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9 Detoxification Mechanisms of Heavy 60
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Metals by Algal–Bacteria Consortia 63
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15 Enrique J. Peña-Salamanca, Ana Lucia Rengifo and Neyla Benitez-Campo 67
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Applied Plant Biology Research Group, Department of Biology, Universidad del Valle, Cali, Colombia
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22 28.1 Introduction 3 Modifying the active conformation of biomolecules, es- 74
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23 pecially enzymes and polynucleotides. 75

Heavy elements are defined as chemical elements whose
24 76
density is at least five times heavier than that of water. 4 Disrupting the integrity of biomolecules.
25 77
Among 35 widely occurring metals, 23 are heavy elements
26 5 Modifying some other biologically active agents. 78
or metals including Ag, As, Au, Bi, Cd, Ce, Cr, Co, Cu,
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27 79
Fe, Ga, Hg, Mn, Ni, Pb, Pt, Te, Th, Sb, Sn, U, V, and Zn
28 The basis for the biological disruption by metal activ- 80
(Kvesitadze et al., 2006). In small amounts, most of these
29 ities is basically based on their ability to bind strongly to 81
elements are indispensable for many organisms, but their
30 oxygen, nitrogen and sulfur atoms, due to their abundance 82
enhanced doses induce acute or chronic poisoning. Ions
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31 in biological systems, and their role to act as ligands to 83

most essential for life are the representative metal ions:
32 84 Is the
Na+ , K+ , Mg2+ and Ca2+ , and the transition metals: Mn, all essential metal ions (Nordeberg et al. 2005). In addi- Au:
33 tion, toxic metal ions can coordinate to essential functional 85
spelling
Fe, Co, Ni, Cu, Zn, Mo, and V. The essential metal ions have
34 groups of proteins which can render the protein inactive. Nordberg
86 or
a variety of functions in biological systems. Their functions Nordeberg?
35 87
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range from regulators of biological processes to important This is especially true of Hg, which has a tremendously high
36 affinity for sulfur, a common ion used to form amino-acid 88
structural components in proteins (Nordberg et al., 2005).
37 residues. In fact, the mechanisms of metal ion toxicity are 89
The toxicity of heavy metals is apparent in reducing
38 directly related to the modes of metal ion binding in bio- 90
growth and development in microorganisms and plants,
39 logical systems. A biomolecule that can bind a metal ion 91
which seriously harm the health of animals and humans.
40 must possess a number of chemical characteristics, such as, 92
The deleterious effects of metal ions can be manifested in
41 a region that has a high concentration of oxygen, nitrogen 93
many ways, but their toxicity can be divided into five general
42 or sulfur atoms, the number of donor atoms to stabilize the 94
groups (Kvesitadze et al., 2006):
43 metal ion, and sufficient space in the metal ion space that 95
44 allows an appropriate three-dimensional geometry about 96
45 1 Displacing essential metal ions from biomolecules and 97
other biologically functional units. the metal ion (Gaur and Rai, 2001).
46 There are three major sources of heavy metals in most 98
47 terrestrial ecosystems: the underlying parent material, the 99
2 Blocking essential functional groups of biomolecules, in-
48 atmosphere, and the biosphere. Biotic sources of metals are 100
cluding enzymes and polynucleotides.
49 101
50 102
51 Handbook of Marine Macroalgae: Biotechnology and Applied Phycology, First Edition. Se-Kwon Kim. 103
52 © 2011 John Wiley & Sons, Ltd. Published 2011 by John Wiley & Sons, Ltd. 104
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442 DETOXIFICATION MECHANISMS OF HEAVY METALS BY ALGAL–BACTERIA CONSORTIA

1 originally obtained from one of the other two sources. Par- 3 The inhibition of respiratory oxygen consumption. 53
2 ticularly, inputs to a system from existing vegetation occur 54
3 in different ways: inputs from above-ground biomass, from 4 The disruption of nutrient uptake processes. 55
4 roots, and from below-ground biomass (Wang and Lewis, 5 Enzyme inhibition, due to displacement of essential metal 56

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5 1997; Perales-Vela et al. 2006). These inputs are also con- ions 57
6 sidered fluxes within the food chain of an ecosystem, since 58
7 plants are the base of the uptake, transport, and accumula- 6 The inhibition of protein synthesis 59

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8 tion of metal in biological systems (Kvesitadze et al., 2006). 60
9 The need for a cost-effective process and safe methods 7 Abnormal morphological development and ultraestruc- 61
10 for removing heavy metals from discharging effluents has tural changes. 62
11 resulted in search for other unconventional materials such 8 The impairment of motility and loss of flagella in certain 63
12 as organic or inorganic sorbents (Loy et al., 2004). The use 64

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microalgae
13 of microbial biomass such as, fungi (Bang et al., 2000), algae 65
14 (Perales-Vela et al., 2006), and bacteria (Loy et al., 2004) for 66
On the other hand, eukaryotic algae have developed
15 removal of heavy metals from aqueous solutions is gain- 67
some tolerance and detoxification mechanisms to allow
16 ing increasing attention. Recently, microbial systems have 68
them to resist metal ions inside their cells, which are shown
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been successfully used as adsorbing agents for the removal of
heavy metals. Microbial populations in metal polluted envi-
ronments adapt to toxic concentrations and become metal
in Figure 28.1.
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20 resistant. Recently, there has been interested in the study 28.2.1 Production of extracellular 72
21 of the metabolic capacity of plant-associated bacteria used 73
binding-polypetides
22 for phytoremediation strategies. In the rhizosphere, many 74
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23 pesticides as well as trichloroethylene, polycyclic aromatic One of the primary mechanisms observed in micro- and mi- 75
24 compounds, and petroleum hydrocarbons are degraded at croalgae was related to the production of peptides capable to 76
25 accelerated rates. Although plant-associated bacteria have bind heavy metals (Nies, 1999). These molecules are further 77
26 dynamic and possess varied metabolic capacities, current partitioned inside vacuoles to facilitate appropriate con- 78
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27 strategies on algal–bacteria consortia are little known and trol of the cytoplasmic concentration of heavy metal ions 79
28 during the last years there have given a special attention to (Cobbett and Goldsbrough, 2002). Those polypeptides are 80
29 those interactions. commonly named specific ion-chelators or siderophores. 81
30 In this chapter, the strategies of algal–bacteria consortia The complexing capacity of those ligands has been demon- 82
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31 to detoxify heavy metals and their potential of biotechnolog- strated primarily in cyanobacteria and freshwater microal- 83
32 ical applications on heavy metal treatments are discussed. gae (Butler et al., 1980). They form large extracellular ag- 84
33 Special attention to transformation processes of metal ions gregates and posses anionic properties that are capable of 85
34 by algal associated bacteria is given. binding metal cations. The peptides discussed can be 86
35 grouped into two categories: 87
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28.2 Mechanisms used byalgae 1 Short-chain polypeptides named phytochelatins (PCS) 89
38 in heavy metals tolerance (class III metallothioneins, MT), found in higher plants, 90
39 and removal algae, and certain fungi (Nicholas et al., 2003). 91
40 92
41 The ability of eukaryotic algae to survive and reproduce in 2 Specific proteins; class II MT (identified in cyanobac- 93
42 metal-polluted habitats may depend on genetic adaptation teria, algae and higher plants), and class I MT found 94
43 over extended time periods by mutation, genetic exchange, in most vertebrates, observed in Neurospora and Agar- 95
44 selection, and changes in physiology, resulting from metal icus bisporus (not reported in algae) (Robinson, 1989; 96
45 exposure (Shaw, 1989; Peña et al., 2005). The effects of heavy Rauser, 1990). 97
46 metal toxicity in algae may include: 98
47 1 An irreversible increase in plasmalemma permeability, The metal-binding polypeptides produced in algae are 99
48 leading to the lost of cell solutes (e.g., K+ ) and changes abundant in both sulfhydryl and carboxyl groups and could 100
49 in cell volume. have affinity for a wide range of metal ions. In the case 101
50 of MT they are low molecular weight, cysteine-rich metal 102
51 2 A reduction in photosynthetic electron transport and binding proteins. Class I and II MT are proteins which are 103
52 photosynthetic carbon fixation. encoded by structural genes (Rauser, 1990; Cobbett and 104
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MECHANISMS USED BYALGAE IN HEAVY METALS TOLERANCE AND REMOVAL 443

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Nontransformed Organic
2 Contominant 54
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Soluble conjugated of
5 Contaminant in 57
Excretion
6 Vacuole 58
7 Compartmentation 59

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Functionalization Contaminant with
8 CO2 60
Organic functional group
9 Contamination 61

n
Conjugation

tio
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11 Conjugated of Contaminant 63

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12 with cell compounds 64

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Insoluble conjugates of
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Plant cell Contaminant in Cell
14 Wall 66
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16 Figure 28.1 Suggested mechanisms involved in plant detoxification for metal ions. Specific details are given in the case of 68
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algal–bacterial consortia, especially for the algal processes. 69
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20 Goldsbrough, 2002). Recently, the genes encoding for PCS 28.2.3 Internal detoxification 72
21 activity were isolated (Perales-Vela et al., 2006). Cysteine 73
is part of the MT II chelating core and is an activator of The study of internal detoxification of heavy metal in algae
22 74
has received little attention than surface binding and trans-
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23 the gene PCS. MT I and II biosynthesis can be induced by 75

heavy metals such as Cd2+ , Ag+ , Bi3+ , Pb2+ , Zn2+ , Cu2+ , port. However, algae are able to activate a definite set of bio-
24 76
Hg2+ and Au2+ . MT III are synthesized in the cytosol and chemical and physiological processes to resist the toxic ac-
25 77
are subsequently transported into the chloroplast and mi- tion of environmental contaminants (Gaur and Rai, 2001).
26 78
In microalgae such as the diatoms Amphora and Navicula
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27 tochondria. This was first observed in Euglena organelles 79

where almost 60% of the accumulated Cd2+ present in- copper is localized intracellularly in electron-dense spher-
28 80
side the chloroplast was due to the Cd–MT III complexes ical bodies corresponding to polyphosphate granules and
29 81
(Schmitt et al., 2001, Soldo et al., 2005). in a dense irregular body containing sulfur, calcium, and
30 82
copper (Ahner and Morel, 1990; Soldo et al. 2005). The
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main processes of detoxification are included: conjugation
32 84
of the heavy metal with intracellular compounds, and fur-
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28.2.2 Exclusion mechanism ther compartmentation of conjugates, degradation to com-
34 86
mon cell metabolites, and finally to carbon dioxide. Those
35 The slow phase of heavy metal accumulation by “binding 87
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processes can be regulated by environmental factors such

36 sites” is often related with intraprotoplast uptake, or cell 88
as temperature, salinity, pH, and others, which implies the
37 exclusion in contrast “rapid” physical binding or biosorp- 89
significance of those interactions with their self-resistance
38 tion (Robinson, 1989, Mehta et al., 2002). Indeed, changes 90
mechanisms (Ospina-Alvarez et al., 2006).
39 in the affinity of binding sites within the cell wall matrix 91
40 reflects, in part active (metabolism-dependent) uptake. Ac- 92
41 tive transport systems have been described for several heavy 93
metals in algae (Schiewer and Wong, 2000). Some metal-
28.2.4 Metal transformation
42 94
43 tolerant strains of microalgae may operate an exclusion Algae can carry out chemical transformations of heavy met- 95
44 mechanism, when reducing the internal accumulation ca- als such as oxidation, reduction, methylation, and demethy- 96
45 pacity (Whitton, 1984). This process implies metal ions lation. Those ones act as mechanisms of resistance, and 97
46 suffer antagonism, such as the case of iron and cadmium, most of them involved selective processes to eliminate non- 98
47 in the marine diatom Thalassiosira weissflora (Stauber and essential metal ions for growth metabolism (Perales-Vela 99
48 Florence, 1987). The exclusion mechanism may implicate a et al., 2006; Lenis et al., 2007). Methylation has been ob- 100
49 metal ion transport system in cadmium but an inhibition served in brown algae, to detoxify Hg form aqueous solu- 101
50 of iron. Consequently, a decreased internal accumulation tion (Davis et al. 2003). The process implies degradation to 102
51 in iron appears as a tolerance of the metal by the alga (Peña less toxic compounds, as in the case of the transformation 103
52 et al., 2004). of tributyltin by Chlorella. The compound is debutylated 104
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444 DETOXIFICATION MECHANISMS OF HEAVY METALS BY ALGAL–BACTERIA CONSORTIA

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3 BIOSORPTION M2+ 55
BIOMINERALIZATION
M2+
4 M2+ 56

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5 L– 57
L– H2S + M2+ MS
6 M2+ 58
7 L– 59

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9 2H+ M2+ + CO32– MCO3 61
microorganisms
10 + OH– M(OH)2 62
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12 M2+ 64
chemical absortion by microorganisms

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BIOTRANSFORMATION
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Figure 28.2 Main biological mechanisms involved in the detoxification of metals by plant-associated bacteria.
20 72
21 73
22 to di- and monobutyltin molecules, although it should be efflux pumps, or intra- and extracellular sequestration. In 74
stressed that light-stimulated photolysis also occurs (Toumi bacteria, they involve particularly enzymatic transforma-
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23 75
24 et al., 2000). The transformation of those methyltin deriva- tion, toxic chemical species by redox reactions, methylation, 76
25 tives are also related with bacterial transformation. It has alkylation/dealkylation and reduction in the sensitivity of 77
26 also been suggested that the decomposition products of cellular targets to metal ions (Nies, 2003). In some plant 78
arseno sugars from macroalgae lead to the formation of ar- bacterial association, these organisms underwent a vari-
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27 79
28 senobetaine. This compound is a ubiquitous component of ety of plasmid-mediated adaptation (Brinza et al., 2007). 80
29 marine animal tissues and the importance of algae as the The understanding of how those consortia resist metals can 81
30 initial source of this molecule in the marine environment is provide insight into strategies for detoxification or removal 82
widely discussed (Davis et al., 2003). Additionally, arsenic of pollutants from the environment. Both microorganisms
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32 can be taken up by certain marine algae and converted and algae have adapted to the presence of different metal- 84
33 to various organoarsenicals and such conversion may be a toxic environments by developing a variety of mechanisms 85
34 detoxification strategy (Schiewer and Wong, 2000). (Loutseti et al., 2009). A number of mechanisms are pro- 86
35 posed to explain how this consortium regulates the detox- 87
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36 ification and transformation of essential and non-essential 88

37 28.3 Algal–bacterial mechanisms metal ions along with their biotechnological applications 89
38 involved in heavy metal (Figure 28.2). 90
39 91
40
detoxification 92
28.3.1 Biosorption
41 Recent studies have demonstrated that algal–bacterial con- 93
42 sortia have an important role in the cycling of toxic metals Recently microbial systems like fungi, bacteria, and algae 94
43 and pollutants. Advances have been made in understanding have been successfully used as adsorbing agents for re- 95
44 metal–microbe interactions and new applications of these moval of heavy metals (Lee et al., 2002; Wang and Cheng, 96
45 processes to the detoxification of metal and radionuclide 2009). Microbial populations in metal-polluted environ- Au: In
97the
46 contamination have been developed (Lloyd, 2003). ments adapt to toxic concentrations of heavy metals and reference
98 list
47 Overall, algae and bacteria share a variety of strate- become metal resistant. Different species of Aspergillus, Cheng99
is Chen -
which is
48 gies for heavy metal tolerance. In some cases, metal tol- Pseudomonas, Sporophyticus, Bacillus, Phanerochaete, etc., 100
correct?
49 erance is the outcome of their metabolism or is an in- have been reported as efficient chromium and nickel reduc- 101
50 trinsic property related to their cell wall structure or the ers (Bapat et al., 2003). Bacteria were used as biosorbents 102
51 presence of extracellular polymeric substances. In other because of their small size, ubiquity, and ability to grow 103
52 cases, the resistance mechanisms include active transport under controlled conditions; and their resilience to a wide 104
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ALGAL–BACTERIA CONSORTIA IN THE RED ALGA BOSTRYCHIA CALLIPTERA (RHODOMELACEAE) 445

1 range of environmental situations. Bacteria and algae have preacidification by sulfur-oxidizing bacteria and the subse- 53
2 the ability to act as biological materials to accumulate heavy quent immobilization of the metals through organic acid 54
3 metals through metabolically mediated or physicochemical production. In some bacteria, the transformation mech- 55
4 pathways of uptake or binding (Vieira and Volesky, 2000). anism involves the presence of genes that form a spe- 56

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5 These bioprocesses involve biosorptive (passive) uptake by cific ion-resistance operon (HgII), that not only detoxifies 57
6 dead biomass or bioaccumulation by living cells. this ion but also transports and self-regulates resistance 58
7 The main drawback in the use of algal–bacterial con- (Bruins et al., 2000). This same set of genes also encodes the 59

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8 sortia as biosorptive materials is their ability to interact as production of a periplasmic binding protein that regulates 60
9 ion-exchange synthetic resins and cell surface sequestration the biomineralization of mercury compounds to less toxic 61
10 for metal ions. Nevertheless, biosorption methods seem to molecules which can be easily transport to cytoplasm for 62
11 be more effective than their physicochemical counterparts detoxification (Loy et al., 2004). 63
12 in removing dissolved metals at low concentrations (be- 64

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13 low 2–10 mg/l) (Peña et al., 2004) and demonstrate higher 65
14 specificity, which avoids overloading of binding sites by 66
15 alkaline-earth metals (Bunke and Buchholz, 1999). 28.4 Algal–bacteria consortia in 67
16 the red alga Bostrychia 68
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28.3.2 Bioaccumulation
D calliptera (Rhodomelaceae)
During the last 20 years, the potential uses of macroalgae
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Nickel-resistant bacterial populations isolated from the
20
green alga Rhizoclonium riparium (Cladophorales) exhib- epiphytic on mangrove aerial roots have been studied as 72
21
ited reduced bioaccumulation when cells were in stationary biomonitors of estuarine contamination (Peña, 1998; Peña, 73
22
phase (Peña et al., 2004). In contrast, during the mid-log et al. 1999, 2005). Particularly, the metal concentrations 74
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phase of cellular growth, metal uptake rate was higher, of macroalgae and associated bacterial populations have 75
24
demonstrating the enhancing of Ni(II) removal by Micro- extensively studied in the Buenaventura estuary, on the Pa- 76
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coccus sp. The initial condition was a Ni(II) concentration cific coast of Colombia (Peña et al., 2004; Ospina-Alvarez 77
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of 50 mg/l, pH 7, temperature: 35 ◦ C. Similar studies have et al. 2006; Peña, 2008; Rengifo, 2010). More recently, the 78
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been reported by Enterobacter cloacae (Leung et al., 2000), percentage of chromium removal in the alga–bacterium as- 79
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Bacillus circulans (Tian-Wei et al., 2004) and Asperigillus sociation exposed to a set of different metal concentrations 80
29
sp. (Nasseri et al., 2002). All studies demonstrated that in vitro conditions were studied (Rengifo, 2010). 81
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Micrococcus isolate were more effective for the removal of The monitoring of the estuarine pollution was motivated 82
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Cr(VI) and Ni(II) when compared with other microbial by the increase concerning of heavy metal pollution in the 83
32
biomass reported. The initial metal ion concentration bay. The metals of concern, specifically chromium, cop- 84
33
plays a role in determining the bioaccumulative capacity of per, and lead, among others, enter waterways from a wide 85
34
bacterial isolate species. As the heavy metal concentrations range of both natural and anthropogenic sources (Peña 86
35 et al., 2004). While external inputs of metals into estuaries 87
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increased, the cellular growth of all the isolates can be

36
inhibited, depending on the metal ion kinetics (Prasenjit are important, estuarine sediments themselves may become 88
37
and Sumathi, 2005). the main sources of contaminants to estuarine waters. Bue- 89
38 naventura bay is surrounded by extensive mangrove habi- 90
39 tats that contain macroalgae attached to roots, tree trunks 91
40 28.3.3 Biotransformation and and mud surfaces (Peña, 1998). Despite the continual expo- 92
41 sure of these algae to the high contaminant concentrations 93
biomineralization found within estuarine ecosystems, few studies have ex-
42 94
43 In microbial populations, the most widely studied bio- amined the effects of contaminants on the interactions in 95
44 transformation mechanism involves enzymatic reduction algal–bacterial communities in these tropical habitats. 96
45 of metal ions to less toxic, volatile elemental (Nies, 1999). Wild plants of Bostrychia calliptera associated with bac- 97
46 In addition to metal reduction, another strategy is the pro- terial populations collected from Dagua River were moni- 98
47 duction of organic acids, and the generation of sulfuric tored in the laboratory. The trial was conducted in synthetic 99
48 acid through bioxidation of sulfur (e.g., by Thiobacillus seawater with two levels of chromium, 5 and 10 ppm, us- 100
49 spp.) (Gadd, 2000). A recent development has been the se- ing bioreactors according to four treatments: unprocessed 101
50 quential extraction of copper by bacterial associated to root plant material (algae–bacteria), plant material with antibi- 102
51 plants on macrophytes (e.g., by Eichornia crassipess) (Peña otic (alga–antibiotic), sediment and/or suspended matter in 103
52 et al., 2005; Kumar-Rai, 2008). The mechanism involves surface algae (natural bacterial consortium or CBN), and 104
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446 DETOXIFICATION MECHANISMS OF HEAVY METALS BY ALGAL–BACTERIA CONSORTIA

1 5 (A) 100 53
2 4.5 CBN [10] 90 54
3 80 AB 55
4
4 56

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3.5 70
Log (UFC/mL)

% remotion
5 CBN 57
3 60
6 58
AB [10] 50
7 2.5 59

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8 2 60
30 AA
9 1.5 61
10 20 62
1 CONTROL
11 AB [5] 10 63
0.5 CBN [5]
12 0 1 64

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1 3 7
14 Days 66
Days
15 67
(B) 90
16 Figure 28.3 Bacterial growth at different metal concentra- 68
80
17
18
19
(isolated algal–bacteria strains).
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tions (5 and 10 mg/l). CBN (natural bacterial consortia); AB
70 AB
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% remotion
20 72
the control without the presence of B. calliptera or bac- 50 AA
21 73
22
teria. The experimental design followed a model of two 40 CBN
74
factors (Concentration of chromium × Types of combina-
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23 30 75
24
tion) with repeated measures using one factor. The behav- 76
20 CONTROL
25
ior of microbial populations and the chromium decrease 77
concentration percentage was monitored by using atomic 10
26 78
absorption spectroscopy (AAS). 0
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27 A 79
28
Results showed greater bacterial growth at higher 0 1 3 7
80
29
chromium concentrations (10 mg/l) compared to those Days
81
30
with the treatment exposed at 5 mg/l. Additionally, signif- 82
icant differences were obtained for both, bacterial popula- Figure 28.4 Percent of heavy metal removal by algal–bacteria
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31 consortia exposed to different chromium concentrations. (A) 83

32
tion to the total concentration of chromium in the algae- 84
Percent removal at 5 mg/l. (B) Percent removal at 10 mg/l.
33
bacteria systems and CBN, algae–bacteria being the most 85
34
efficient treatment to remove the metal at the highest metal 86
35
concentration (Figure 28.3). bioprecipitation and biomethylation. These techniques aim 87
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The natural consortia bacteria associated with the red to change the speciation of the heavy metals, making them 88
37
alga (CBN–alga) showed higher chromium removal (Fig- either more mobile in order to improve their removal, or 89
38
ure 28.4) suggesting their active role in the transformation decreasing their toxicity and mobility. Phytoremediation 90
39
processes of this metal in aqueous marine solutions at en- is a special situation in which plants and their associated 91
40
vironmental levels. microorganisms are used to assimilate and remove con- 92
41 taminants from the environment. Phytoremediation of 93
42 heavy metals comprises several processes (Salt et al., 1995). 94
43 28.5 Biological treatment of Although phytoremediation is a promising method, it is re- 95
44 stricted to contamination at shallower depths and requires 96
45
heavy metals longer times compared to other methods (Peña et al., 97
46 Conventional methods of heavy metal treatment are often 2005). Microorganisms can help plants to overcome heavy 98
47 expensive, hence alternative cost-effective technologies metal toxicity stress, either by decreasing metal toxicity or 99
48 generally based on biological processes are being developed by counteracting the plant’s stress response. In addition, 100
49 to remediate heavy metal pollution (Vieria and Volesky, they can assist the plants by rendering heavy metals more 101
50 2001). Bioremediation exploits microorganisms to deal bioavailable, so improving their uptake. Recent advances 102
51 with heavy metal pollution in a variety of methods such demonstrated that all bioremediation/phytoremediation 103
52 as bioleaching, biosorption, oxidation/reduction reactions, technologies rely on the genetic and biochemical capacities 104
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BIOTECHNOLOGICAL APPLICATIONS 447

1 of the interactions of plant and microorganism to protect bacteria could have aided the biodegradation of aromatic 53
2 themselves against the toxic effects of heavy metals (Wang compounds. Algae degradation of these chemicals has been 54
3 and Cheng, 2009). An understanding of the ways how recently reported and this is a growing field of research in 55
4 algal–bacteria consortia cope with toxic concentrations of environmental microbiology (Davis et al., 2003). 56

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5 heavy metals is therefore essential in order to exploit them The use of both living and death algal–microbial biomass 57
6 for detoxification and removal of heavy metals. for removal of heavy metals from aqueous solutions us- 58
7 ing biosorptive mechanisms is gaining increasing attention. 59

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8 Biosorption is regarded as a potential cost-effective biotech- 60
9 28.6 Biotechnological applications nology for the treatment of high volume low-concentration 61
10 complex wastewaters containing heavy metals (Wang and 62
The efficiency of any biotechnological applications on heavy Chen, 2009). It has been found that development of biore-
11 63
metal bioremediation depends on the activity of the mi- actors with living cells for improving biosorption activity
12 64

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croorganisms involved which is, in turn, affected by envi- depends on properties of adsorbent and molecules in the
13 65
ronmental conditions, operational parameters and the lo- transfer from the solution to the solid phase. It has also
14 66
cal composition of the overall algal–microbial community been reported that biosorption capacities for heavy met-
15 67
(Ospina-Alvarez et al., 2006; Perales-Vela, 2006). When opt- als are strongly pH sensitive and that adsorption increases
16 68
ing for a biological remediation strategy, important ques-
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tions to be answered include:

r Are the organisms with the desired characteristics and

as solution pH increases (Ospina-Alvarez et al., 2006). It
has been found that the plant-associated bacteria possessed
maximum sorption capacity for the cationic metal ions at
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20 pH values between 4 and 6. At pH below 3, uptakes of cop- 72
activities present at the contaminated site? per, nickel and zinc were negligible, probably due to the 73
21
22 r What is their activity? cation competition effects with oxonium (hydronium) ion 74
H3 O+ (Klimmek et al., 2001).
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23 75
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r How is the algal–microbial community composition and In commercial applications, another factor affecting 76
25 function influenced by environmental parameters and biosorption activity, beside pH are the multi-omponent 77
26 process conditions? metal solutions (Volesky and Naja, 2005). The sorption pro- 78
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27 cesses were found to be slower in a mixed-metal solution 79

28 Algae-bacterial associations have been traditionally used than in the single-component metal solutions, and equi- 80
29 for pollution control, especially for the removal of inor- librium was reached after 5 h of the experiments. Reach- 81
30 ganic nutrients (Toumi et al., 2000). The most common ing the equilibrium point, copper and zinc were bound 82
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31 arrangements used are high rate algal ponds (HRAP) and 46 %, nickel 30 % and chromium 20 %. Moreover, during 83
32 the patented Algal Turf Scrubber (ATS), which employs the next 5 hours there was no evidence in further uptake 84
33 suspended biomass of common green algae (Chlorella, of metal ions (Volesky and Naja, 2005; Wang and Cheng, 85
34 Scenedesmus, Cladophora), and bacteria (Cyanobacteria 2009). It can be concluded that the kinetics of biosorption 86
35 such as Spirulina, Oscillatoria, Anabaena) or consortia of appears to be faster in the single-component systems in 87
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36 both. The above mentioned algal systems have been tested the comparison with the multicomponent one. It is prob- 88
37 for heavy metal removal (Peña et al., 2005). Toumi et al. ably due to the absence of competitive processes between 89
38 (2000) compared the heavy metal removal rates of tradi- metals and biomass. Generally, for the future of biosorp- 90
39 tional waste stabilization ponds (WSP) and a HRAP where tion technology, there are two trends of biosorption de- 91
40 both were receiving urban polluted water with trace concen- velopment for metal removal. One trend is to use hybrid 92
41 trations of Zn2+ , Cu2+ and Pb2+ . It was found that HRAP technology (algae/bacteria biomass) for pollutants removal 93
42 had a higher removal rate per unit volume per day, with (Tsezos, 2001), especially using living cells. Another trend 94
43 values up to 10 times more efficient in the case of Cu2+ . is to develop good commercial biosorbents, just like a kind 95
44 The values obtained could have resulted from the high pH of ion exchange resin, and to exploit the market with great 96
45 achieved as a result of algal photosynthesis that enhances endeavor (Volesky, 2007). 97
46 metal precipitation. Recently, molecular and non-molecular methods for 98
47 Adey et al. (1996) developed a system using consortia of the identification and characterization of plant-associated 99
48 filamentous cyanobacteria and suspended green algae for bacteria and their specific properties have been used to 100
49 treating polluted underground waters. This research proved assess the composition and activity of those consortia 101
50 their advantage for the efficient removal of heavy metals, found at heavy metal-contaminated sites. These techniques 102
51 and also the removal of chlorinated and aromatic organic promise to become complementary tools to classic chem- 103
52 compounds was observed. The authors hypothesized that ical and physiological analysis (heavy metal concentrations 104
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448 DETOXIFICATION MECHANISMS OF HEAVY METALS BY ALGAL–BACTERIA CONSORTIA

1 and speciation, redox potential, etc.) for monitoring Brinza, L, Dring, M.J., and Gavrilescu, M. (2007) Marine 53
2 spatial and temporal changes in microbial community micro- and macro-algal species as biosorbents for heavy 54
3 composition and function. Advances in understanding of metals. Environ. Eng. Manage. J., 6, 237–251. 55
4 the roles of these interactions in such processes, especially Bunke, G., and Buchholz, R. (1999) Metal Removal by 56

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5 for algal–bacterial consortia, together with the ability Biomass: Physico-Chemical Elimination Methods. Wiley- 57
6 to fine-tune their activities using the tools of molecular VCH Verlag, Weinheim. 58
7 biology, has led to the development of novel or improved Butler, M., Haskew, A. E., and Young, M. (1980) Copper 59

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8 metal bioremediation processes during the last years (Lloyd tolerance in the green alga Chlorella vulgaris. Plant Cell. 60
9 and Lovley, 2001). Environ., 3, 119–128. 61
10 Bruins, M.R., Kapil, S. and Oehme, F.W. (2000) Micro- 62
11 bial resistance to metals in the environment. Ecotox. Env. 63
12 28.7 Conclusions and future Safety, 45, 198–207. 64

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13 remarks Cobbett, C. and Goldsbrough, P. (2002) Phytochelatin and 65
14 metallothioneins: Roles in heavy metal detoxification and 66
15
Significant advances have been made in understanding the homeostasis. Annu. Rev. Plant Biol., 53, 159–182. 67
16
roles of algae and bacteria in mineral cycling, and in the Davis, T., Volesky, B. and Mucci, A. (2003) A review of the 68
application of these processes to the bioremediation of
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metals. Additional advances are expected in the study of
algal–bacterial interactions, focused on the use of new tech-
biochemistry of heavy metal biosorption by brown algae.
Water Res., 37, 4311–4330.
Gadd, G. (2000) Bioremedial potential of microbial mech-
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niques, such as genomic approaches, which will undoubt- anisms of metal mobilization and immobilization. Curr. 72
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22
nology. Gaur, J.P. and Rai, L.C. (2001) Heavy metal tolerance in al- 74
Extensive surveys of heavy metal tolerant algal-
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23 gae. In: Algal Adaptation to Environmental Stresses. Physi- 75

24
associated bacteria are needed in order to obtain new data ological, Biochemical and Molecular Mechanisms (eds L.C. 76
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for specific strains that can be isolated for biotechnological Rai and J.P. Gaur). Springer-Verlag, Berlin, pp. 363–388. 77
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applications such as biosorptive commercial designs. Stud- Klimmek, S., Stan, H.J., Wilke, A., Bunke, G. and Buchholz, 78
ies that revise particular detoxification/resistance mecha-
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they can involve in commercial applications for remediation 35, 4283–4288. 81
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of heavy metals in aqueous solutions. Especial attention is Kumar-Rai, P. (2008) Heavy metal pollution in aquatic 82
needed to identify candidate enzymes for genetic manipu-
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31 ecosystems and its phytoremediation using wetland 83

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lation, responsible for the production and transportation plants: an eco-sustainable approach. Int. J. Phytoreme- 84
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of specific molecules involved in uptake and detoxification diation, 10, 133–160. 85
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processes in algal–bacteria consortia. Kvesitadze, G., Khatisashvili, G., Sadunishvili, T. and Rams- 86
35 den, J.J. (2006) Biochemical Mechanisms of Detoxification 87
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