
Webster, 1995) and produce substances that promote the growth of microorganisms, such as the AM
fungi, that are beneficial to the local plant life (Fracchia et al., 2000). The analysis of some
biological parameters can give more complete information on the manner in which agricultural
practices affect soil quality and health (Avidano et al., 2005). Importantly, soil microorganisms are
able to respond immediately to environmental changes and are therefore more efficient indicators of
soil quality and health than physical and chemical parameters (Benedetti and Dilly, 2006). In our
research we observed that the AM fungi, saprobe fungi and SS significantly increased dry shoot
weight. The AM fungi induced a significant increase in Fluorescein diacetate (FDA) activity but did
not increase β-glucosidase activity. Addition of SS to AM-inoculated soil did not affect either FDA
or β-glucosidase activities in plants from soil that was either uninoculated or inoculated with the
saprobe fungi (Arriagada et al., 2009a). On the other hand, the E. globulus colonized by six AM
fungi and with application to SS promoted the plant growth and can be regarded as a successful
biotechnological tool for the greenhouse plant production (Arriagada et al., 2009c).
In general we observed that the municipal wastewater SS used in different studies with AM and
saprobe fungi improved soil fertility due to its high content of organic and inorganic plant nutrients.
In this order, Arriagada et al., (2009a), have suggested that the application of SS to AM or saprobe
fungi inoculated E. globulus variably affected plant biomass production and enzyme activities. In
some cases, inoculation with saprobe fungi in the presence of SS improved plant biomass, and
141
treatment of sewage sludge with saprobe fungi could affect FDA activity in the plant rhizosphere
soil. In contrast, β-glucosidase activity was primarily affected by the application of saprobe fungi
inoculated sewage or by inoculation of the plants with the AM fungi.
Financial support was provided by the Fondo Nacional de Desarrollo Científico y Tecnológico,
FONDECYT 1060390 grant and Vicerrectoría de Investigación y Postgrado Universidad de La
Frontera.
Arriagada, C., Sampedro, I., García-Romera, I. and Ocampo. JA. 2009a. Improvement of growth of
Eucalyptus globulus and soil biological parameters by amendment with sewage sludge and
inoculation with arbuscular mycorrhizal and saprobe fungi. Science of the Total Environment.
(407): 4799–4806.
Arriagada, C., Aranda, E., Sampedro, I., García-Romera, I. and Ocampo. JA. 2009b. Differential
effect of Trametes versicolor and Coriolopsis rigida on the action of the arbuscular mycorrhizal
fungus Glomus deserticola on the copper tolerance of Eucalyptus globulus. Chemosphere. 77:273–
278.
Arriagada, C., Pacheco, P., Pereira, G., Machuca, A., Alvear, M. and J A Ocampo. 2009c. Effect of
arbuscular mycorrhizal fungal inoculation on Eucalyptus globulus Labill. seedlings and some soil
enzyme activities under application of sewage sludge amendment. Journal of Soil Science and Plant
Nutrition. 9(2): 89-101.
Avidano L, Gamalero E, Cossa GP, and Carraro E. 2005. Characterization of soil health in an Italian
polluted site by usingmicroorganisms as bioindicators. Appl Soil Ecol. 30:21–33
Caravaca, F., Figueroa, D., Alguacil, M.M., and Roldán, A. 2003. Application of composted urban
residue enhanced the performance of afforested shrub species in a degraded semiarid land.
Bioresource Technology 90: 65–70.
Dix NJ and Webster J. 1995. Fungal Ecology. England: Chapman and Hall. 594 pp.
Fracchia S, García-Romera I, Godeas A, and Ocampo JA. 2000. Effect of the saprophytic fungus
Fusarium oxysporum on arbuscular mycorrhizal colonization and growth of plants in greenhouse
and field trials. Plant Soil.223:175–84.
142
USE OF INDUSTRIAL WASTE FROM PULP MILLS IN AGRICULTURE

Gallardo F.
Department of Chemical Science, De La Frontera University, Temuco-Chile. fgallar@ufro.cl
Cellulose is the main Chilean product of forest exportation. In 2000, cellulose exports grew 50%
compared to previous years. The wastewater treatment plants in the pulp and paper industry produce
large quantities of sludge that require disposal. Disposal of sewage sludge’s include landfilling or
incineration, but the high cost associated at these practice have led to an evaluation of viable and
cost-effective alternatives. Today, utilization of sewage sludge in agricultural field or degraded soil
is gaining popularity as a means of waste disposal and as alternative to soil mineral fertilizer.
Sewage sludge can enhance plant productivity as it contain high amounts of essential nutrients such
as C, N, P and trace elements. On the other hand, the sludge generally contains high organic matter
levels, microorganisms and inorganic substances (silt, clay and calcium carbonate) therefore, sludge
application to the soil can promotes moreover, the improvement of soil structure, aggregate stability,
enhance the biological activity and the adjustment of pH. In Chile, there are approximately 3.1
million ha of soil derived from volcanic ash (Andisols and Ultisols), whose main characteristics are
their high P fixation capacity and acid pH. Extractable Al is generally high in these soils and is
associated with P fixation level, which increases when pH decreases. In addition, toxicity produced
by aluminum is considered to be the most important limiting factor in plant growth and development
in soils with high acidity. Several countries have regulated the use of sewage sludge in agriculture to
avoid harmful effects on soil, vegetation, animals and human being. In Chile, the use of kraft mill
sludge is not regulated yet, but studies have been conducted with the finality of know the feasibility
to be applied as soil amendment. Knowing of chemical composition of kraft mill sludges is of great
importance when developing recommendations for the rates of sludge applications on agricultural
land. The successive application of sludge to degraded soil will increase the nutrient availability for
the plants and will modify the microbial biodiversity, thus increasing the productivity of soil. On the
other hand, the best accumulation of these nutrients by crops will avoid losses through the soil
profile.
The effect of successive application of kraft mill sludge on agricultural soils (Andisol and Ultisol)
has been assessed for the long time. An increase of organic matter, N, P content was observed with a
sludge application of 10, 20 and 30 t ha-1 in each application. On the other hand, decrease aluminum
saturation was observed. The main increment of micronutrients was observed for Mn and Zn in both
soils. However, through a study done in pots showed with ryegrass, an accumulation of these
elements in the foliage was obtained; provably decreasing the leachates of Mn and Zn through the
soil depth, leachates studies in the field could to confirm the real effect of sludge application in the
agricultural soils. The sludge application at different rates modify the soil activity, studies through
microbial respiration, fluorescien diacetate (FDA) and phosphatase have shown greater activity
when different rates of sludge have been applied. Studies through molecular techniques will permit
know the effect of sludge on microbial diversity. As preliminary conclusion, the sludge applications
contribute to increase the soil levels nutrients and fertility.
Acknowledgments: This study is financed by Fondecyt proyect 1080427.
143
ROLE OF ENZYMES IN THE REMEDIATION OF POLLUTED ENVIRONMENTS

Liliana Gianfreda
Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali,
Università di Napoli, Federico II, Via Università 100, 80055 Portici, Napoli, Italy,
liliana.gianfreda@unina.it
Environmental pollution is more and more growing for the indiscriminate and frequently deliberate
release of hazardous, harmful substances. In 2007, the Blacksmith Institute of New York launched
the second annual review of the most polluted places in the world – sites where human health is
severely affected. The Institute has listed the 2007 World’s Worst Polluted Places - The Dirty Thirty
Summary Matrix - which includes also the top ten polluted sites. Most of them are located in poor
countries where pollution continues to be a major cause of death, illness, and long-term
environmental damage. Several types of polluting substances including inter alias Pesticides, PAHs,
PCBs, Dyes, Phenols, Chlorophenols, Nitrocompounds and others as well as heavy metals (Arsenic,
Cadmium, Chromium, Copper, Lead) and cyanide are present, very often as mixtures not only of
different organic but also of organic and inorganic compounds. Different are the origins and sources
of pollution: industrial activities such as mining and metal processes, petrochemical and industrial
complexes, industry effluents, chemical weapons, pulp and paper industry, dye industry and
industrial manufacturing; anthropogenic activities such as traffic, agricultural practices, and others.
Research efforts have been devoted to develop new, low-cost, low-technology, environment-
friendly treatments capable of reducing and even eliminate pollution in the atmosphere, the
hydrosphere and the soil environment.
Among biological agents, enzymes have a great potentiality to effectively transform and
detoxify polluting substances. The main reasons are that enzymes: are the main effectors of all the
transformation occurring in the biota (for detoxification microorganism and plants); are catalysts
with either narrow (chemi-, region and stereo-selectivity) or broad specificity to be applicable to a
large range of different compounds also in mixture; may produce extensive transformations of the
structure and toxicological properties of the contaminants, and even their complete conversion into
innocuous inorganic end products; may perform processes for which no efficient chemical
transformations have been devised; may present advantages over traditional technologies, and also
over microbial remediation;
Enzymes may act intracellularly, i.e. in the presence and inside their originating cells, or
extracellularly, i.e. as free or immobilized molecules. The most representative enzymatic classes in
the remediation of polluted environments are: hydrolases, dehalogenases, transferases and
oxidoreductases. For many of these enzymes the transformation of different xenobiotic substances
has been tested mainly under laboratory conditions.
Some classes of pollutants and of enzymes capable to effectively transform them and restore
polluted environments will be examined. Particular attention will be devoted to pollutants with a
high polluting potential such as chlorophenols, polyphenols, PAH, arsenic and cyanide. The
enzymatic processes developed and implemented in some of these detoxification treatments will be
examined in details. The main advantages as well as the main drawbacks still present in the
extensive application of enzymes in the in situ restoration of polluted environments will be
discussed.
144
Simposio 5.
Biotecnología Ambiental y Manejo de Contaminantes
145
146
EFFECT OF RHIZOSPHERE ON THE DEGRADATION OF PENTACHLOROPHENOL

BY BIOAUGMENTATION OF Anthracopyllum discolor
1
Urrutia, C., 1Paredes, C., 2Rubilar, O., and 2Diez, M.C.
1
Graduate Program in Natural Resources Science, Universidad de La Frontera, Temuco,
c.urrutia04@ufromail.cl. 2Department of Chemical Engineering, Universidad de La Frontera,
mcdiez@ufro.cl.
Chlorophenols are xenobiotic compounds can be found in wastewater and soil.

Pentachlorophenol (PCP) is a broad-spectrum biocide was used widely such as wood preservative,
preharvest herbicide, molluscicide, and other applications. Different methods can be use for
remediation of pentachlorophenol (PCP) includes land disposal, incineration, soil washing, and
chemical extraction. Some of these strategies can be expensive and inefficient for the elimination of
contaminants in environment; the searching of other biotechnological applications specially the
biodegradation by microorganisms and plants presents a new alternative for bioremediation.
The process of phytoremediation of xenobiotics compounds is based on beneficial effects

between roots and microbial activity of soil to enhance the biodegradation of contaminants.
Different properties of soil can be change for the presence of roots. These include Fisico-
chemical and biological characteristics of soil principally the pH, texture, colloids, microbial
diversity, enzyme activities. The microbial dynamics in soil can be influence by chemical
compounds from plants especially by roots exudates, these compounds in large quantities could be
change the biochemical characteristic of rizosphere.
The utilization of biological methods to increase the contaminants biodegradation through the
non-native microorganism has been presented a new alternative of bioaugmentation. The use of
different microorganism such bacteria, fungi especially white-rot fungi has been studied because the
potential to increase the bioremediation process. Theses fungi possess an extracellular enzymatic
system, can degrade insoluble chemicals, an extremely diverse range of very persistent or toxic
environmental pollutants. The Chilean native fungus Anthracophyllum discolor a native Chilean
fungus previously isolated from temperate forests of southern Chile, have demonstrated ligninolytic
activity, mainly of manganese peroxidase (MnP), accelerating degradation rates of PCP in soil.
In this context, the main objective of this project will study the removal of PCP by the effect of
rhizospheric activity of Lolium multiflorum an allophonic soil (Andisol), by bioaugmentation of A.
discolor. The main activities will be to determine the remotion of PCP in the rizhosphere whit A.
discolor. After that will be identify some exudates of Lolium multiflorum influenced by change of
pH of soil. Finally will be to determinate the enzyme activities of A. discolor in soil.
Therefore, this study provides knowledge about the Phytoremediation increased by a native
strain A. discolor used for biodegradation of PCP giving a new biotechnology application.
Acknowledgements: Investigation financed by FONDECYT 1050614, CONICYT-CSIC 2007-

144 and DIUFRO GAP-2007 projects.
147
BIOESTIMULATION, BIOAUGMENTATION AND ENZYMATIC BIOREMEDIATION IN

SPIKED SOIL WITH POLYCYCLIC AROMATIC HYDROCARBONS
Acevedo F.1,2 and Diez M.C.2
1
Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera, Temuco,
Chile. 2Environmental Biotechnology Center, Scientifical and Technological Bioresource Nucleus
Department of Chemical Engineering, Universidad de La Frontera, Temuco, Chile.
facevedo@ufro.cl
A promising strategy for environmental clean-up is the use of microorganisms for removing,
transforming or mineralizing any xenobiotic compound to a less toxic form by any process.
Alternatively, any attempt to manipulate contaminated environmental areas by adding nutrients,
water and air (biostimulation) or by adding micro-organisms (bioaugmentation) can be classified as
accelerated bioremediation. Another form of bioremediation is enzymatic treatment utilizing free or
immobilized enzymes, which have a minimal impact on ecosystems.
The aim of this work was to evaluate three different bioremediation techniques (biostimulation,
bioaugmentation and enzymatic bioremediation) of a spiked soil with polycyclic aromatic
hydrocarbons (PAH). To achieve this objective, PAH removal by native microflora of soil, by
adding the Chilean white-rot fungus Anthtracophyllum discolor and by adding free or immobilized
manganese peroxidase (MnP) in spiked soil were evaluated.
A high PAH removal was obtained by the action of native microflora after 60 days,
biostimulated by adding wheat grains as C and N supply, water and air. 98.3% phenanthrene, 83.1%
anthracene, 82.5 % fluoranthene, 82.5% pyrene and 14.2% benzo(a)pyrene were removed in this
soil. The presence of A. discolor (bioaugmentation) provoked a less efficient removal of anthracene
(83.1% vs. 61.5%), fluoranthene and pyrene (82.5% vs. 43.1%) after the same period of incubation,
showing a possible inhibition and competition of native microbes with the fungus. The colonization
of soil by A. discolor may require a higher demand for substrate compared to the control (autoclaved
soil). In absence of endogenous microorganisms, A. discolor showed biodegradation capability in
soil, particularly benzo(a)pyrene (66% of degradation efficiency), demonstrating clearly a positive
correlation with ligninolytic enzymes production such as MnP. On the other hand, about 65% of
removal efficiency for anthracene was obtained in soil by nanoclays-immobilized manganese
peroxidase after 24 h, being about 3-fold higher compared with the control (free MnP). The action
of endogenous microorganisms in the PAH degradation was not observed during this period.
Furthermore, in this study, the biostimulation in spiked soil with PAH was successful compared
with the bioaugmentation strategy adding A. discolor. However, this scenario could be different on
aged PAH contaminated soil where microbial communities are normally less diverse compared with
a recently contaminated soil. This fact could be a key issue when studying A. discolor inoculation in
aged contaminated soil. In addition, the results of enzymatic remediation suggest the possible use
of immobilized MnP as a suitable and effective biocatalyst for environmental remediation purposes.
Acknowledgements: to FONDECYT Nº 1050614 project, PhD. scholarship from CONICYT, Chile

and the Department of Microbiology of the Swedish University of Agricultural Sciences, Sweden.
148
USE OF BIOBED SYSTEM FOR CHLORPYRIFOS DEGRADATION

Tortella, G. R1,2., Rubilar, O1,2., Cea, M2., Avendaño1, M., Fernandez-Alberti, S1. and Diez,
M.C 1,2.
1
Department of Chemical Engineering, Universidad de La Frontera, P. O. Box 54-D. Temuco, Chile.
2
Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Av. 01145.
Temuco, Chile. gtortell@ufro.cl
Studies in several parts of the world, mainly in Sweden, have demonstrated that biobeds can
effectively treat pesticide waste arising from accidental spillages. The biobed is a simple biological
system composed by biomixture of top soil, peat and straw, providing a matrix to adsorb the
pesticides and facilitate their biodegradation. The straw stimulates the growth of lignin-degrading
fungi and the formation of the ligninolytic enzymes; the soil provides sorption capacity and should
have a humus and clay content that promote microbial activity, and is also an important source of
pesticides degrading microorganisms, that can act synergistically with the fungi; the peat contributes
to sorption capacity, moisture control and also abiotic degradation of pesticides. Therefore, the aim
of this study was performed to determine whether biomix of biobeds system with allophanic top soil
(Andisol) can degrade effectively the organophosphorous insecticide chlorpyrifos.
The biomix was conformed of straw (50% v/v), peat (25% v/v) and topsoil (25% v/v). The
biomix was pre- incubated for 0, 15 and 30 day at 20 ± 1 °C before to be contaminated with
chlorpyrifos (160 mg kg-1) and the chlorpyrifos degradation was evaluated during 30 days. Besides,
the degradation of chlorpyrifos in biobed system at the concentration of 320 and 480 mg kg-1 was
evaluated. CO2 evolution, enzymatic activities (manganese peroxidase (MnP) and fluorescein
diasetate hydrolysis-FDA), residual concentration of chlorpyrifos and TCP (3,5,6-trichloro-2-
pyridinol) formation were periodically evaluated in both studied.
Degradation of chlorpyrifos (160 mg kg-1) was quicker with 15 day of pre-incubation of the
biomix (90%). Instead, the degradation with 0 and 30 days of pre-incubation were 83 and 80%,
respectively. The maximum of TCP formation occurs after 10 days of incubation, in the tree pre-
incubation period evaluated, with values of 49, 27 and 47% of initial clorpyrifos concentration.
However, over this time, degradation of TCP was observed in all assays. MnP activity was highest
in 0 day of pre-incubation period (11 U Kg-1 of soil), instead at 15 and 30 days the activities were 8
and 3 U Kg-1 of soil, respectively. Initial FDA activity was 9.5, 6.3 and 2.5µ g g-1 h-1 at 0, 30 and 15
days of pre-incubation period, respectively. No significant differences were observed in C02
formation.
The degradation of chlorpyrifos 320 and 480 mg kg-1 occurred efficiently in biobed system (>
90%) after 40 days of incubation period. In both assays TCP formation was detected, however, over
10 days degradation of TCP was observed in both assays. Significant differences were not found in
C02, MnP and FDA activities, but them increased in the time.
In conclusion, the results of this study demonstrate that pre-incubation time of biomix with
allophanic soil between 0-30 days had no major effect on the chlorpyrifos degradation. High
concentration of chlorpyrifos can be degraded in biobed. TCP was formed in the biobed system, but
it also can be also degraded by this system.
Keywords: Biobeds; Allophanic soil; chlorpyrifos.
Acknowledgements: Investigation financed by Fondecyt 1090678, Fondecyt 3090049,
149
EFFECT OF DAIRY MANURE AMENDMENTS ON ATRAZINE AND DIMETHENAMID

LEACHING IN A SOUTHERN CHILEAN ANDISOL
Oscar Candia , Gabriela Briceño1, Paula Aguilera1, Rolando Demanet2, María de La Luz
1
Mora1, and Graciela Palma.1

1
Chemistry Department, Universidad de La Frontera, Temuco, Chile. Email: ocandia@gmail.com
2
Agricultural Production Department, Universidad de La Frontera, Temuco, Chile.
Dimethenamid and atrazine herbicides are widely used for pre-emergence control of broad-leaved weeds in
corn and several other crops. They have been frequently detected in ground water, near agricultural field,
where they had been applied [1]. Also, livestock slurries are applied in forage maize during soil preparation,
which increase both the population and aggressiveness of weeds, then decrease the efficacy and increase the
requirements of herbicides [2]. Considering this problematic, a field study was carried out during 2008 year
and the aim of this work has been to evaluate the influence of dairy manure rate in dimethenamid and atrazine
leaching. The field results will be compared with some ones obtained for us under controlled conditions. The
plot size was 3x5 m and the slurry was applied on September, one month before planting, in order to
incorporate it evenly to the soil. The rates of slurry manures were 0, 100,000, 200,000 and 300,000 L ha-1,
corresponding to treatments S-0, S-100, S-200 and S-300, respectively. The application of herbicides was
performed one day after sowing at the 1.5 kg ha-1 and 1.3 kg ha-1 rate of the commercial formulations
(atrazine 90 GW and frontier EC). The trial was conducted in a randomized block design with three
replications. Before the application of herbicide suction lysimeters were installed at a depth of 60 cm and
these were sampled at regular intervals, associated with rain events. Water samples were frozen for later
residue herbicide analysis by HPLC. The samples were taken during 6 months, after the application of the
herbicides. 40 days after herbicides application 2.4, 1.3, 2.5 and 4.4 µg of atrazine were found for plot S-0, S-
100, S-200 and S-300 respectively. These results can be correlated with the results obtained in our laboratory
under controlled conditions [3], which indicate that atrazine is adsorbed less when 300,000 L ha-1 of liquid
manure was applied, showing a high leaching potential compared to soil (GUS index [4] of 3.5 and 3.9
respectively). For dimethenamid the values were 3.3, 2.1, 1.2 and 0.7 µg for plots S-0, S-100, S-200 and S-
300 respectively. These results can be discussed based on a relative high adsorption of dimethenamid in soil
without manure (Koc 140), but the dissipation of dimethenamid increased when the soil was amended with
higher doses of manure, probably due to external addition of dissolved organic matter [5][6]. These results
could be correlated with the degradation studies performed in our laboratory where we determined the half-
life of dimethenamid for these treatments, calculating the rate of GUS, which resulted from 3.0, 2.9, 2.8 and
2.6 for S-0, S -100, S-200 and S-300, respectively.
[1] Crawford, J., Sims, G., Simmons, F., Wax, L., and Freedman, D. 2002. Dissipation of the herbicide [14C]
dimethenamid under anaerobic conditions in flooded soil microcosms. J. Agric. Food Chem., 50: 1483 -
1491.
[2] Moorman, T., Cowan, J., Arthur, E. and Coats, J., 2001. Organic amendments to enhance herbicide
biodegradation in contaminated soils. Biology and Fertility of Soils, 33: 541–545.
[3] Briceño, G., Demanet, R., Mora, M. y Palma G. 2008. Effect of Liquid Cow Manure on Andisol
Properties and Atrazine Adsorption. J. Environ. Qual., 37:1519-1526.
[4] Gustafson, D., Environmental Toxicology and Chemistry, 1989, 8:339-357.
[5] Archangelo, E., Karam, D., Ferreira, F., Prates, H., Ferreira, L., Cardoso, A. 2004. Sorption, Desorption,
and Leaching Potential of Dimethenamid in Brazilian Soils Planta Daninha, Viçosa-MG, 22: 467-477.
[6] Vasilakoglou, I., Eleftherohorinos, I., & Dhima, K. 2001. Activity, adsorption and mobility of
three acetanilide and two new amide herbicides. Weed Research, 41:535-546.
Acknowledgments: FONDECYT Project Nº1070568
150
EFECTO DEL TIEMPO DE INCUBACIÓN DE UN LECHO BIOLÓGICO SOBRE LA

DEGRADACIÓN DE CLORPIRIFOS
(Effect of incubation time of a biobed on chlorpyrifos degradation)
Fernández-Alberti, S.1, Tortella G R2, Diez M.C.2
1
Programa de Magíster en Ciencias de la Ing. mención Biotecnología, Universidad de La Frontera,
Temuco. 2Environmental Biotechnology Center, Scientific and Technological Bioresource Nucleus.
Universidad de La Frontera, Temuco
Los plaguicidas desempeñan un papel importante en la agricultura moderna y la producción de

alimentos. Sin embargo, las malas prácticas agrícolas y el manejo inadecuado de los plaguicidas,
puede contaminar el medio ambiente, especialmente las aguas subterráneas y superficiales. Las
fuentes puntuales como el llenado y lavado de los equipos de aspersión, son las que más contribuyen
a la contaminación del ambiente. Estudios en Dinamarca, Alemania y Suecia han señalado que los
lechos biológicos han demostrado ser un método simple y eficaz para reducir al mínimo la
contaminación ya que pueden retener y degradar plaguicidas producidos por derrames accidentales.
En este contexto, se usó el insecticida Clorpirifos O,O-dietil O-(3,5,6-tricloro-2-piridinil)
fosforotioato, perteneciente al grupo químico de los organofosforados, siendo un insecticida de
amplio espectro. El objetivo de este estudio fue determinar el efecto de una elevada concentración
de Clorpirifos (160 mg/kg) sobre la actividad respiratoria, enzimática y su degradación en la
biomezcla del lecho biológico.
La biomezcla consistió en paja de trigo, suelo agrícola Andisol (serie Gorbea) y turba
magallánica, todo en proporción 2:1:1. Se evaluó el efecto de 3 tiempos de pre-incubación de la
biomezcla (0, 15 y 30 días) a 20 °C en oscuridad. Se evaluó periódicamente la actividad respiratoria
(CO2); actividad manganeso peroxidasa (MnP), hidrólisis de la fluoresceína diacetato (FDA) y la
degradación de clorpirifos.
Los resultados obtenidos muestran que la actividad respiratoria fue superior en la biomezcla pre-
incubada a 15 y 30 días (> 400 mg CO2/100g de suelo) respecto de la biomezcla no pre-incubada
(230 mg CO2/100g de suelo). Asimismo, no se observó diferencias significativas de la actividad
respiratoria entre la biomezcla contaminada y el control (sin contaminar), en los 3 tiempos de pre-
incubación evaluados. Resultados similares fueron obtenidos con la actividad MnP, donde no se
observó diferencias entre la biomezcla contaminada y el control. Sin embargo, la actividad FDA
disminuyó por la presencia de clorpirifos en la muestra no incubada.
La degradación de Clorpirifos fue mayor en la biomezcla con 15 días de pre-incubación
alcanzando una remoción > 70%. En orden de eficiencia de degradación se señala que pre-
incubación de 15 días > 0 días > 30 días.
Por lo tanto, de este estudio se concluye que los lechos biológicos pueden ser capaces de tratar
altas concentraciones de plaguicidas y que la presencia de clorpirifos en una dosis elevada no afecta
la actividad biológica de la biomezcla de un lecho biológico, siendo de este modo, una alternativa
viable de tratamiento de residuos de plaguicidas.
Financiamiento. Trabajo financiado por los proyectos FONDECYT Nº 1090678 y 3090049.
151
ENZYME ACTIVITIES AND DYE DEGRADATION BY COMPLEX PELLETS OF

WHITE-ROT FUNGUS TRAMETES VERSICOLOR
Elgueta S1, Cuevas R2, Rubilar O 2, and Diez M.C2
1
International Doctorate in "Enviromental Resource Science" Universidad de La Frontera, Chile-
Universidad de Napoles Federico II, Italia.
2
Centro de Biotecnología Ambiental, Scientifical and Technological Bioresource Nucleus,
Universidad de La Frontera, Chile. sebaelgueta@ufro.cl
Effluents containing textile dyes are usually discharged in large quantities worldwide into natural
water bodies. The presence of these compounds can cause problems due to the possible entrance
into de food chains of humans and animals. Once in the environment, they can show their toxic and
genotoxic effects on organisms. Therefore, is necessary to remove dyes before effluent discharge.
Different treatments can be used for dyes removal; however, some of them can cause toxicity to the
microorganisms of the biological system.
In recent years several studies have been demonstrated that white-rot fungi are able to decolorize
and remove a wide variety of structurally diverse pollutants including synthetic dyes. The fungi
present the advantage over other microorganisms due to their extracellular ligninolytic enzyme
systems composed by manganese peroxidase (MnP), manganese-independent peroxidase (MiP),
lignin peroxidase (Lip) and laccase (Lac). Therefore, the main objective of this study was to
evaluate the synthetic dye degradation by pellets of white rot fungus Trametes versicolor.
Complex pellets of white-rot fungus Trametes versicolor was formulated with mycelium,
activated carbon and sawdust (2:1:1) for the synthetic dye degradation. The pellets were formed by a
center of activated carbon and sawdust surrounded by fungal mycelium.
The degradation was development in modified Kirk liquid medium with an initial concentration
of each dye of 100 mg L-1 of Reactive Black 5 (RB5), Acid Orange 6 (AO6), Reactive Orange 16
(RO16), Basic Violet 4 (BV4) and Blue Remazol Brilliant (BRB). The flasks were incubated at 25 º
C, under agitation at 100 rpm, by 15 days. During the incubation period color degradation and
ligninolytic enzyme activity (lignin and manganese peroxidase, laccase and manganese-independent
peroxidase) were evaluated.
The results obtained showed that the dyes degradation by complex pellets of Trametes versicolor
was: 93, 82, 95, 16, 75 and 95% for Reactive Black 5, Acid Orange 6, Reactive Orange 16, Basic
Violet 4 and Blue Remazol Brilliant respectively. A higher activity was obtained for manganese
peroxidase and laccase with 150 and 60 U L-1 respectively. Manganese-independent peroxidase was
30 U L-1 and less activity was obtained with lignin peroxidase (1,4 U L-1).
The faster degradation was for the RB5 and BRB dyes. In conclusion, the application of
complex pellets of Trametes versicolor can be potentially used for the biodegradation of synthetic
dyes.
Keywords Trametes versicolor; complex pellet; synthetic dye, ligninolytic enzymes
ACKNOWLEDGMENTS: Financed by FONDECYT 1090678
152
PRODUCTION OF AN ECOFERTILIZER USING BIOCHAR AS RAW MATERIAL

González M.E1, Diez M.C.2, 3, Navia R2, 3.
1
Doctoral Program in Sciences of Natural Resources, Universidad de La Frontera, Temuco, Chile.
2
Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Temuco, Chile.
3
Department of Chemical Engineering, Universidad de La Frontera, Temuco, Chile.
ma.eugenia.g.q@gmail.com
Worldwide experiences show that the use of charcoal – also known as biochar or “terra
preta” – could be a bioenergy source, partially avoiding global warming and positioning itself as a
tool for promoting agriculture in unproductive zones, enhancing current productivity. In this context,
due to its high surface area and negative surface charge, biochar has a high cation exchange
capacity. Similarly, it has been demonstrated that it is able to trap anions such as phosphate and
nitrate, among others. Therefore, given its physicochemical properties, the hypothesis established in
this research is that biochar has the ability to adsorb high concentrations and a controlled liberation
of nitrogen in the ground. Consequently, the objective of this research is to evaluate the use of
biochar for the generation of an ecofertilizer, in order to obtain a sustainable product.
In this research, the physicochemical properties of biochar obtained from lignocellulosic
biomass by means of pyrolysis will be evaluated, as well as biochar’s ability of adsorption and
desorption of a nitrogen source and the feasibility for pelletizing the biochar based fertilizer using
different polymers for its covering. Finally, relevant aspects for commercial applications of
pelletized biochar, such as storage conditions and quality control over storage time, will be
evaluated
Keywords: Ecofertilizer, biochar, nitrogen.
Acknoweledgments: Investigation financed by project FONDEF D07I1096 .
153
INFLUENCE OF SOYA LECITHIN ON THE ENZYMATIC SYSTEM OF WHITE-ROT

FUNGI Anthracophyllum discolor
Bustamante, M1., González, M.E 1., Cartes, A2. and Diez, M.C2.
1
Doctoral Program in Sciences of Natural Resources, University of La Frontera. 2 Department of
Chemical Engineering, Environmental Biotechnology Center, University of La Frontera, Avda.
Francisco Salazar 01145, Casilla 54 – D, Temuco, Chile. ybustama@ufro.cl
Natural and synthetic surfactant have the potential to enhance the bioavailability of hydrophobic
organic pollutants in contaminated sites. Interestingly, it has also been proposed that this surface
activity agent can be used as a growth substrate, resulting in an increased microbial biomass, thereby
promoting a mare efficient bioremediation process. Biosurfactants are less toxic and more
environmentally benign than synthetic surfactant. However, some these compounds can be toxic,
recalcitrant and exert inhibitory effects on the biodegrading microorganism. Until now, most studies
have been conducted mainly to evaluate the effects of chemical surfactant on bacteria, and some
white-rot fungi, as Phanerochaete chrysosporium and Trametes versicolor. But few researches have
addressed to analyze the effects of phytogenic surfactant on the ligninolytic activity of white-rot
fungus. In this context, the aim of this study was to evaluate the effects of soya lecithin (SL) on the
production of ligninolytic enzymes, laccase (Lac), lignin peroxidase (LiP), manganese peroxidase
(MnP), and manganese independent peroxidase (MiP), produced by the white-rot fungi
Anthracophyllum discolor.
Studies previous have demonstrated that the optimum culture conditions of A. discolor for
maximizing ligninolytic enzyme production in modified Kirk medium were 26ºC; pH 5.5; and C/N
ration of 250. The modified Kirk medium was supplemented with SL in the range 0-1 gL-1. The
flasks with 50 ml of medium were incubated for 31 days in stationary cultivation, and the
ligninolytic activity in the extracellular fluid was evaluated periodically.
The results showed that A. discolor predominantly produced MnP in the evaluated
concentrations of SL, with a maximum activity of 30.64 UL-1 ± 4.61 UL-1 when the medium was
supplemented with 1 gL-1 of SL after 23 days of incubation. On the other hand, LiP present a
maximum of 30.11 UL-1 ± 0.13 UL-1 with 30 days of culture, when the medium was supplemented
with 0.15 and 0.30 gL-1 of SL. While the amount of Lac and MiP produced did not exceed 5 UL-1.
Financiamiento: Fondecyt Nº 1090678
154
DEGRADACIÓN DE METHYL ORANGE UTILIZANDO NANOPARTÍCULAS DE FE-

IMOGOLITA COMO UN CATALIZADOR HETEROGÉNEO DEL TIPO FENTON.
Nicolás Arancibia-Miranda1, Mauricio Escudey2, Karen Manquián1, Mauricio Molina3.
1
Programa de Doctorado y Magíster en Ciencias de los Recursos Naturales, Depto. Ciencias
Químicas, Universidad de La Frontera, Av. Francisco Salazar 01145, Casilla 54-D, Temuco, Chile.
E-mail:naranci@ufro.cl. 2Facultad de Química y Biología, Universidad de Santiago de Chile, Av.
B. O’Higgins, 3363 Santiago, Chile. 3Departamento de Industrias, Universidad Técnica Federico
Santa María, Av. Santa María 6400, Santiago, Chile.
El objetivo de este trabajo fue evaluar la capacidad de degradación de Methyl Orange (MO)
catalizada por nanopartículas de imogolita sintéticas, modificada con Fe estructural (Fe-imogolita).
La síntesis de la imogolita se realizó según lo descrito por Denaix, et al., 1999 y Ookawa, et al.,
2006 [1, 2], reemplazando un 10% de Al por Fe(III), obteniéndose una razón (Al+Fe)/Si de 2. La
Fe-imogolita fue caracterizada por TEM, DRX, IR y PZC. Se estudió la capacidad de catalizar la
reacción de Fenton, a través de la descomposición de peróxido de hidrógeno, midiendo el O2
producido a pH controlado de 5.8. Se usó 30 mg de la muestra de Fe-imogolita y se comparó la
respuesta frente a imogolita no modificada.
La oxidación de MO, implicó en una primera etapa el estudio de adsorción de los compuestos en
Fe-imogolita, la que se realiza en ausencia de H2O2, mientras que, de manera paralela la segunda
etapa evalúa a la degradación del azocompuesto mediada por H2O2, ambas etapas se realizaron en
duplicado y fueron monitoreadas a intervalos de 2 minutos durante 3 horas, mediante el uso de
espectroscopia UV/Vis 465 nm para methyl Orange [3]. Los resultados indicaron que Fe-imogolita
catalizó la descomposición de H2O2, mediante una reacción de tipo Fenton, encontrándose
diferencias sustanciales frente a imogolita no modificada. Los estudios de degradación establecieron
que Fe-imogolita es capaz de degradar a los MO, presentando una eficiencia de degradación en
methyl Orange cercana del 70% de compuesto degradado en los primeros 30 minutos de reacción,
tiempo en el cual se logra la máxima eficiencia de degradación de Fe-imogolita.
]1] L.I. Denaix Laurence, Bottero Jean Yves, Colloids and Surfaces A: Physicochem. Eng.
Aspects 158 (1999) 11.
]2] I.Y. Ookawa Masashi, Watanabe Masako, Suzuki Masaya and Yamagushi Tsutomu, Clay
Science 2 (2006) 5.
]3] M.C. Pereira, C.M. Tavares, J.D. Fabris, R.M. Lago, E. Murad, and P.S. Criscuolo, Clay
Minerals 42 (2007) 299-306.
155
PELLETIZATION OF WHITE-ROT FUNGUS Anthracophyllum discolor FOR LIGNIN

DEGRADATION IN AIRLIFT REACTOR
O. Rubilar 1, G. Tortella1, S. Elgueta1, L. Gianfreda2 and MC. Diez1
(1)
Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, PO Box 54-D
Temuco, Chile. (2)Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni
Animali, Universita` di Napoli Federico II, via Universita` 100, 80055 Portici, Naples, Italy.
Corresponding author: orubilar@ufro.cl
The white-rot fungi (WRF) have been studied because are organisms able to tolerate and degraded
higher concentrations of diverse persistent organic compounds. The ability of WRF to degrade
pollutants has been attributed to the action of non-specific extracellular ligninolytic enzyme system,
composed principally of laccases, lignin peroxidases (LiP) and manganese peroxidases (MnP).
The WRF can be grown in submerged cultures by several different morphological forms:
suspended mycelia, clumps or pellets. However, pellet form becomes the desirable morphology for
industrial process, because this form not only reduces the medium viscosity, but also allows the
possibility of biomass reuse and thereby continuous operation of the process. Nevertheless, one of
the main factors leading to treatment effectiveness of wastewater is the use of the appropriate
inoculum that can be used under different conditions and in large scale. Several investigations have
shown that different types of organic and inorganic supports allow the fungi pellets formation
providing a structural and nutritional function.
In this context, pellets of white-rot fungus Anthracophyllum discolor with carbon activated and
sawdust (complex pellets) were formulated for the lignin degradation. The lignin degradation was
development in an airlift reactor at initial concentration of 1000 mg/L and the pellets were re-used
for two times in batch operations.
The complex pellets were formed by a center of activated carbon and sawdust surrounded by
fungal mycelium, whereas simple pellets consisting of only mycelium were hollow spheres.
Degradation of lignin was performed in an airlift reactor at initial lignin concentration of 1000 mg/L
and the fungal pellets were re-used two times in batch operations. Complex pellets degraded lignin
by 87 and 72% in the first and second cycle, respectively, and they were significantly more effective
than simple pellets. In addition, complex pellet showed higher manganese peroxidase activity in the
reactor.
Sadwust and activated carbon appear to be suitable carriers of A. discolor for degradation of
organic pollutants in wastewater and soil.
Keywords: White-rot fungi; pellet; lignin; airlift reactor
ACKNOWLEDGMENTS: The authors would like to thank FONDECYT Project No. 3080013 and
1090678.
156
CARACTERIZACIÓN DEL POTENCIAL CONTAMINANTE DE PURINES GENERADOS

EN PLANTELES BOVINOS
Morales, E.
Doctorado y Magíster en Ciencias de Recursos Naturales. Universidad de La Frontera. E-mail para

correspondencia: eduardomoralesr@gmail.com.
Resumen (Plan de trabajo)
La constante demanda de alimento para una población mundial siempre creciente ha

intensificado la actividad ganadera, con lo que se ha logrado un aumento de la producción de leche y
carne, pero al mismo tiempo, estos sistemas de producción animal intensivos están generando
grandes volúmenes de desechos, ocasionando graves problemas medioambientales y sanitarios, si
éstos son mal manejados. Actualmente, el mal manejo de los purines es el mayor problema que
enfrentan los sistemas ganaderos intensivos, siendo el causante del mayor deterioro ambiental.
Tradicionalmente los purines bovinos se han utilizado como fertilizante, y son aplicados a la
pradera para aumentar la producción de forraje, existiendo una lixiviación de nutrientes y patógenos
a través del suelo. En este sentido, cabe señalar que el purín bovino es un residuo que puede ser
reciclado en el predio al ser utilizado como fertilizante, pero cuidando de no causar un impacto
negativo sobre el ambiente, para lo cual es necesario conocer realmente cuál es el potencial
contaminante de este residuo ganadero en planteles de la zona sur de Chile.
El objetivo del presente trabajo es caracterizar el potencial contaminante del purín bovino
generado en lecherías y en planteles de engorda a corral (feedlots) en la zona sur de Chile.
Se colectará muestras de purín bovino de 4 predios ubicados en los alrededores de la ciudad de

Temuco, Región de La Araucanía, dos de planteles de lechería y dos de planteles de engorda.
Posteriormente, se determinará los siguientes parámetros físico-químicos y microbiológicos, de
acuerdo a los protocolos establecidos para el análisis de aguas residuales por la normativa chilena
respectiva: Demanda Biológica de Oxígeno (DBO5), Demanda Química de Oxígeno (DQO),
Fósforo Total (P), Nitrógeno Total Kjeldahl (NTK), Nitrógeno Total, Sólidos Suspendidos Totales
(SST) y Volátiles (SSV), Sólidos Sedimentables (SD), Sólidos Totales disueltos (ST), Coliformes
totales, Temperatura y pH. Todos los análisis serán realizados en triplicado para cada muestra, y se
efectuarán en dependencias de la Universidad de La Frontera.
Finalmente, las distintas variables del estudio serán analizadas estadísticamente. Para ello se
utilizará los paquetes estadísticos Statistica v.7.0 (2005) y Minitab Statistical Software v.15.1.3
(2009).
157
EFFECT OF DAIRY MANURE RATE AND THE STABILIZATION TIME OF

AMENDED SOILS ON ATRAZINE DEGRADATION
Paula Aguilera a, Gabriela Briceño a, Maribel Candia a, Maria de la Luz Mora a,
Rolando Demanet b, Graciela Palma a
a
Departamento de Ciencias Químicas, Universidad de La Frontera, Casilla 54-D, Temuco, Chile
b
Departamento de Producción Agropecuaria, Universidad de La Frontera, Casilla 54-D,
Temuco, Chile. paguilera@ufro.cl
In the south of Chile, there are edaphoclimatics conditions suitable for the production of milk
and meat, with 75% of the cattle located in this region. The milk production systems have been
intensified in the last few years, increasing the amount of liquid cow manure (LCM) effluents that
are stored in open air lagoons for their application on grasslands or fodder corn crops. The large
volume of LCM accumulated in a reduced storage capacity provokes frequent and higher application
rates to the field per year reaching an amount up to 300,000 L ha-1.
The fodder maize (Zea mayz L.) is cropped on Andisol, characterized with high organic carbon
content and acidic pH. The maize management includes the application of LCM and atrazine as pre-
plant herbicide. Specifically, for the corn crop, the LCM is applied from August to October, before
planting the corn, and its end total amount of application is highly variable. Another associated
problem with LCM application is there is no clear indication on the amount of time needed between
the last applications of LCM and herbicide application. As a consequence, inefficient weed control
can usually be observed in the field. At present the use of the animal and agricultural residues in
soils is a common agronomical practice throughout the world to improve the physico-chemical and
biological soil properties. However, the application of amendments may modify these properties
strongly, because of their content and quality of organic matter (OM) and pools of microorganisms,
affecting the efficacy and fate of pesticides.
The application rate of LCM in the field and the stabilization time of amended soils before
application of pre-plant herbicides are factors that determine their efficiency.
This study includes evaluation of residual atrazine (2-chloro-4-ethylamino-6-isopropylamino-
1,3,5-triazine) in soil and amended soils with equivalent rate of 100,000; 200,000; and 300,000 L ha-
1
of LCM and the effect of pre-incubation time of amended soils on atrazine degradation. The study
was carried out under controlled conditions using an Andisol with previous historical application of
atrazine. The respiratory activity and fluorescein diacetate (FDA) studies indicated that the time
necessary for stabilization of amended soils is over 20-30 d. During the measurement of respiratory
and FDA activity, no significant differences were observed when atrazine was applied. The half-life
of atrazine ranged from 5 to 8 d and the relative distribution of degradation products seem to be
affected by the application of LCM. The pre-incubation time of amended soil and LCM dose would
not affect atrazine degradation rate, when the soil has a history of herbicide application.
However, repeated applications of LCM in a long period of time could change the soil pH and
increase the content of dissolved organic carbon DOC which could further contribute to a faster
degradation of atrazine. Both effects would reduce the effectiveness of atrazine in weed control.
This study was financed by the project FONDECYT 1070568.
158
Simposio 6.
Valorización de Residuos, Bioenergía y Energías Renovables
Presentaciones Orales
Moderador: Dr. Rodrigo Navia
1. Dr. Rodrigo Navia. Universidad de La Frontera, Chile: “Valorización de

Residuos, Bioenergía y Energías Renovables”.
2. Dr. Gustavo Ciudad. Universidad de La Frontera, Chile: “Avances en procesos

biotecnológicos para la producción de biodiesel”.
3. Dr. Claudio Toro. Universidad de La Frontera, Chile: “Glicerol como materia

prima para la elaboración de poliésteres y su uso en la refinación de
biodiesel”.
4. Dra. Mariella Rivas. Universidad de Antofagasta, Chile: “Microalgas como

fuente de energía renovable”.
5. Dr. David Jeison. Universidad de La Frontera, Chile: “Biogás como fuente de

energía renovable”.
6. Dr. Renato Hunter. Universidad de La Frontera, Chile: “Turbinas eólicas de

baja potencia: una apuesta para apoyar la independencia energética en
sectores productivos aislados”.
159
160
WASTE TO ENERGY, BIOENERGY & RENEWABLE ENERGY

Rodrigo Navia
Department of Chemical Engineering & Scientific and Technological Bioresources Nucleus,

University of La Frontera, Temuco, Chile
The use of waste and biomass as renewable energy source is a must in a society which intends to be
sustainable, environmentally friendly and efficient. Municipal and industrial wastes are an important
source of energy in developed countries, as well as agricultural and forestry biomass.
The Araucanía Region of Chile consumes about 1600 GWh per year electricity, but generates
only 582 GWh per year, indicating that the Region needs to consume a high energy percentage from
other regions of the Country. This issue is of particular relevance as the energetic potential of the
Region is much higher than its consumption.
The hydraulic potential (only considering less than 20 MW installations) for electricity
generation is about 279 MW, power that corresponds to about two times the current total installed
electricity power of the Region.
Regarding municipal solid waste, the energetic potential of Temuco city considering landfilling
is 1 MW, while gasification or incineration of municipal solid waste may achieve at least 3 MW
only in the city of Temuco. In addition, the Region has an electrical power potential of about 6 MW
only considering organic cattle slurry.
The energetic generation potential from agricultural residues in the Araucanía Region ranges
between 110 and 231 MW. These figures are mainly considering residual straw, and residual
biomass from cereals harvesting and processing. In addition, the use of forestry residual biomass for
electricity production may achieve a range between 120 and 450 MW.
Moreover, waste and biomass are also suitable for the production of solid, liquid and gaseous
biofuels, applying suitable technologies for the generation of biochar and pellets (solid), biomass to
liquid fuel (BTL), bio-oil, biodiesel and bioethanol (liquid), as well as biogas and synthesis gas.
Concluding, the Araucanía Region has a high potential for a sustainable long term development,
considering environmentally and efficient sound technologies for energy and biofuels production.
161
ADVANCES IN BIOTECHNOLOGY PROCESS TO PRODUCE BIODIESEL

Ciudad, G1., Azócar, L1., Muñoz, R1., Reyes., I1., Wick, Y. L2 Heipieper, H2., and Navia, R1,3.
Scientific and Technological Bio-resource Nucleus, Universidad de La Frontera, Casilla 54-D,

Temuco, Chile1. Department of Environmental Biotechnology, Centre for Environmental Research -
UFZ, Permoserstr., 15, 04318 Leipzig, Germany2. Departamento de Ingeniería Química,
Universidad de La Frontera, Casilla 54-D, Temuco, Chile3. *Corresponding author: Dr.
gciudad@ufro.cl
Abstract
The use of conventional alkaline catalyzed processes to produce biodiesel from alternatives raw
materials such as waste frying oil (WFO) and algae oil, has several drawbacks, due their high free
fatty acid (FFA) content. Lipase catalyzed processes avoid these problem by direct esterification of
FFA to biodiesel. In this work is reviewed the use of Novozym 435 (immobilized lipase) and whole
cell catalyst (lipase producing microorganism) as effectives alternatives to produce biodiesel,
including the use of purification process by membrane, valorization of glycerol as by-product and
the development of a novel three phase bioreactor whole cell.
Acknowledgments
This work was sponsored by FONDECYT Projects 3080021 and the Chilean PBCT-CONICYT
project TPI-16.
162
GLYCEROL AS RAW MATERIAL FOR POLYESTER MANUFACTURE AND THEIR

USE IN BIODIESEL REFINING
Toro C.a,b, Hidalgo P.a,b, Sanhueza S.a,b, Catril C.a,b, Navia R.a,b
a
Scientific and Technological Bioresource Nucleus, University of La Frontera, Temuco, Chile
b
Department of Chemical Engineering, University of La Frontera, Temuco, Chile. E-mail:
ctoro@ufro.cl
Transesterification of triglycerides by means of alcohol in the presence of a suitable catalyst

produces biodiesel and glycerol as by-product. The transesterification reaction can be catalyzed by
both alkalis and acids catalysts. Basic catalysis is the most common, since the process is faster and
the reaction conditions are moderated; however, this type of catalysts produces soap of sodium in
the course of the reaction. After the transesterification has been completed both the soap and the not
reacted catalyst are concentrated on the glycerol phase, nevertheless, a small quantity of these
compounds stay in the biodiesel phase. These components should be removed because the content of
ashy and possible emissions of particulated matter could increase in the biodiesel operated engines.
In addition, the sodium coming from the not reacted catalyst, depending on their concentration, it
can originate serious troubles, such as corrosion of pieces in motors and deactivation of catalytic
converters, therefore environmental damages. Additionally, these components should be removed in
order to fulfill strict standards for commercial biodiesel (ASTM D6751 and/or EN 14214).
Nowadays, two main methods exist for biodiesel purification: wet and dry washing. However,
water in the system generates time and production costs increased, besides highly polluting waste
water. Dry washing replace water with an adsorbent material (clay, carbon, magnesium silicate,
ionic exchange resin, silicone based adsorbent, etc.).
The global objective of this investigation has been to develop polyester starting from glycerol, a
by-product of biodiesel production process, an adipic acid, to use them as an adsorbent material in
biodiesel purification. This hypothesis is based on evidence of the presence of a huge number of
carboxylic and hydroxide functional groups on the structure of these materials, whose number
depending on the glycerol/adipic acid ratio in the initial reaction. These groups can be able to retain
and/or exchange soaps groups and not reacted catalyst existing in biodiesel by both ion exchange
and polarity mechanisms. In addition, due to gel type structure of the polyesters are able to swelling
in a solvent dispersed through, and then an adsorption exchange and catalytic activity is possible.
Polyesters have been manufactured starting from glycerol and adipic acid, by means of the
policondensation reaction. In this work three types of polyesters were manufactured, for which, the
molar ratio glycerol/adipic acid (G/Ad) was varied to obtain polymers with different structural
characteristics (POLA I: 1/1; POLA II: 1/1.5; POLA III: 1/2). To determinate Na cations from soap
and catalyst in both purified and non purified biodiesel atomic absorption spectroscopy was used.
Besides polyesters was characterized by means light microscopy, specific surface area (SBET), pore
volume, pore size distribution, and FTIR analysis. By means structural analysis it was demonstrated
the adsorption capacity of the polymers is mainly due to its gel type structure, by means which the
polymer is able to swell in presence of a solvent and to present adsorption capacity. Finally by
atomic absorption was demonstrated the polyesters manufactured starting from glycerol have a
removal capacity of soaps and not reacted catalyst similar to traditional adsorbents, being POLA I
the one that reached better results.
This work was supported by CONICYT-Chile, through FONDECYT Nº: 3090072 and Nº:
1090382.
163
MICROALGAE BIOFUELS
Rivas M.*, Bahamondes C., Bazaes J., González L., Marticorena P., Sepúlveda C., Silva F.,
Varas R., Vargas P., Riquelme C.
Laboratorio de Ecología Microbiana, Facultad de Recursos del Mar, Universidad de Antofagasta.

*E-mail: mrivas@uantof.cl
The idea of produce biodiesel from any biomass emerged during the beginning of the crisis
petroleum due to the uncertainty of the non-renewable resources in the future, plus the increase
petroleum prices. The world’s primary energy needs in the Reference Scenario are projected to grow
by 55% between 2005 and 2030, at an annual rate average of 1.8% per year. Demand reaches 17.7
billion tonnes of oil equivalent, compared with 11.4 billion tons in 2005. Fossil fuels remain the
dominant source of primary energy, accounting for 84% of the overall increase in demand between
2005 and 2030 (World Energy Outlook. 2007).
The search and study of new renewable energy sources arise with the objective of minimizing
the current oil problem and the problems inherent in their production and consumption. In this sense,
the production of biodiesel from microalgae has great advantages as feedstock over other types of
energy production: it is an activity of a low energy demand which does not require fertile soil and
water of quality, is possible to be used waters of secondary treatments (STS) for its culture,
therefore does not compete with other agrarian activities. Microalgae are an alternative for the
biodiesel production; however, it has to be considered that the use of these microorganisms implies
the search and handling of the optimal parameters for biomass production under controlled
conditions which are also economically sustainable (Patil et al., 2008; Reijnders, 2009).Microalgae
have been the subject of studies about the use of microalgae to generate bio-energy as biodiesel,
biomethane and biohydrogen, or combined uses for the production of biofuels and CO2 mitigation
(Scragg et al., 2003, Miao et al., 2004 ; Miao and Wu 2004, Kruse et al., 2005; Tsukahara and
Sawayama 2005, Rupprecht et al., 2006, Xu et al., 2006; Chisti 2007; Hankammer et al., 2007,
Huntley and Redalje 2007, Li et al., 2007, Ono and Cuello 2007).
In present time certain microalgae are the research base for the production of biodiesel; this is
the case of Botryococcus braunii microalgae that is studied in our university. B. braunii is a colonial
green alga, of slow growing and it`s found in freshwater lakes, ponds and brackish waters, it is
recognized as a powerful renewable resource for production of liquid hydrocarbons (Metzger and
Largeau, 2005). In our country, described the presence of B. braunii in Chungara Lake, where in
some periods was the dominant microalgae biomass with the highest percentage (Dorador et al.,
2003). B. braunii produce a substantial quantity of liquids hydrocarbons and it is classified in 3
races A, B and L, depending on the produced hydrocarbons (Metzger et al., 1985). The race A
synthesizes long chain hydrocarbons with an odd number carbons between C25 to C33, mainly n-
alkadenos and alkatrienos (Metzger et al., 1985; Metzger et al., 1991). The hydrocarbons content in
the different strains from A vary between 15 to 61 wt.% (Valderrama and Téllez-Sosa, 2004). The B
race produce 2 types of triterpenoidshydrocarbons, through the isoprenoids pathway,
botryococcenes (CnH2n-10, n=30-37) as the major component and small quantities of methylated
squalenes (Metzger et al., 1985), this race is a potential source of renewable fuel and/or valuable
chemicals and so the quantity of produced triterpens can be amongst 10 to 86% of the algal dry-
weight (Brown et al., 1969). The race L produces lycopadiene, which is an acyclic triterpenoid of 40
carbons, with only between 2 to 8% wt.% of the total composition (Metzger et al., 1990;
Valderrama and Téllez-Sosa, 2004).
164
TURBINAS EÓLICAS DE BAJA POTENCIA: UNA APUESTA PARA APOYAR LA

INDEPENDENCIA ENERGÉTICA EN SECTORES PRODUCTIVOS AISLADOS
*Dr. Ing. R. Hunter A., +Mg. Ing. B. Pavez L.
*Departamento de Ingeniería Mecánica

+Departamento de Ingeniería Eléctrica
Hoy en día la generación de electricidad con energía eólica ha tomado un papel fundamental en
países desarrollados. Este hecho queda patente en España, tercer país con la mayor capacidad eólica
instalada en el mundo, ya que hace un par de semanas la generación de energía eléctrica con energía
eólica alcanzo el 53% del total de la energía generada en el país. En Chile, la energía eólica ha
experimentado un crecimiento acelerado en los últimos años, pasando de 2MW a más de 168 MW
de capacidad instalada, contemplando la puesta en macha de Canela II, El Totoral y Monte
Redondo, a fines de este año. Sin embargo, para el próximo año no se prevé la incorporación de
nuevas centrales eólicas al Sistema Interconectado Central (SIC).
Por otro lado, cabe destacar que un campo poco explorado es el aprovechamiento de pequeñas
fuentes de energía eólica con turbinas de baja potencia. La utilización de este tipo de turbinas
permitirá aprovechar recursos eólicos que no pueden ser explotados por grandes parques eólicos,
dada la gran cantidad de superficie de terreno a utilizar en la instalación de las turbinas, aportando
una solución sustentable para la mejora de la calidad de vida de los habitantes de zonas rurales y a la
vez aumentando la competitividad del sector frutícola y ganadero de la macro zona sur.
165
166
Simposio 6.
Valorización de Residuos, Bioenergía y Energías Renovables
Presentación de Paneles
167
168
IMPROVING FATTY ACID METHYL ESTERS PRODUCTION YIELD IN A LIPASE-

CATALYZED PROCESS BY USING WASTE FRYING OILS AS FEEDSTOCK
Azócar, L1., Ciudad, G1., Heipieper, H2., Muñoz, R1 and Navia, R1,3.
Scientific and Technological Bio-resource Nucleus, Universidad de La Frontera, Casilla 54-D,

Temuco, Chile1.
Department of Environmental Biotechnology, Centre for Environmental Research - UFZ,
Permoserstr., 15, 04318 Leipzig, Germany2.
Departamento de Ingeniería Química, Universidad de La Frontera, Casilla 54-D, Temuco, Chile3.
*Corresponding author: Ph.D. student lazocar@ufro.cl
The application of waste frying oil (WFO) mixed with rapeseed oil as feedstock for an effective
production of fatty acid methyl ester (FAME) in a lipase-catalyzed process was investigated.
Response surface methodology (RSM) was used to optimize the interaction of four variables: The
percentage of WFO in the mixed feedstock, the methanol to oil ratio, the dosage of Novozym 435 as
catalyst, and the temperature. Furthermore, the addition of methanol to the reaction mixture in a
second step after 8 hours of reaction was shown to be an effective technique to diminish enzyme
inhibition. Using this technique, the model predicted optimal conditions to reach 100% FAME at
methanol to oil molar ratio of 3.8:1, 100 wt% WFO, 15 wt% Novozym 435 and 44.5°C at 12 hours
of reaction time and 200 rpm, and further verification experiments confirmed the validity of the
model. According to the model, the addition of WFO increased FAME production yield. Major
reason for this positive effect of WFO was its higher monoacylglycerols, diacylglycerols and free
fatty acids content (in comparison to rapeseed oil) which seem to be more available substrates for
the enzymatic catalysis. Therefore, the replacement of rapeseed oil by WFO in Novozym 435
catalyzed processes could diminish biodiesel production costs by using a less expensive feedstock;
increases the production yield and could be a potential alternative to FAME production at industrial
scale.
169
PETROLEUM COKE FLY ASHES AND THEIR POTENTIAL REUSE

González A1,2., Moreno N3. & Navia R2,4
1
Ph.D. Program in Sciences of Natural Resources, University of La Frontera, P.O. Box 54-D,
Temuco, Chile. 2Scientific & Technological Nucleus in Bioresources, University of La Frontera,
P.O. Box 54-D, Temuco, Chile. 3Department of Geosciences, IDAEA-CSIC, C/ Jordi Girona, 18-26,
E-08034 Barcelona, Spain. 4Department of Chemical Engineering, University of La Frontera, P.O.
Box 54-D, Temuco, Chile. Corresponding mail: aixa@ufro.cl
Acid wastewaters may cause severe impacts in the environment by acidifying and discharging
large amounts of salts and heavy metals into aquifers. Among the remediation options, the most
used and cost-effective is the addition of neutralizing agents. Nevertheless, their use implies a high
environmental and economic cost, due to the use of natural resources as raw materials. Several
studies have proposed the direct addition of alkaline secondary raw materials such as petroleum
coke fly ashes (PCFA), which are a by-product from petroleum coke circulated fluidized bed
combustion (CFBC). This work deals with the characterization of a Chilean CFCB-PCFA and its
potential use in neutralization and Cu2+ removal from acid wastewaters.
CFBC-PCFA characterization included determination of loss on ignition, grain size distribution,
specific surface area, major and trace elements content and mineral phases. Cu2+ removal kinetics
test was performed at 25°C, 100 rpm, Cu2+solution of 100 mg/L at pH 4 and at different liquid/solid
ratios. Residual Cu2+ was analyzed by means of AAS. Neutralizations batch tests were performed by
the addition of 1 L distilled water with different doses of CFBC-PCFA, ranging between 0.2 and 3
g/L. Supernatant was collected and analysed for S, Ca, Ni, V and Mo concentrations by means of
ICP-MS and ICP-AES. Additionally the final pH value and electric conductivity were also
determined.CFBC-PCFA has a heterogeneous morphology with presence of unburned carbon,
calcite and anhydrite particles, corroborating the main crystalline minerals detected by means of
XRD. It has been also detected smaller particles containing Ca-V-S-O bearing minerals. The acid
digestion show that CaO (44%), SO3 (24%) and traces such as V and Ni (0.6%) are the main
elements in CFBC-PCFA, accompanying with high content of unburned carbon (LOI=30%).
The dosages applied showed Cu2+ removal efficiencies between 90 and 99%. Using the 0.2 g/L
dose the equilibrium was reached at 4 h, detecting two different stages (1) between 0 and 8 min,
visually detecting flock formation in the solution and (2) starting on 16 min until 20 h, observing
flock disappearance. CFBC-PCFA arising from removal kinetics before 8 min and at 20 h showed
the formation of a crystalline mineral phase of posnjakite (Cu4SO4(OH)6H2O). The differences
between both CFBC-PCFA are the crystallization degree and morphology, concluding that during
Cu2+ removal, the main involved mechanism could be precipitation.
During neutralization test was observed an increase of pH with increasing CFBC-PCFA doses,
due to the dissolution of water soluble minerals such as portlandite and anhydrite in acid condition.
However their dissolution did not neutralize the added H+ (0.2-2.2 g/L). Starting from the 2.6 g/L
CFBC-PCFA dose, pH increased sharply from 2.6 to 6.7. Levels of V in leachates show a similar
behavior as calcium concentrations, indicating that the V associations may be V-Ca-O and Ca-V-
SO42-.
It is proposed the reuse of CFBC-PCFA in the neutralization of acidic waste water (for the
consequent heavy metals removal) replacing raw alkaline materials. Removal kinetics shows high
removal efficiencies for Cu2+, being the possible main mechanisms precipitation and pH increase
was observed during neutralization at pH 2.
170
EVALUACIÓN DE LA ACTIVIDAD METANOGÉNICA EN LODOS ANAEROBIOS POR

MÉTODO MANOMÉTRICO
Valdés G., Freddy1; Jeison N., David2
1
Scientific and Technological Bioresource Nucleus, Doctorado en Ciencias de Recursos Naturales,
Universidad de La Frontera. freddyvaldesg@gmail.com. 2Departamento de Ingeniería Química,
Universidad de La Frontera.
Antecedentes Generales
Diversas actividades productivas generan residuos en diversas formas. Estos desechos deben
tratarse y/o disponerse de manera tal que su impacto ambiental sea lo menos dañino posible, e
idealmente darles un uso alternativo que los revalorice.
La digestión anaerobia es un sistema de tratamiento de residuos orgánicos que presenta

favorables características, como son: requerir un bajo consumo energía para operar, poseer la
capacidad de tratar altas cargas orgánicas, generar una reducida cantidad de lodos, producir biogás,
entre otras.
Como producto de este proceso se obtiene más microorganismo y biogás (una mezcla de CH4,
CO2, vapor de agua y traza de otros gases), que tiene propiedades de combustible. La utilización
energética del biogás presupone el conocimiento de la cantidad de metano a generarse a partir de un
sustrato determinado. Tal valor puede determinarse teóricamente realizando un balance de demanda
química de oxígeno (DQO) o experimentalmente por medio de la determinación de potencial
metanogénico.
La determinación analítica del potencial metanogénico, se realiza generalmente mediante un

método gasométrico. La forma clásica de determinar la generación de biogás es por medio del
desplazamiento de líquido, sin embargo, también existen métodos basados en el incremento de la
presión del espacio de cabeza de micro-reactores. Este ofrece ventajas relacionadas con la
posibilidad de automatizar la medición, permitiendo adquisición de datos en línea. Sin embargo
existen una serie de condiciones de medición que deben ser definidas de manera de poder generar
datos confiables y comparables. El presente trabajo pretende avanzar de manera significativa en tal
dirección.
Objetivo General:
Implementar un modelo de ensayo para la determinación precisa y reproducible del potencial
metanogénico de muestras de residuos por medio del monitoreo en línea de la presión del espacio de
cabeza.
Objetivos Específicos:
Comparar la reproducibilidad y precisión del método desarrollado con aquél presentado por métodos
tradicionales.
171
172
CONFERENCIA DE CLAUSURA/CLOSING LECTURE
UTILIZATION OF COAL COMBUSTION PRODUCTS (CCPS) FOR ENVIRONMENTAL

REMEDIATION
Nanthi Bolan , Ravi NaiduA,B and Balaji SeshadriA, B
A,B
A
CRC-Contamination Assessment and Remediation of Environment (CRC CARE);
Nanthi.Bolan@unisa.edu.au
B
CERAR, University of South Australia, Mawson Lakes, SA-5095
Coal combustion products (CCPs)

The coal combustion products are grouped into four broad categories that include fly ash, boiler
slag, and bottom ash and flue gas desulphurisation (FGD) materials. Fly ash produced from the
burning of coal has become a generic term for all types of CCPs. Specifically, fly ash refers to the
portion of the ash stream composed of particles small enough (1.0 – 100µm) to be carried from the
boiler in the flue gas. These particles are either mechanically/ electrostatically captured or emitted
via the stack. Bottom ash and boiler slag are residues found in the furnace after burning either coal
alone or in the presence of other amendments (e.g. lime to trap sulphur oxide) and are common to all
types of coal combustion process. With the introduction of Clean Air Act in many countries, a
number of new types of value-added products are being produced depending upon the type of the
coal combustion process and the methodology used to reduce gaseous emission such as sulphur
oxide and nitrous oxide. These include wet and dry FGD products, and dry fluidised bed
combustion (FBC) products.
FGD products result from the post-combustion treatment of the flue gas in the coal fired power
stations with a dry or wet alkaline absorbent (usually a liming material) aimed at reducing the
gaseous emission of sulphur and nitrogen. The simultaneous combustion of coal and an alkaline
liming material in the FBC process results in end products that are very different from those
produced from the combustion of coal alone. FGD and FBC products invariably contain calcium
sulphate resulting from the reaction of sulphur oxide with the liming material and some residual
unreacted liming material.
Beneficial use of CCPs

Approximately 80% of CCPs are treated, stored, or disposed of by means of land management, and
the remaining 20% are recycled. Land management involves the use of surface impoundments,
landfills, mines, and quarries. The overall cost incurred in the management of CCPs in USA ranges
from $2.20 to $34.14 per ton, depending on the type and size of the waste facility and the
characteristics of CCPs. This cost is expected to rise steadily mainly because of the environmental
regulations of waste disposal and the limited availability of landfill sites. Hence there has been
increasing efforts in the safe and sustainable management of CCPs through recycling.
CCPs are used extensively in the agricultural and construction industries. In the agricultural
industries they are used primarily as an amendment to improve the physical and chemical properties
of soil, as a source of liming material to ameliorate sub-soil and surface soil acidity and as a nutrient
173
source to supply calcium and sulphur. In the construction industry they are used mainly as a source
of concrete, roofing material and road surface sealing.
Environmental remediation using CCPs

Increasingly CCPs are used in the remediation of contaminated environments. Some of these uses
include, control of acid mine drainage, mitigating phosphorus leaching in farm lands and
immobilization of toxic metals in mine sites and agricultural soils. CCPs are generally alkaline in
nature and hence are found suitable for neutralizing acid mine drainage and the reclamation of mine
sites for revegetation and carbon sequestration. Co-composting of CCPs with organic wastes such
as poultry manure has been shown to be very effective in conserving phosphorus in these manure
sources. The high concentration of calcium, iron and aluminium in CCPs enhances the
immobilization of phosphorus in manures and soils. Similarly CCPs are used to immobilize heavy
metals, thereby enhancing their recovery in industrial and domestic wastewater streams, and
minimising their entry to ground water resources.
CCPs contain toxic heavy metals such as mercury (Hg) which may accumulate in soils if excessive
amounts of CCPs are used to ameliorate degraded soils. It is therefore important to understand and
quantify the environmental risk associated with the regular use of CCPs for environmental
remediation and ecosystem development.
174

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Scientific and

Doctorado en Ciencias Technological

AVANCES EN CIENCIA Y TECNOLOGÍA

A los estudiantes del Doctorado en Ciencias de Recursos Naturales de la Universidad de La Frontera

A la Secretaria del Programa de Doctorado en Ciencias de Recursos Naturales de la Universidad de

Dra. Carolina Shene, Univ. Frontera

Dra. Liliana Gianfreda, U. Federico II, Italy

SIMPOSIO 1. AVANCES EN BIOTECNOLOGÍA AGROALIMENTARIA 1

PRODUCCIÓN MICROBIANA DE LÍPIDOS 3

ACTIVIDAD BIOLÓGICA DE COMPUESTOS POLIFENÓLICOS Y SUS APLICACIONES EN LA 6

ADVANCED TECHNOLOGY IN FOOD COLOR: DESCRIPTION OF THE KINETIC 9

TECHNOLOGICAL INNOVATION IN BIOPROCESS 18

INMOVILIZACIÓN DE PAPAÍNA EN ALUMINOSILICATOS NANOTUBULARES SINTÉTICOS 25

SOLVENT EXTRACTION OLIGOSACCHARIDES OF LUPINE FLOUR 26

INCORPORACIÓN DE FLAVONOIDES AGLICONA Y GLICOSILADOS EN UNA MATRIZ 27

MECHANICAL PROPERTIES, CHEMICAL INTERACTIONS AND STRUCTURE OF 28

ZEBRAFISH (Danio rerio) LARVAL REARING: PROGRESS IN ESTABLISHING A MODEL 29

OPTIMIZATION OF AN AQUEOUS EXTRACTION OF MUCILAGE FROM FLAXSEED CAKE 30

SIMPOSIO 2. ECOLOGÍA QUÍMICA COMO HERRAMIENTA BIOTECNOLÓGICA 31

CHEMICAL ECOLOGY: AN INTERDISCIPLINARY DISCIPLINE 33

BIOTECHNOLOGY OF CROP PROTECTION 35

ECOLOGICAL AND EVOLUTIONARY GENOMICS OF HERBIVORE INSECTS 36

INSECTICIDE RESISTANCE IN PEST OF FRUITS IN CHILE 37

PHARMACOLOGICAL AND PHYSIOLOGICAL PROPERTIES OF EXTRACTS AND PURE 38

MÉTODOS PARA DETERMINACIÓN DE CAPACIDAD ANTIOXIDANTE EN PLANTAS 45

BEHAVIOURAL RESPONSES OF CLOVER ROOT BORES (Hylastinus obscurus) 47

EVIDENCE OF SEXUAL PHEROMONE IN THE RASPBERRY WEEVIL Aegorhinus superciliosus 48

RESISTANCE TO PYRETHROIDS IN Haematobia irritans (DIPTERA: MUSCIDAE) AND 49

ELECTROANTENNOGRAPHIC RESPONSE OF CLOVER ROOT BORER Hylastinus obscurus 50

BIOLOGICAL AND CHEMICAL FACTORS ASSOCIATED IN THE INTERACTION BETWEEN 51

ANTIFUNGAL ACTIVITY OF VOLATILES EMITTED BY MICELIAL CULTURES OF 52

ESTUDIO DE COMPUESTOS VOLÁTILES, CAPACIDAD ANTIOXIDANTE Y 54

SIMPOSIO 3. MICROBIOLOGÍA Y RIZOSFERA 55

ARBUSCULAR MYCORRHIZAE IN AGRICULTURAL AND FOREST ECOSYSTEMS OF 57

ARBUSCULAR MYCORRHIZAL FUNGI AND ITS ROLE IN STABILIZING ECOSYSTEMS 58

PAPEL DE LAS MICORRIZAS ARBUSCULARES EN LA PROTECCIÓN DE LA PLANTA 59

EFECTIVIDAD DE RESIDUOS AGROINDUSTRIALES BIOTRASFORMADOS Y SU 60

MYCORRHIZAS OF TEMPERATE FORESTS IN SOUTH-CENTRAL CHILE 61

CONSTRUCTION OF VECTORS TO IMPROVE BOTH PHOSPHATE AND PHYTATE 62

PHYTATE-DEGRADING BACTERIA AND THEIR BIOTECHNOLOGICAL POTENTIAL IN 63

THE ECOLOGY OF MICROBIAL COMMUNITIES IN THE PLANT RHIZOSPHERE 64

DETECTION AND QUANTIFICATION OF INDOLE ACETIC ACID IN RHIZOBACTERIA 69

EFFECT OF THE APPLICATION OF NATURAL PRODUCTS PROMOTERS OF THE VEGETAL 70

ABIOTIC STRESS AFFECTING THE MYCORRHIZAL OF FOREST ECOSYSTEMS FROM 72

NOVEL BACTERIA AND MICROBIAL COMMUNITIES INVOLVED IN SIMAZINE 73

ISOLATION AND SCREENING OF POTENTIAL PLANT GROWTH PROMOTING 74

DIFFERENT PATTERNS OF LMWOA EXUDATION IN METALLOPHYTE AND 75

TWO PHYTASE PRODUCING BACTERIA WITH BIOTECHNOLOGICAL POTENTIAL FOR 76

SCREENING AND ISOLATION OF PHOSPHATE SOLUBILIZING AND MINERALIZING 77

MICROBIAL BIOFERTILIZERS: PREPARATION OF MYCORRHIZAL INOCULANTS ON 78

CHANGES IN THE DENSITY OF MYCORRHIZAL FUNGI PROPAGULES ASSOCIATED TO 79

SIMPOSIO 4. SUELOS, FISIOLOGÍA Y NUTRICIÓN VEGETAL 81

SOIL-PLANT INTERACTIONS IN VOLCANIC SOILS 83

ABIOTIC STRESS EFFECTS ON PHYSIOLOGICAL AND BIOCHEMICAL TRAITS IN 86

CARBOXYLATION ACTIVITY OF RUBISCO IN Arabidopsis thaliana UNDER CD AND CU 89

FACTORS AFFECTING THE MOBILITY AND BIOAVAILABILITY OF (MICRO) NUTRIENTS, 93

MULTICOMPONENT ADSORPTION OF CU AND ZN ON ALLOPHANIC CLAY 99

NUTRIENT DYNAMIC IN RESPONSE TO NITROGEN AND PHOSPHOROUS FERTILIZATION 100

ANTIOXIDANT ACTIVITY AND TOTAL PHENOLIC AND ANTHOCYANINS CONTENTS IN 101

EFFECT OF NITROGEN FERTILIZATION ON SOIL PHOSPHOROUS DYNAMICS AND 102

IDENTIFICACIÓN DE ARCILLAS EN SUELOS DE LA REGIÓN DE ANTOFAGASTA Y SU 103

IRRIGACIÓN DE UN SUELO SALINO CON AGUA RESIDUAL PROVENIENTE DE LA 104

DISPONIBILIDAD Y MOVILIDAD DE NITRÓGENO EN UN ANDISOL Y SU INFLUENCIA 105