World J Microbiol Biotechnol (2012) 28:22492256 DOI 10.

1007/s11274-012-1032-3

ORIGINAL PAPER

Extracellular enzymes produced by microorganisms isolated from maritime Antarctica

Lyliam Loperena Veronica Soria Hermosinda Varela Sandra Lupo Alejandro Bergalli Mairan Guigou Andres Pellegrino Angela Bernardo Ana Calvino Federico Rivas Silvia Batista

Received: 18 September 2011 / Accepted: 25 February 2012 / Published online: 14 March 2012 Springer Science+Business Media B.V. 2012

Abstract Antarctic environments can sustain a great diversity of well-adapted microorganisms known as psychrophiles or psychrotrophs. The potential of these microorganisms as a resource of enzymes able to maintain their activity and stability at low temperature for technological applications has stimulated interest in exploration and isolation of microbes from this extreme environment. Enzymes produced by these organisms have a considerable potential for technological applications because they are known to have higher enzymatic activities at lower temperatures than their mesophilic and thermophilic counterparts. A total of 518 Antarctic microorganisms, were isolated during Antarctic expeditions organized by the In stituto Antartico Uruguayo. Samples of particules suspended in air, ice, sea and freshwater, soil, sediment, bird and marine animal faeces, dead animals, algae, plants, rocks and microbial mats were collected from different sites in maritime Antarctica. We report enzymatic activities

present in 161 microorganisms (120 bacteria, 31 yeasts and 10 lamentous fungi) isolated from these locations. Enzymatic performance was evaluated at 4 and 20C. Most of yeasts and bacteria grew better at 20C than at 4C, however the opposite was observed with the fungi. Amylase, lipase and protease activities were frequently found in bacterial strains. Yeasts and fungal isolates typically exhibited lipase, celullase and gelatinase activities. Bacterial isolates with highest enzymatic activities were identied by 16S rDNA sequence analysis as Pseudomonas spp., Psychrobacter sp., Arthrobacter spp., Bacillus sp. and Carnobacterium sp. Yeasts and fungal strains, with multiple enzymatic activities, belonged to Cryptococcus victoriae, Trichosporon pullulans and Geomyces pannorum. Keywords Maritime Antarctica Psychrophile microorganism Psychrotroph microorganism Enzymatic activity

Electronic supplementary material The online version of this article (doi:10.1007/s11274-012-1032-3) contains supplementary material, which is available to authorized users.
L. Loperena (&) V. Soria H. Varela A. Bergalli M. Guigou A. Pellegrino A. Bernardo A. Calvino F. Rivas Departamento de Bioingeniera, Facultad de Ingeniera, Instituto de Ingeniera Qumica, Julio Herrera y Reissig 565, 11300 Montevideo, Uruguay e-mail: lilianl@ng.edu.uy S. Lupo Laboratorio de Micologa, Facultad de Ciencias, Instituto de Biologa, UdelaR, Igua 4225, 11400 Montevideo, Uruguay S. Batista Unidad Microbiologa Molecular, Instituto de Investigaciones Biologicas Clemente Estable (MEC), Avenida Italia 3318, 1600 Montevideo, Uruguay

Introduction Developments in genetics and microbial physiology have had a profound impact on enzyme production technologies and the search for new organisms with unusual activities remains an important area in process engineering and biotechnology. Enzymes from extremophilic microorganisms offer versatile tools for sustainable development in a variety of industrial applications as they show important environmental benets due to their biodegradability, specic stability under extreme conditions, improved use of raw materials and decreased amounts of waste products (Brenchley 1996; Antranikian et al. 2005; Margesin et al. 2005; Tosi et al. 2010). The classication in facultative and obligate microorganisms has been established according to their growth

123

2250

World J Microbiol Biotechnol (2012) 28:22492256

temperature requirements. Those organisms able to grow only in a narrow range of low temperature (stenothermal) are usually designated obligate psychrophiles. Another group, facultative psychrophiles or psychrotrophs, are able to grow at wider range of temperatures (eurythermal). Psychrotrophs are thought to be more abundant than obligate psychrophiles in cold environments like maritime Antarctica, possibly because they have developed the ability to tolerate large variations in temperature. Several psychrophilic or psychrotrophic bacteria from Antarctica were previously identied as belonging to the genera Pseudoalteromonas, Moraxella, Psychrobacter, Polaromonas, Psychroexus, Polaribacter, Moritella, Vibrio, Arthrobacter, Bacillus and Micrococcus. Methanogenium, Methanococcoides and Halorubrum were also identied in Antarctica as psychrophilic archaea. Maritime Antarctica has a rich mycoora including fungi of the genera Candida, Cryptococcus, Geomyces, Penicillium, Mortierella, Cladosporium, Thelebolus, Phoma among others (Feller and Gerday 2003; Ruisi et al. 2007). The mechanisms used by these microorganisms to grow at low temperatures are associated with diverse cellular processes. Microorganisms isolated from low temperature environments typically have increased proportions of unsaturated fatty acids, particularly PUFAs, methyl branched fatty acids, polar carotenoids and the ratio of anteiso- to iso-branched fatty acids, as well as a decrease in the average chain length of fatty acids and in the ratio of sterols to phospholipids. These alterations in the relative composition in these microbes are thought to be necessary for maintaince of membrane uidity at lower temperatures (Margesin and Miteva 2011; Fogliano et al. 2010). The expression of coldadapted enzymes has been also observed. These enzymes can exhibit ten times higher activity at low temperatures compared to their mesophilic counterparts (Aghajari et al. 1996; Feller and Gerday 2003; Margesin et al. 2005). This study describes screening of Antarctic microorganisms for the ability to produce extracellular enzymes for specic applications in industry and is an attempt to contribute with the research on extremophilic organisms as a source of cold active enzymes for industrial use. Bacteria, yeasts and lamentous fungi isolated were tested for proteases, amylases, pectinases, xylanases, cellulases, lipases and ligninases.

Instituto Antartico Uruguayo in December 2006 and February 2007. One hundred seventy-four samples were collected at different sites on the Fildes peninsula, King George Island (KGI) (62020 S, 58210 W), Deception Island (62580 S, 60390 W, Port Lockroy (64490 S, 63300 W, Cuverville Island (64410 S, 62380 W) and Cape Kaiser (64140 S, 62000 W). Samples of air with suspended material collected on plates with solid media sea and freshwater ice, soil, sediment, bird and marine animal faecal sediments, dead animals, algae, plants, rocks and microbial mats were aseptically collected. Aliquots of 0.2 ml of seawater, freshwater and melted ice were streaked directly on plates with Marine Agar medium or Tryptone Soy Agar (TSA). Each solid sample (ca. 10 g) was suspended in sterile water or Tryptone Soy Broth and incubated at 4C. Aliquots of the suspension were spread on the surface of TSA and Malt Yeast extract agar plates. The plates were incubated at 4C and the bacterial and fungal colonies with distinct morphologies were repeatedly isolated until pure.

Enzymatic activity The production of extracellular enzymes was determined using a diffusion method involving colonies grown on solid media with a specic substrate. Each isolate was inoculated and tested at 4 and 20C, assays were performed by duplicate. Zones of clearing around the colonies were used as an indication of enzymatic activities and measured in mm as the difference between the diameter of the halo and the colony. In some cultures, the halos could be measured daily. If the addition of a chemical compound was required, the plates were incubated for at least a week before the incorporation of the developing agent. The production of extracellular proteases was determined using two culture media, one with casein (Wang et al. 2007) and the other with gelatin (Seeley et al. 1991). Measures of extracellular amylases, xylanases and cellulases were done incorporating, respectively, starch, xylan or carboxymethylcellulose into the growth medium (Seeley et al. 1991; Paterson and Bridge 1994; Gonzales et al. 2004; Villalba et al. 2004; Rivas et al. 2007). Pectinolytic activity was analyzed including pectin in the growth media prepared at two different pHs because exopectinases are usually most active at pH 5.0 and endopectinases at pH 7.0 (Paterson and Bridge 1994; Rodrguez et al. 2007). Tributyrin was included in the medium for the detection of lipolytic activity in bacteria and Tween 80 in the case of yeasts and fungi (Paterson and Bridge 1994). Lignilolitic activity was determined in a medium containing Remazol Brilliant Blue R (Mtui and Nakamura 2004).

Materials and methods Sampling sites and microorganism isolation Samples from which microorganisms isolated were collected during Antarctic expeditions organized by the

123

World J Microbiol Biotechnol (2012) 28:22492256

2251

Isolate identication Nineteen bacterial isolates were identied by sequence analysis of a portion of the 16S rDNA gene. Genomic DNA was extracted from cultures grown on TSA plates for seven days at 4C by a phenolchloroform method (Alippi and Aguilar 1998). PCR amplication was done in 20 ll reaction mixtures containing 10 ll of Fast PCR Master Mix (2X) (Fermentas), 2 ll of each primer solution (10 lM of each universal primer 27F and 1492R), 1 lg of DNA and water up to nal volume. PCR reaction was done in a Palm-Cycler TM (Corbett Research UK Ltd). The amplication conditions were: incubation at 95C for 2 min, followed by 35 cycles of 95C, 15 s, 50C, 30 s, 70C, 1 min 30 s. The reaction included a nal extension at 70C, 5 min. Amplied products of expected size were puried and sequenced at Macrogen Inc. (Korea). DNA sequences obtained from each isolate using primers 27F, 518F and 1492R were aligned by CLUSTALW (Higgins et al. 1994) using MEGA version 5.1 (Tamura et al. 2007). Assembled DNA sequence data was analyzed by BLASTn (Altschul et al. 1990) and compared with non-redundant nucleotide sequence database (nr/nt) at the National Center for Biotechnology Information (NCBI) (http://blast.ncbi. nlm.nih.gov/Blast.cgi). The identication of yeast isolates was done by DNA sequence analysis of the ITS region. DNA extraction was performed from cells growing in Yeast Extract Peptone Dextrose Agar (Lupo et al. 2006). The ITS region was amplied by PCR using the primers ITS4 and ITS5 (White et al. 1990) and reaction conditions were described by Lupo et al. (2006). The amplication product was puried and sequenced by Macrogen, Inc. (Korea). The analysis was done using BLASTn (http://www.ncbi.nlm.nih.gov) and compared with the sequences deposited in the

GenBank database noted above. Filamentous fungal isolates were identied using micro-morphological criteria. Phylogenetic analysis Multiple alignments were generated using CLUSTALW (Higgins et al. 1994). Phylogenetic distance trees were inferred by Maximum-Parsimony (MP, heuristic search factor of 2) and Neighbour-Joining (NJ, p-distance matrix) analyses, using MEGA 5.1 (Tamura et al. 2007). Condence in topologies was assessed using bootstrapping (1,000 replicates).

Results and discussion A total of 518 microorganisms were isolated: 421 bacteria, 46 yeasts and 51 lamentous fungi. The collection of isolates used in this work was deposited in the Culture Collection of Microorganisms of the Bioengineering Department and Mycology Laboratory at the Faculty of Engineering (UdelaR, Uruguay). For screening of enzymatic activities we worked with 120 bacteria, 31 yeasts and 10 lamentous fungal isolates (Table 1). The size of halos and colonies were used to compare enzymatic activities and colony growth. Several investigators have described the sensitivity of this method for semi-quantitative determinations and it has been used successfully for identication of novel activities in envi ronmental isolates of microorganisms (Wickstrom 1983; Leon et al. 2007; Li et al. 2009). The enzymatic activities most frequently found among bacterial isolates were: amylase, caseinase, lipase and gelatinase. Some bacterial clones had ligninase, xylanase and cellulase activities. Yeasts and lamentous fungal isolates more frequently had

Table 1 Percentage of clones with enzymatic activity detected at 4 and 20C Temperature Bacteria 4C Proteolytic activity with casein Proteolytic activity with gelatin Lipolytic activity Amyolytic activity Pectinolytic activity at pH 5.0 Pectinolytic activity at pH 7.0 Cellulolytic activity Xylanolytic activity Lignilolytic activity 33 18 35 16 16 0 1 3 18 20C 36 29 26 51 12 3 1 13 7 Both temp. 31 17 17 8 5 0 1 2 7 Yeasts 4C 3 6 13 3 0 n.d. 6 3 n.d. 20C 3 10 16 3 6 n.d. 10 6 n.d. Both temp. 3 6 10 3 0 n.d. 2 3 n.d. Fungi 4C 0 70 40 0 0 n.d. 50 0 n.d. 20C 0 70 30 0 0 n.d. 40 10 n.d. Both temp. 0 50 30 0 0 n.d. 20 0 n.d.

Total clones analyzed: 120 bacteria, 31 yeasts and 10 fungi n.d. not determined

123

2252

Table 2 Enzymatic activity at 4 and 20C of bacterial isolates

Origin Caseinase 4C Sediment sample collected next to Collins glacier (KGI) Bird remains on the beach in front of Drake sea (KGI) Bird remains on the beach in front of Drake sea Krill remains on the beach in front of Drake sea String sample collected next to Artigas Base (KGI) Sediment sample collected next to Sufeld Point (KGI) 8.5 2 11 0 10 1 14 1 n.a. 10 1 n.a. 15 1 11 1 n.a. 61 n.a. n.a. 26 1 24 0 2.5 1 11 1 n.a. 14 0 14 3 n.a. 34 3 30 0 7.5 1 n.a. n.a. 18 3 26 2 20 2 61 30 2 33 2 20 3 22 3 36 2 32 1 17 2 n.a. n.a. 20 1 36 2 30 0 n.a. n.a. 18 1 3 n.a. n.a. n.a. 22 1 n.a. n.a. 17,5 2 17 1 22 2 n.a. 17 0 21 0 30.5 1 20 0 Ice with algae collected between Drake passage and Artigas Base Microbial mat from the beach in front of Drake sea Grass collected next to Artigas Base Benthic mat from a creek between Collins glacier and Artigas Base Bryophytes collected next to Artigas Base Sediment sample collected next to Sufeld Point Bryophytes collected on KGI Bird remains (KGI) Bryophyte sample (Deception Island) Microbial mat (KGI) Piece of wood collected next to Artigas Base Sediment and lichens collected next to Artigas Base 15 1 n.a. n.a. n.a. 31 60 n.a. n.a. n.a. 23 2 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. 41 n.a. 61 41 31 61 50 91 41 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. 22 2 n.a. n.a. n.a. 16 1 12 3 83 41 5.5 0 71 41 17 1 61 42 n.a. 73 19 2 14 2 81 18 1 30 1 n.d. n.d. n.a. n.a. 91 25 1 28 1 30 1 n.d. n.d. n.a. 42 n.d. n.d. n.a. n.a. n.a. 62 n.a. 82 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. 11 0 23 1 32 2 n.a. n.d. n.d. 52 10 3 n.d n.d. n.d. n.d. n.a. n.a. n.a. 23 2 n.a. n.a. 31 3 21 2 n.a. n.a. n.a. n.a. 31 4 n.a. 20C 4C 20C 4C 20C 4C 20C 4C 20C Gelatinase Lipase Amylase Pectinase at pH 5.0 Temperatures Pectinase at pH 7.0 20C n.d. n.d. n.d. n.a. n.a. n.a. 22 2 n.a. n.a. n.a. 24 2 n.a. n.a. n.a. n.a. n.a. n.a.

123
n.a. n.a. n.a. n.a. 14 2 n.a. n.a. 40 n.a. n.a. n.a.

Clone nameAccession number

Database microorganism with highest similarity (% identity)

c1-HM165451

Antarctic bacterium R25 (99%)

c2-HM165450

Pseudomonas sp. P1 (99%)

c3-HM165449

Pseudomonas sp. KOPRI 25402 (99%)

c4-HM165448

Psychrobacter luti NF11 (99%)

c5-HM165447

Arthrobacter lactophilus KNOUC403 (99%)

c6-HM165446

Pseudomonas sp. Gd3F (99%)

c7-HM165445

Antarctic bacterium GAOF (99%)

c8-HM165452

Pseudomonas sp. tsz07 (99%)

c9-HM165453

Pseudomonas uorescens MS300 (99%)

c10-HM165454

Pseudomonas sp. tsz07 (99%)

c11-HM165455

Pseudomonas uorescens FB25 (99%)

c12-HM165456

Arthrobacter oxydans S32212 (99%)

c13-HM165457

Thermoleophilum minutum YS-4 (99%)

c14-HM165458

Pseudomonas sp. LD126 (99%)

C15-HM165459

Arthrobacter sp. KOPRI 25519 (99%)

C16-HM165460

Pseudomonas sp. AW6 (99%)

C17-HM165461

Arthrobacter psychrochitiniphilus JCM 13874 (99%)

World J Microbiol Biotechnol (2012) 28:22492256

C18-HM165462

Bacillus sp. Nj-19 (99%)

World J Microbiol Biotechnol (2012) 28:22492256

Pectinase at pH 7.0

2253

lipase and cellulase activities. Fungal isolates had little or no detectable pectinase, xylanase or amylase activities. The four bacterial isolates with the highest activity for each of the following enzymes: caseinase, gelatinase, amylase, lipase and pectinase, are shown in Table 2. Isolates with highest caseinase, gelatinase and pectinase activities belonged to the genus Pseudomonas, and were isolated from bird remains, algae, bryophyte and microbial mat samples. The isolates with the highest lipase activity were assigned to Psychrobacter, Pseudomonas and Arthrobacter. These bacteria were recovered from krill remains, sediment and bryophyte samples. The highest amylase activities were found in isolates identied as Arthrobacter. These bacteria were recovered from sediments and pieces of wood. Pseudomonas are well known for their high genetic and physiological diversity and ability to produce extracellular enzymes (Zhang and Zeng 2011). In agreement with this, 9 of the 19 isolates analyzed belonged to this genus and had the highest enzyme activities in ve of the six tests done in this study. We also selected two yeast and two lamentous fungal clones that showed the highest activities for cellulase, lipase, xylanase, caseinase and gelatinase. Yeast clone 80-2, identied as Trichosporon pullulans, was isolated from a creek. This isolate exhibited high caseinase activity at 4C (20 1 mm) and gelatinase activity at 4C and at 20C (18 1 mm and 21 2 mm, respectively). This clone also had high xylanase activity at 20C (25 2.5 mm). Yeast clone 65-2, identied as Cryptococcus victoriae, was isolated from water of Lake Uruguay. This clone had lipase, cellulase and xylanase activities, but activity was found only at 20C (22.5 9, 12 3 and 22.5 1.5 mm respectively). Fungal clones 14-2 and 63-2, identied as Geomyces pannorum, were isolated from decomposing algae collected near Drake Passage and from water of Lake Uruguay. These isolates exhibited high gelatinase activity (20 3.5 and 22.5 2 mm) as well as lipase activity (27.5 2.5 and 24.5 2 mm respectively) at 4C. The potential use of a number of these isolates in industrial microbial fermentation could be evaluated in more detail, Psychrobacter sp. c4, Arthrobacter sp. c15 and the yeast C. victoriae 65-2) could be considered as a source of lipases, and Arthrobacter ssp. c12 and c17 a source of amylases. The production of gelatinase, caseinase and pectinase could also be analyzed using Pseudomonas spp. c2, c8, c10, c11 and c16. T. pullulans 80-2 and Cryptococcus victoria 65-2 both could be a good source of xylanases. In particular, yeast isolate 65-2 exhibited cellulase and xylanase activities, which are of potential use for the pre-treatment of lignocellulose for ethanol production and of forage crops to improve nutritional quality and digestibility, and also for the treatment of water

20C 20C 4C 4C 20C

Pectinase at pH 5.0

Amylase

n.a. Sediment between Artigas Base and Sufeld passage n.a. n.a. n.a. n.a. n.a. n.a.

11 2 Activities were measured as the difference between the diameter of the enzymatic activity and the colony halo in mm; n.d. not determined, n.a. no activity

Lipase

Gelatinase

Temperatures

Caseinase

Origin

4C

20C

4C

20C

4C

20C

n.a.

n.a.

n.a.

The highest activities at 4 or 20C are indicated in bold

Database microorganism with highest similarity (% identity)

Table 2 continued

Clone nameAccession number

C19-HM165463

Carnobacterium sp. Nj-46 (99%)

123

2254 Fig. 1 Rooted phylogenetic tree showing sequences with highest similarity from the GenBank database of the NCBI and the 19 operational taxonomic units that were used in this analysis. The evolutionary history was inferred using the NeighborJoining method. The percentage of replicate trees ([50%) in which the associated taxa clustered together in the bootstrap test is shown next to the branches. Synechococcus sp. SRODG098 was used as an outgroup taxon

World J Microbiol Biotechnol (2012) 28:22492256

Pseudomonas frederiksbergensis strain DSM 13022 Pseudomonas sp. PR3-5 91 Pseudomonas sp. AW6 Pseudomonas sp. W6 c 16 c1 Pseudomonas sp. INK1 68 Antarctic bacterium R25 Pseudomonas congelans SS157 c 13 c 14 81 Thermoleophilum minutum YS-4 Pseudomonas brenneri FB22 Pseudomonas sp. 8H1 Pseudomonas sp. 3(2008b) Pseudomonas sp. LD126 Antarctic bacterium GA0A 65 Antarctic bacterium GA0F c 10 c7 c9 99 Pseudomonas sp. J83 Pseudomonas fluorescens CCM 2115 ATCC13525 Pseudomonas sp. YXE3-18 c8 65 Pseudomonas fluorescens FB25 Antarctic bacterium R-7616 99 Pseudomonas fluorescens MS300 c 11 Pseudomonas sp. tsz07 c2 c3 Pseudomonas sp. KOPRI 25400 99 Pseudomonas sp. KOPRI 25402 Pseudomonas syringae Lz4W 99 64 Pseudomonas sp. a101-18-2 Pseudomonas sp. Nj-59 Pseudomonas sp. NJ-22 Pseudomonas sp. P1(2010) Pseudomonas sp. ArSA 99 69 Pseudomonas sp. BAM288 c6 Pseudomonas sp. Gd3F 85 99 95 Psychrobacter frigidicola type strain: DSM 12411 Arctic seawater bacterium Bsw20370 c4 Psychrobacter okhotskensis MD17 NCIMB 13931 Psychrobacter luti NF11 LMG 21276 Psychrobacter sp. BSw21070 Carnobacterium funditum pf3 DSM 5970 99 Carnobacterium. funditum R-36987 1 c 19 99 Sporosarcina globispora 785 DSM 73 Bacillus sp. Nj-19 99 c 18 Sporosarcina globispora AIC11-11 90 Sporosarcina globispora OS-253 Arthrobacter sp. KOPRI 25422 61 Arhtrobacter sp. SS-2009-PA1 PA1 Arthrobacter psychrochitiniphilus JCM 13874 Arthrobacter sp. SH-82B Arthrobacter psychrolactophilus KNOUC403 c5 c 17 99 Arthrobacter sp. KOPRI 25535 Arthrobacter sp. KOPRI 25486 Arthrobacter oxydans BG1-7 c 12 99 94 Arthrobacter sp. DSP-S2 Arthrobacter oxydans S32212 Arthrobacter oxydans Asd M5-7 Arthrobacter oxydans Asd M3-4 c 15 99 Arthrobacter sulfureus DSJ12 Arthrobacter sp. Tibet-ITa1 Synechococcus sp. SRODG098

0,01

123

World J Microbiol Biotechnol (2012) 28:22492256

2255

efuents (Pathan et al. 2010; Antranikian et al. 2005; Mi kan Venegas and Castellanos Suarez 2004). There is current interest in the use lipases and proteases in laundry detergents and as well as in the dairy industries. Pectinase, amylase and cellulase have been shown to be useful in clearing the appearance of and increasing the sweetness of fruit juices. Amylases are used to hydrolyze starch for the production of ethanol, and also to synthesize high-fructose corn sweeteners and syrups (Nigam and Singh 1995; Pandey et al. 2000; Vihinen and Mantsala 1989). When activity was analyzed at 4C, Psychrobacter sp. c4 and Arthrobacter sp. c15 had the highest lipase activity, Arthrobacter sp. c12 the highest amylase activity, Pseudomonas spp. c3 and c9, the highest caseinase activity and Pseudomonas spp. c1 and c8 the highest gelatinase activities. G. pannorum 14-2 and 63-2 could be also considered as a source of gelatinase and lipase. These microorganisms could be evaluated for the design of inoculants for organic matter biodegradation, e.g., for efuent treatments (Gratia et al. 2009). In temperate climates, low temperatures achieved during the cold seasons may negatively affect the metabolic activity of native microorganisms (Brenchley 1996), leading to a poor biotransformation rate of pollutants and to their accumulation in case of continuous input. In this respect, the use of psychrophilic microorganisms could provide a useful alternative since psychrophiles are adapted to both low and moderate temperatures (Margesin et al. 2002). In addition, such microorganisms have in some cases been shown to produce cold-activated enzymes possessing a much higher specic activity than those of their mesophilic counterparts (Feller and Gerday 2003). We compared the colony diameters of 103 bacterial, 10 yeast and 10 lamentous fungal isolates expressing enzymatic activities, grown on the same solid media at 4 and 20C. About 80% of the bacteria grew better at 20C than at 4C and only 8% had a reduced growth at 4C. In relation to bacterial enzymatic activities (see Table 1), most of the activities were expressed at 20C. Our tests suggest that most of bacterial isolates from maritime Antarctica were psychrotrophs, similar results were described in related studies (Feller and Gerday 2003; Lo Giudice et al. 2010; Selbmann et al. 2010), conrming that psychrotrophs are usually more abundant than obligate psychrophiles in cold ecosystems. In this sense, psychrotrophs are usually preferred for enzyme production than psycrophilic microorganisms because industrial fermentations could proceed at ambient temperatures. Comparable results were obtained with yeasts as well: nine grew better at 20C than at 4C. However, only 40% of lamentous fungal isolates grew better at 20C than at 4C, and three could be considered obligate psychrophiles. Comparative sequence analysis of 16S rRNA gene indicated that some were closely related to previously

described bacteria, (similarity of 99%). BLASTn analyses of isolates c1, c2, c3, c4, c7, c8, c10, c12, c16, c17, c18 and c19 exhibited high similarity (within the 5 best scores) with other Antarctic bacteria identied previously. The afliation of each isolate, based on the BLAST analyses, is presented in Table 2: Pseudomonas spp. (c2, c3, c6, c8, c9, c10, c11, c14, c16), Psychrobacter sp. (c4), Arthrobacter spp. (c5, c12, c15, c17), Bacillus sp. (c18), Carnobacterium sp. (c19). Isolate c13 had highest similarity with Thermoleophilum minutum (Genbank locus: HQ223108.1). Some physiological properties described for this species, however, did not match those of the isolate including growth temperature and carbon source requirements. The location of c13 in the phylogenetic tree suggests that it is most similar to Pseudomonas and that the identication of T. minutum should be revised. As such, the NJ distance tree shown in Fig. 1 includes reference sequences with highest similarity from strains of the GenBank database of the NCBI. Both NJ and MP algorithms gave similar topologies (Fig. 1 and Figure 1S as supplemental material). Two of the clones grouped with Arthrobacter, clones c5 and c17, showed a similar pattern of amylase activity, in agreement with the close relation inferred from the distribution of sequences observed in the phylogenetic trees (Fig. 1 and Figure 1S). However, clone c5 exhibited amylase activity at 4 and 20 C, whereas c17 expressed this activity only at 20C. Clones related to Pseudomonas showed complex patterns of enzymatic activities. In particular, clones c8, c9 and c11, closely related according to phylogenetic analysis like clones c7 and c10, showed properties that did not correspond the nearest phylogenetic relation (see Table 2).
Acknowledgments We are grateful to Dr. Paul R. Gill for English corrections and valuable comments. This study was supported by a grant from the Comision Sectorial de Investigacion Cientca Universidad de la Republica and by Instituto Antartico Uruguayo.

References
Aghajari N, Feller G, Gerday C, Haser R (1996) Crystallization and preliminary X-ray diffraction studies of a-amylase from the Antarctic psychrophile Alteromonas halplanctis A23. Protein Sci 5(10):21282129. doi:10.1002/pro.5560051021 Alippi AM, Aguilar OM (1998) Characterization of isolates of Paenibacillus larvae subsp. larvae from diverse geographical origin by the polymerase chain reaction and Box primers. J Invertebr Pathol 72:2127. doi:10.1006/jipa.1998.4748 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403410. doi: 10.1016/S0022-2836(05)80360-2 Antranikian G, Vorgias CE, Bertoldo C (2005) Extreme environments as a resource for microorganisms and novel biocatalysts. Adv Biochem Eng Biotechnol 96:219262

123

2256 Brenchley JE (1996) Psychrophilic microorganisms and their coldactive enzymes. J Ind Microbiol Biotechnol 17:432437. doi: 10.1007/BF01574774 Feller G, Gerday C (2003) Psychrophilic enzymes: hot topics in cold adaptation. Nat Rev Microbiol 1:200208. doi:10.1038/nrmicro773 Fogliano V, Andreoli C, Martello A, Caiazzo M, Lobosco O, Formisano F, Carlino PA, Meca G, Graziani G, Di Martino Rigano V, Vona V, Carfagna S, Rigano C (2010) Functional ingredients produced by culture of Koliella antarctica. Aquaculture 299:115120. doi:10.1016/j.aquaculture.2009.11.008 Gonzales JA, Gallardo CS, Combar A, Rego P, Rodrguez LA (2004) Determination of enzymatic activity in ecotypic Saccharomyces and non-Saccharomyces. Electron J Environ Agric Food Chem 3(5):743750 Gratia E, Weekers F, Margesin R, DAmico S, Thonart P, Feller G (2009) Selection of a cold-adapted bacterium for bioremediation of wastewater at low temperatures. Extremophiles 13:763768. doi:10.1007/s00792-009-0264-0 Higgins D, Thompson J, Gibson T, Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specic gap penalties and weight matrix choice. Nucl Acids Res 22:46734680. doi:10.1093/nar/ 22.22.4673 Leon J, Liza L, Soto I, Cuadra DL, Patino L, Zerpa R (2007) Bioactive Actinomycetes of marine sediment from the central coast of Peru. Rev Peruana Biol 14(2):259270 Li J, Chi Z, Wang X, Peng Y, Chi Z (2009) The selection of alkaline protease-producing yeasts from marine environments and evaluation of their bioactive peptide production. Chin J Oceanol Limnol 27(4):753761. doi:10.1007/s00343-009-9198-8 Lo Giudice A, Casella P, Caruso C, Mangano S, Bruni V, De Domenico M, Michaud L (2010) Occurrence and characterization of psychrotolerant hydrocarbon-oxidizing bacteria from surface seawater along the Victoria Land coast (Antarctica). Polar Biol 33:929943. doi:10.1007/s00300-010-0770-7 Lupo S, Bettucci L, Perez A, Martnez S, Cesari C, Escoriaza G, Gatica M (2006) Characterization and identication of the basidiomycetous fungus associated with hoja de malvon grapevine disease in Argentina. Phytopatholog Mediterr 45: S110S116 Margesin R, Miteva V (2011) Diversity and ecology of psychrophilic microorganisms. Res Microbiol 162:346361. doi:10.1016/ j.resmic.2010.12.004 Margesin R, Feller G, Gerday C, Russell NJ (2002) Cold-adapted microorganisms: adaptation strategies and biotechnological potential. In: Bitton G (ed) Encyclopedia of environmental microbiology, vol 2. Wiley, New York, pp 871885 Margesin R, Fauster V, Fonteyne PA (2005) Characterization of coldactive pectate lyases from psychrophilic Mrakia frigida. Lett Appl Microbiol 40:453459. doi:10.1111/j.1472765X.2005. 01704.x Mikan Venegas JF, Castellanos Suarez DE (2004) Screening for isolation and characterization of microorganisms and enzymes with useful potential for degradation of cellulose and hemicellulose. Rev Colomb Biotecnol VI(1):5871 Mtui G, Nakamura Y (2004) Lignin-degrading enzymes from mycelial cultures of Basiodiomycete fungi isolated in Tanzania. J Chem Eng Jpn 37(1):113118. doi:10.1252/jcej.37.113 Nigam P, Singh D (1995) Enzyme and microbial systems involved in starch processing. Enzym Microb Tech 17:770778. doi: 10.1016/0141-0229(94)00003-A

World J Microbiol Biotechnol (2012) 28:22492256 Pandey A, Nigam P, Soccol C, Soccol V, Singh D, Mohan R (2000) Advances in microbial amylases. Biotechnol Appl Bioc 31:135152. doi:10.1042/BA19990073 Paterson RRM, Bridge PD (1994) Biochemical techniques for lamentous fungi. IMI technical handbook 1, CAB Int Pathan AAK, Bhadra B, Begum Z, Shivaji S (2010) Diversity of yeasts from Puddles in the vicinity of Midre Love0 nbreen Glacier, Arctic and Bioprospecting for enzymes and fatty Acids. Curr Microbiol 60:307314. doi:10.1007/s00284-009-9543-3 Rivas R, Garca-Fraile P, Mateos PF, Martnez-Molina E, Velazquez E (2007) Characterization of xylanolytic bacteria present in the bract phyllosphere of the date palm Phoenix dactylifera. Lett Appl Microbiol 44:181187. doi:10.1111/j.1472-765X.2006. 02050.x Rodrguez K, Echer Ferreira MK, Salamoni Pinto S, Cofre Barragana V, Van Der Sand S (2007) Perl da atividade enzimatica de actinomicetos isolados de proceso de compostagem. Sinaferm XVI, BAM 0626 Ruisi S, Barreca D, Selbmann L, Zucconi L, Onofri S (2007) Fungi in Antarctica. Rev Environ Sci Biot 6:127141. doi:10.1007/ s11157-006-9107-y Seeley HW Jr, Vandemark PJ, Lee JJ (1991) Microbes in action, 4th edn. Freeman WH and Company, New York Selbmann L, Zucconi L, Ruisi S, Grube M, Cardinale M, Onofri S (2010) Culturable bacteria associated with Antarctic lichens: afliation and psychrotolerance. Polar Biol 33:7183. doi: 10.1007/s00300-009-0686-2 Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:15961599. doi:10.1093/molbev/msm092 Tosi S, Kostadinova N, Krumova E, Pashova S, Dishliiska V, Spassova B, Vassilev S, Angelova M (2010) Antioxidant enzyme activity of lamentous fungi isolated from Livingston Island, Maritime Antarctica. Polar Biol 33:12271237. doi: 10.1007/s00300-010-0812-1 Vihinen M, Mantsala P (1989) Microbial amylolytic enzymes. Crit Rev Biochem Mol Biol 24:329418. doi:10.3109/10409238 909082556 Villalba LS, Mikan JF, Sanchez J (2004) Actividades hidrolticas y caracterizacion enzimatica de poblaciones microbianas aisladas del patrimonio documental del Archivo General de Colombia. Nova 2(2):4958 Wang HY, Liu DM, Liu Y, Cheng CF, Ma QY, Huang Q, Zhang YZ (2007) Screening and mutagenesis of a novel Bacillus pumilus strain producing alkaline protease for dehairing. Lett Appl Microbiol 44:16. doi:10.1111/j.1472-765X.2006.02039.x White TJ, Bruns T, Lee S, Taylor J (1990) Amplication and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, San Diego, pp 315322 Wickstrom MB (1983) Detection of microbial proteolytic activity by a cultivation plate assay in which different proteins adsorbed to a hydrophobic surface are used as substrates. Appl Environ Microb 45(2):393400 Zhang J, Zeng R (2011) Molecular cloning and expression of an extracellular a-amylase gene from an Antarctic deep sea psychrotolerant Pseudomonas stutzeri strain 7193. World J Microb Biot 27:841850. doi:10.1007/s11274-010-0526-0

123