**3. Biosorption and general characteristics of absorption of metals with** *Synechocystis* **strains**

Heavy metals are discharged from various industries, such as smelters, electroplating facilities, metal refineries, textile, mining, ceramic and glass industries. Some of the chief metals studied in terms of biosorption are those that have the potential to cause most pollution and include lead, antimony, copper, mercury, cadmium, chromium and arsenic as well as radionuclides of elements such as Cobalt, Strontium, Uranium and Thorium [1]. These all have different properties, may exist as complexes, have different oxidation states and their nature may depend on the pH of the medium. The remediation of trace amounts of metals can be carried out via electrolytic extraction, separation processes such as reverse osmosis or dialysis, chemical precipitation or solvent extraction, evaporative methods, or absorption methods such as carbon ion-exchange resin adsorption. However, because of the global problem of metal remediation and the cost of clean-up, new methodologies have been investigated and bio‐ sorption falls into this category.

Biosorption offers the following advantages: the volume of chemical and biological sludge can be minimised, there are potentially low operating costs, the possibility of metal recovery and regeneration of the biosorbent afterwards. In recent years, there has also been a significant effort to search for new methods of metallic trace element removal that can be used *in situ* at contaminated sites. The mechanisms by which metal ions can attach to microbial surfaces can include van der Waals forces, electrostatic interactions, precipitation extracellularly, covalent bonding, redox interactions leading to oxidation or volatilisation (as with mercury) and precipitation, or a combination of such mechanisms. The negatively charged groups (carboxyl, hydroxyl and phosphoryl) of the bacterial cell surface can adsorb metal cations. Cation exchange capacity or ability to bind metals, which can be useful in predicting microorganismmetal interactions, has been determined from pH titration curves for the cyanobacteria *Anacystis nidulans* and *Synechocystis aquatilis* and the green alga *Stichococcus bacillaris*. The results suggest that the exchange capacity is dependent on the external pH of the environment [49]. Thus the physico-chemical environment plays a major role in addition to the binding materials themselves. Many organisms capable of secreting EPS are potential candidates [41] but the ability of cyanobacterial species to grow photoautotrophically in contaminated oligotrophic marine or fresh water environments together with the potential biomass availa‐ bility, their high sorption characteristics and their non-pathogenic nature makes them ideal candidates for such studies. The cyanobacterial cell surface with EPS consisting of polysac‐ charide, protein and lipid, together with adsorbed material make them ideal candidates. In addition, model strains such as *Synechocystis* PCC 6803 offer a tool kit of genomic techniques to explore biosorption and examine potential genetic improvements that may be possible.

#### **3.1. Absorption of Cr(VI) and CD(II) by** *Synechocystis*

Ozturk *et al* recently reported the removal of Chromium, Cr (VI) and Cadmium, Cd (II) by *Synechocystis sp*. BASO671 [50]. In their experiments, strains with a biomass density of 2.5 at OD665nm were exposed to 10 ppm Cr(VI), Cd(II) and a Cr(VI) + Cd(II) mixture for 7 days in BG11 medium (the standard laboratory growth medium for *Synechocystis*), at 25o C with a light (3000 lux) and a dark cycle of 12/12 h, with shaking. Metal removal was determined as metal in the medium, metal adsorbed onto the surfaces of the cells, and metal accumulated within the cells determined by atomic absorption. Consequently, around 90% of the 10 ppm Cd(II) was absorbed onto the cell surfaces and none accumulated intercellularly. With Cr(VI), some 14% of the 10 ppm Cr(VI) was found to be intercellular with none adsorbed onto the cell surface [50]. In the case of Cd(II), there was an extremely fast adsorption to the surface layers. When mixed metal solutions were added, the preference for Cd(II) binding was confirmed with less binding of Cr(VI). The results suggest that competition for functional groups on the surface of cells may favour one type of metal species over another and suggests that biosorption may be highly dependent on the initial binding kinetics. This study also highlighted a number of interesting issues relating to the production of EPS. The productivity of EPS in strains exposed to Cd(II) compared to strains exposed to Cr(VI) was superior with both strains producing less than controls without metal exposure [50]. However, higher metal exposures (beyond 10 ppm) appeared to enhance the production of EPS, further suggesting the possibility of a stress response to the metal species.

The nature of the monomer composition of *Synechocystis* EPS was monitored as a function of the addition of the single metals [Cd(II) and Cr(VI)] and the mixture of both [50]. Relative to the control, Cr(VI) decreased the uronic content (~25%) of the EPS, while Cd(II) and the mixture increased the uronic acid content (~25%). There was no change in glucose content, a general reduction in rhamnose content, an increase in xylose content (~100%) with Cr(VI), which reduced to zero with Cd(II). Glucuronic and galacturonic levels were increased by the presence of both metals [50]. These results suggest that not only is EPS induced in response to metals but that the nature of the EPS alters and that this alteration may be metal specific, at least in the case of chromium and cadmium in *Synechocystis*.

contaminated sites. The mechanisms by which metal ions can attach to microbial surfaces can include van der Waals forces, electrostatic interactions, precipitation extracellularly, covalent bonding, redox interactions leading to oxidation or volatilisation (as with mercury) and precipitation, or a combination of such mechanisms. The negatively charged groups (carboxyl, hydroxyl and phosphoryl) of the bacterial cell surface can adsorb metal cations. Cation exchange capacity or ability to bind metals, which can be useful in predicting microorganismmetal interactions, has been determined from pH titration curves for the cyanobacteria *Anacystis nidulans* and *Synechocystis aquatilis* and the green alga *Stichococcus bacillaris*. The results suggest that the exchange capacity is dependent on the external pH of the environment [49]. Thus the physico-chemical environment plays a major role in addition to the binding materials themselves. Many organisms capable of secreting EPS are potential candidates [41] but the ability of cyanobacterial species to grow photoautotrophically in contaminated oligotrophic marine or fresh water environments together with the potential biomass availa‐ bility, their high sorption characteristics and their non-pathogenic nature makes them ideal candidates for such studies. The cyanobacterial cell surface with EPS consisting of polysac‐ charide, protein and lipid, together with adsorbed material make them ideal candidates. In addition, model strains such as *Synechocystis* PCC 6803 offer a tool kit of genomic techniques to explore biosorption and examine potential genetic improvements that may be possible.

Ozturk *et al* recently reported the removal of Chromium, Cr (VI) and Cadmium, Cd (II) by *Synechocystis sp*. BASO671 [50]. In their experiments, strains with a biomass density of 2.5 at OD665nm were exposed to 10 ppm Cr(VI), Cd(II) and a Cr(VI) + Cd(II) mixture for 7 days in BG11

lux) and a dark cycle of 12/12 h, with shaking. Metal removal was determined as metal in the medium, metal adsorbed onto the surfaces of the cells, and metal accumulated within the cells determined by atomic absorption. Consequently, around 90% of the 10 ppm Cd(II) was absorbed onto the cell surfaces and none accumulated intercellularly. With Cr(VI), some 14% of the 10 ppm Cr(VI) was found to be intercellular with none adsorbed onto the cell surface [50]. In the case of Cd(II), there was an extremely fast adsorption to the surface layers. When mixed metal solutions were added, the preference for Cd(II) binding was confirmed with less binding of Cr(VI). The results suggest that competition for functional groups on the surface of cells may favour one type of metal species over another and suggests that biosorption may be highly dependent on the initial binding kinetics. This study also highlighted a number of interesting issues relating to the production of EPS. The productivity of EPS in strains exposed to Cd(II) compared to strains exposed to Cr(VI) was superior with both strains producing less than controls without metal exposure [50]. However, higher metal exposures (beyond 10 ppm) appeared to enhance the production of EPS, further suggesting the possibility of a stress

The nature of the monomer composition of *Synechocystis* EPS was monitored as a function of the addition of the single metals [Cd(II) and Cr(VI)] and the mixture of both [50]. Relative to the control, Cr(VI) decreased the uronic content (~25%) of the EPS, while Cd(II) and the mixture

C with a light (3000

medium (the standard laboratory growth medium for *Synechocystis*), at 25o

**3.1. Absorption of Cr(VI) and CD(II) by** *Synechocystis*

58 Advances in Bioremediation of Wastewater and Polluted Soil

response to the metal species.

SEM (Scanning Electron Microscopy) and EDS (Energy Dispersive X-ray Spectroscopy) analysis of *Synechocystis* exposed to Cd(II) and Cr(VI) was also carried out and demonstrated that surface roughness was increased, with direct metal binding observed via EDS [50]. Fourier Transform Infra-Red Spectroscopy (FTIR) was also utilised to determine the nature of the functional groups involved in metal binding. Metal binding changed peaks in various parts of the FTIR spectrum, at 3400 cm-1 (hydroxyl and amino groups), at 2933 cm-1 (aromatic groups), at 1600-1725 cm-1 (carboxylic acid groups) and at 1034-1025 cm-1 (possibly carboxyl groups of polysaccharides), indicating a role for these groups in metal binding [50].

To determine the optimal biosorption process, a comparative study was carried out using dried, immobilised and live cultures of *Synechocystis sp*. with calcium alginate beads used as the immobilization substrate [51]. The removal efficiency by biosorbent was studied as a function of pH (2-8), temperature (20–40°C), initial cadmium ion concentration (50–300 mg/L), and contact time (0–120 min). The maximum biosorption capacities of the dried, immobilized dried, and immobilized live *Synechocystis sp*. and plain Ca-alginate beads were 75.7, 4.9, 4.3, and 3.9 mg.g-1 respectively, under optimum conditions, with the biosorption equilibrium taking 15 min. These results indicated that dried *Synechocystis* biomass was superior for Cd(II) ion removal from aqueous solution by a factor of 15 fold. Interestingly, the dried material could be reused up to 5 times via adsorption and desorption cycles without significant loss in the biosorption capacity [51].

Given the large number of variables that might affect metal biosorption, an approach using response surface methodology (RSM) was employed to study the removal of Cd(II) by *Synechocystis*. RSM allows the study of the effect of several factors influencing the response to metals by varying these factors simultaneously [52]. Utilisation of this approach (RSM) has led to optimization of the critical parameters responsible for higher Cd(II) removal by *Synechocystis pevalekii*. The optimum value of pH, biomass concentration, and metal concentration were pH 6.48, 0.25 mg protein.ml-1 and 5 ug.ml-1 respectively. Modelling data predicted that 4.29 μg.ml −1 Cd(II) would be removed and when experimentally determined, it was found that 4.27 μg.ml −1 Cd(II) removal occurred [52]. This data correlated well with model data and indicated the potential utility of such models for predicting biosorption rates.

#### **3.2. Binding of other important metals by** *Synechocystis*

Binding of EPS from *Synechocystis* to Cu(II) was investigated using fluorescence spectroscopy [53]. Under different test conditions, *Synechocystis sp*. PCC 6803, grown in BG-11 media, with 72 μmol photon. m-2 s-1 of light intensity, a photoperiod of 14 hours light to 10 hours dark at 250 C, was subjected to 0.5-4 μg.ml-1 of Cu (II). Three fluorescence peaks were found in the excitation-emission fluorescence spectra of EPS. Fluorescence of peak A (Ex/Em= 275/452 nm) and peak C (Ex/Em= 350/452 nm) originated from humic-like substances and fluorescence of peak B (Ex/Em= 275/338 nm) was attributed to protein-like substances. Fluorescence of peaks A, B, and C could be quenched by Cu(II). The binding constants indicated that binding to peak A>peak B>peak C, implying that the humic-like substances in EPS have greater Cu(II) binding capacity than the protein-like substances. The binding site number in EPS-Cu(II) complexes for peaks A, B, and C was less than 1 suggesting negative co-operativity between multiple binding sites and the presence of more than one Cu binding site.

Adsorption of metals to cells can be determined through isotherms, which are defined as the amount of adsorbate (in this case metals) bound to adsorbent either as a function of concen‐ tration in liquid phase or pressure in the gas phase at constant temperature. The most common isotherms for the evaluation of adsorption kinetics are listed in Table 3. The reader is referred to [1] for a detailed examination on biosorption isotherms and equilibrium sorption studies in relation to metal biosorption. Absorption isotherms (Table 3) for Cu(II) were determined and indicated that physical adsorption followed Langmuir behaviour with the equilibrium being obtained rather slowly and possibly showing monolayer binding [54]. Absorption was shown to be a function of pH with copper hydroxides limiting absorption at alkaline pH [54]. The results suggested that not only is biomass important in metal absorption but also illustrates the importance of pH dependence with alkaline or acidic conditions promoting complexing of metallic ions rather than biomass absorption. For example, it was observed in the case of Cd(II) that complex forms were less likely to be adsorbed onto EPS of *Synechocystis aquatilis* particularly in the presence of chloride [55]. In mixed metal streams there may be competition between various metal cations for binding onto EPS. Whereas little work has been carried out on this area in *Synechocystis*, various selectivity series have been published which reflect such competition, e.g. binding of Al3+ > Ag+ > Cu2+> Cd2+ > Ni2+ > Pb2+ >Zn2+ >Co2+ > Cr3+ for *Chlorella vulgaris*, and binding of Cu2+ > Sr2+ > Zn2+ >Mg2+ > Na+ for *Vaucheria* sp. [57, 1]. The presence of ions that can complex with the metal may have dramatic effects on the overall biosorption process, again indicating that variability is dependent not only on biomass factors but also on compositional aspects of the effluents being treated.

Many industries, such as coatings, automotive, storage batteries, aeronautical and steel industries generate large quantities of wastewater containing various concentrations of lead. Data from storage battery producers demonstrated that the pH value of wastewater discarded by these industries ranged between pH 1.6 and 2.9, while the concentration of soluble lead was in the range of 5–15 mg.L-1 [57]. The relationship between binding of Pb(II) and Cd(II) on the cell ultrastructure, growth and pigment content of *Synechocystis* PCC 6803 [58] was examined and a dependence on metal concentration was demonstrated. At low level absorption, few growth effects were observed, however as levels of Pb(II) increased to greater than 4 mg.L-1, cell ultrastructure changes were observed including thylakoid deterioration suggestive of high levels of accumulation of intercellular Pb(II). Such accumulation could be useful in Pb contaminated environments. In similar studies, the optimum initial pH of biosorption was found to be pH 4.5 with the equilibrium Pb(II) uptake of 2.265 mg.g-1 at this pH [57].


**Table 3.** Isotherms utilized for adsorption kinetics.

and peak C (Ex/Em= 350/452 nm) originated from humic-like substances and fluorescence of peak B (Ex/Em= 275/338 nm) was attributed to protein-like substances. Fluorescence of peaks A, B, and C could be quenched by Cu(II). The binding constants indicated that binding to peak A>peak B>peak C, implying that the humic-like substances in EPS have greater Cu(II) binding capacity than the protein-like substances. The binding site number in EPS-Cu(II) complexes for peaks A, B, and C was less than 1 suggesting negative co-operativity between multiple

Adsorption of metals to cells can be determined through isotherms, which are defined as the amount of adsorbate (in this case metals) bound to adsorbent either as a function of concen‐ tration in liquid phase or pressure in the gas phase at constant temperature. The most common isotherms for the evaluation of adsorption kinetics are listed in Table 3. The reader is referred to [1] for a detailed examination on biosorption isotherms and equilibrium sorption studies in relation to metal biosorption. Absorption isotherms (Table 3) for Cu(II) were determined and indicated that physical adsorption followed Langmuir behaviour with the equilibrium being obtained rather slowly and possibly showing monolayer binding [54]. Absorption was shown to be a function of pH with copper hydroxides limiting absorption at alkaline pH [54]. The results suggested that not only is biomass important in metal absorption but also illustrates the importance of pH dependence with alkaline or acidic conditions promoting complexing of metallic ions rather than biomass absorption. For example, it was observed in the case of Cd(II) that complex forms were less likely to be adsorbed onto EPS of *Synechocystis aquatilis* particularly in the presence of chloride [55]. In mixed metal streams there may be competition between various metal cations for binding onto EPS. Whereas little work has been carried out on this area in *Synechocystis*, various selectivity series have been published which reflect such

ions that can complex with the metal may have dramatic effects on the overall biosorption process, again indicating that variability is dependent not only on biomass factors but also on

Many industries, such as coatings, automotive, storage batteries, aeronautical and steel industries generate large quantities of wastewater containing various concentrations of lead. Data from storage battery producers demonstrated that the pH value of wastewater discarded by these industries ranged between pH 1.6 and 2.9, while the concentration of soluble lead was in the range of 5–15 mg.L-1 [57]. The relationship between binding of Pb(II) and Cd(II) on the cell ultrastructure, growth and pigment content of *Synechocystis* PCC 6803 [58] was examined and a dependence on metal concentration was demonstrated. At low level absorption, few growth effects were observed, however as levels of Pb(II) increased to greater than 4 mg.L-1, cell ultrastructure changes were observed including thylakoid deterioration suggestive of high levels of accumulation of intercellular Pb(II). Such accumulation could be useful in Pb contaminated environments. In similar studies, the optimum initial pH of biosorption was

found to be pH 4.5 with the equilibrium Pb(II) uptake of 2.265 mg.g-1 at this pH [57].

> Cu2+> Cd2+ > Ni2+ > Pb2+ >Zn2+ >Co2+ > Cr3+ for *Chlorella*

for *Vaucheria* sp. [57, 1]. The presence of

binding sites and the presence of more than one Cu binding site.

60 Advances in Bioremediation of Wastewater and Polluted Soil

competition, e.g. binding of Al3+ > Ag+

*vulgaris*, and binding of Cu2+ > Sr2+ > Zn2+ >Mg2+ > Na+

compositional aspects of the effluents being treated.

**Glossary of Terms:** qe, amount of adsorbate bound to the adsorbent at equilibrium (mg.g-1); Kf, Freundlich isotherm constant (mg.g-1) (dm3 .g-1) related to adsorption capacity; Ce, equili‐ brium concentration (mg.L-1); n, adsorption intensity; Qmax, maximum monolayer coverage capacities (mg.g-1); b, Langmuir isotherm constant (dm3 .mg-1); CBET, BET adsorption isotherm relating to the energy of the surface interaction (L.mg-1); Cs, adsorbate monolayer saturation concentration (mg.L-1); qs, theoretical isotherm saturation capacity (mg.g-1); AT, Tempkin isotherm equilibrium binding constant (L.g-1); B, constant related to heat of sorption (J. mol-1); bT, Temkin isotherm constant; R, Universal gas constant (8.314 J.mol-1. 0 K-2); T, temperature at 2980 K.

In an experimental system treating mixed metal wastes in an algal pond using *Synechocystis salina*, it was shown that 60% Cr(VI), 66% Fe(II), 70% Ni and 77% Hg was removed after 13 days of treatment. This reduction correlated with surface absorption [63], however details of the initial metal concentrations were not given.

Antimony (Sb), a non-essential element in biological systems, poses a major problem in mining areas, particularly in China. Around 80% of the world's reserves are deposited here, leaving aquatic environments in the mining areas polluted by long term leaching [64]. Conventional methodologies to remove Sb are limited to precipitation methods such as alum, lime or ferric salts precipitation. Biosorption using *Synechocystis* has been investigated as a potential economic alternative. Here, the added attraction of using *Synechocystis* lies in the fact that it is a common inhabitant of aquatic environments in the South China region. Absorption of Sb by EPS in *Synechocystis* FASHB898 was examined. It was observed that some 50% of the Sb was absorbed in the first 30 minutes, with equilibrium being reached after 1 hour. Sorption concentrations of 2.61 mg.L-1 of Sb per gram dry weight of biomass were determined [64]. It was shown that using initial Sb concentrations of 100 mg.L-1 that up to 1.92 mg.g-1 was absorbed by EPS, with some 2.64 mg.g-1 being located intercellularly. The results of FTIR analysis confirmed that Sb binds to EPS via protein and carbohydrate group interactions as indicated for many other metals. Again it has been suggested that EPS absorption may act as a stress barrier to protect the cells from such metals [64] in natural environments.

In a study examining resistance to Nickel (Ni), 10 different *Synechocystis* strains were initially examined for nickel resistance. The EC50 values of the 10 isolates ranged from 2.56 to 17.41 mg.L-1, while the EPS concentrations of the 10 isolates ranged from 44 to 143 mg.L-1. *Synecho‐ cystis sp*. BASO403 and *Synechocystis sp*. BASO404 were chosen on the basis of greatest resistance and highest EPS to examine Ni(II) biosorption [65], thus illustrating the potential utility of certain *Synechocystis* strains for (Ni) removal.

Engineered nanoparticles, particularly particles containing titanium dioxide (TiO2) are finding application in industry particularly in paints, cosmetics and as part of solar cells. Although relatively inert, TiO2 can be activated by UV light producing reactive oxygen species which can be antibacterial [66]. Thus with the increased potential use of such nanomaterial's, biological treatment regimens could be compromised by the killing effects on bacterial communities in treatment facilities. It has been demonstrated that *Synechocystis* PCC 6803 has significant ability to biosorb TiO2 [67]. The response of wild-type *Synechocystis,* which pos‐ sesses abundant EPS surrounding the cells, to that of an EPS-depleted mutant was also examined and indicated that the EPS play a crucial role in S*ynechocystis* protection against cell killing caused by TiO2 nanoparticles [67] indicating that it may have potential in remediation of this emerging class of compound.

Manganese (Mn) uptake to cells of *Synechocystis* was measured in cells incubated with Mn solutions. *Synechocystis* cells were shown to be able to take up 150 μM of Mn(II) or Mn(IV) in 48 hours [68]. The predominant accumulation of Mn was associated with the outer membrane for both Mn substrates. Large manganese deposits were found associated with the EPS of *Synechocystis* cells. TEM analysis demonstrated that Mn accumulation occurred on the cell surface and analysis demonstrated that the attached material was manganese phosphate. This bound material withstood multiple washes and appeared to be quite stably bound, indicative of tight binding and its potential as a biosorption material [68].

Arsenic (As) is a widely used component of batteries, a dopant in semiconductors and in optoelectronics. Additionally, it is used in some pesticides and herbicides. Toxicity to humans occurs mainly via drinking water and it is thus important to remove even trace amounts from water. Arsenic is present in two biologically active forms, As(V) and As(III), depending on the redox potential of the environment. Oxidation of As(III) to As(V) is a detoxification process, since As(V) is less toxic than As(III) [69] while arsenate methylation is also a common detoxi‐ fying mechanism in many microbial systems. Examination of the response of *Synechocystis* PCC 6803 to arsenic revealed that the organism can grow and accumulate arsenic to high levels. Biomass of *Synechocystis* could accumulate up to 0.38 g.kg-1 dry weight when treated with 100 μM sodium arsenite over a 14 day period [70]. When treated with arsenate for six weeks, *Synechocystis* produced volatile arsenicals. An *Ars*M homolog of a known arsenic methylases from *Synechocystis* sp. PCC 6803 was purified and shown to play a role in methylating arsenite *in vitro* with trimethylarsine as the end product. This illustrated the potential utility of this organism in detoxification of arsenic compounds. Amongst a number of cyanobacteria examined, *Synechocystis* was shown to have one of the highest levels of tolerance to arsenic and to be able to accumulate arsenic at a high rate [71]. Genomic studies on tolerance to arsenic have shown that arsenic resistance in *Synechocystis* PCC 6803, in addition to *ars*M (the meth‐ ylase), was mediated by the *ars*BHC operon which was regulated by *ars*R and two additional arsenate reductases encoded by the *ars*I1 and *ars*I2 genes [72]. *Ars*B encoded an arsenite transporter, *ars*H an FMN-quinone reductase and *ars*C a FMN-quinone reductase. Using a gene array study, a highly orchestrated response to arsenic was observed in *Synechocystis* with 421 genes involved, of which, 179 were induced while 242 were repressed on arsenic addition based on transcriptomic studies [72]. These arrays of genes, whose expression was modified by arsenic were shown to be associated with the repression of growth, the lowering of energy metabolism and the induction of general stress responses which form part of the core tran‐ scriptional response to stress in many organisms. The most highly induced genes were those for the *ars*BHC operon [72].

In *Synechocystis* PCC 6803 similar systems for detoxification of mercury are observed as found in many other microbial systems. The protein Grx1, annotated as Slr1562 in the *Synechocystis* genome, selectively interacts with the putative mercuric reductase protein, Slr1849, in PCC 6803. Grx1 which is designated *Mer*A- like, appears to play a major role in catalysing NADPHdriven reduction of mercuric and uranyl ions [73]. In addition to a defence role against the toxicity of such metals, the presence of this system may also have a bioremediation role in mixed effluents. However, its potential has not been realised nor have comparative studies been carried out comparing its detoxification abilities with other organisms.

Sorption of caesium (Cs) by *Synechocystis* PCC 6803 has been examined at concentrations between 1 to 100 μM Cs in the presence of three clay types [74]. Binding was found to occur in two distinct phases, the first step was shown to be a rapid uptake not dependent on light to the clay-cell material and a second slower step which was inhibited by metabolic inhibitors. This data indicated a role for cell and energy dependent uptake, which was pH and salt dependent. The data indicated that the clay adsorption played a significant role supplemented by a slower binding step and accumulation by the cyanobacteria. The practical ability to remove caesium using *Rhodobacter* was analysed from contaminated mud in Japan after the Fukushima accident. Approximately 90% of the Cs found in the mud in a swimming pool could be removed by immobilized cells in a 3 day period [75]. The treatment was repeated 3 times and efficiencies remained high with 84% of the remaining material being sorbed on the second treatment and a further 78% sorbed on the third batch treatment. Here Cs attachment was not altered by nitric acid treatment below pH 2 indicating a strong sediment attachment whereas cell sorption showed major utility. This study indicated the potential for cell sorption in dealing with certain tightly attached radionuclides.

#### **3.3. Reactor configurations for biosorption**

a common inhabitant of aquatic environments in the South China region. Absorption of Sb by EPS in *Synechocystis* FASHB898 was examined. It was observed that some 50% of the Sb was absorbed in the first 30 minutes, with equilibrium being reached after 1 hour. Sorption concentrations of 2.61 mg.L-1 of Sb per gram dry weight of biomass were determined [64]. It was shown that using initial Sb concentrations of 100 mg.L-1 that up to 1.92 mg.g-1 was absorbed by EPS, with some 2.64 mg.g-1 being located intercellularly. The results of FTIR analysis confirmed that Sb binds to EPS via protein and carbohydrate group interactions as indicated for many other metals. Again it has been suggested that EPS absorption may act as a stress

In a study examining resistance to Nickel (Ni), 10 different *Synechocystis* strains were initially examined for nickel resistance. The EC50 values of the 10 isolates ranged from 2.56 to 17.41 mg.L-1, while the EPS concentrations of the 10 isolates ranged from 44 to 143 mg.L-1. *Synecho‐ cystis sp*. BASO403 and *Synechocystis sp*. BASO404 were chosen on the basis of greatest resistance and highest EPS to examine Ni(II) biosorption [65], thus illustrating the potential

Engineered nanoparticles, particularly particles containing titanium dioxide (TiO2) are finding application in industry particularly in paints, cosmetics and as part of solar cells. Although relatively inert, TiO2 can be activated by UV light producing reactive oxygen species which can be antibacterial [66]. Thus with the increased potential use of such nanomaterial's, biological treatment regimens could be compromised by the killing effects on bacterial communities in treatment facilities. It has been demonstrated that *Synechocystis* PCC 6803 has significant ability to biosorb TiO2 [67]. The response of wild-type *Synechocystis,* which pos‐ sesses abundant EPS surrounding the cells, to that of an EPS-depleted mutant was also examined and indicated that the EPS play a crucial role in S*ynechocystis* protection against cell killing caused by TiO2 nanoparticles [67] indicating that it may have potential in remediation

Manganese (Mn) uptake to cells of *Synechocystis* was measured in cells incubated with Mn solutions. *Synechocystis* cells were shown to be able to take up 150 μM of Mn(II) or Mn(IV) in 48 hours [68]. The predominant accumulation of Mn was associated with the outer membrane for both Mn substrates. Large manganese deposits were found associated with the EPS of *Synechocystis* cells. TEM analysis demonstrated that Mn accumulation occurred on the cell surface and analysis demonstrated that the attached material was manganese phosphate. This bound material withstood multiple washes and appeared to be quite stably bound, indicative

Arsenic (As) is a widely used component of batteries, a dopant in semiconductors and in optoelectronics. Additionally, it is used in some pesticides and herbicides. Toxicity to humans occurs mainly via drinking water and it is thus important to remove even trace amounts from water. Arsenic is present in two biologically active forms, As(V) and As(III), depending on the redox potential of the environment. Oxidation of As(III) to As(V) is a detoxification process, since As(V) is less toxic than As(III) [69] while arsenate methylation is also a common detoxi‐ fying mechanism in many microbial systems. Examination of the response of *Synechocystis* PCC 6803 to arsenic revealed that the organism can grow and accumulate arsenic to high levels. Biomass of *Synechocystis* could accumulate up to 0.38 g.kg-1 dry weight when treated with 100 μM sodium arsenite over a 14 day period [70]. When treated with arsenate for six weeks,

barrier to protect the cells from such metals [64] in natural environments.

utility of certain *Synechocystis* strains for (Ni) removal.

62 Advances in Bioremediation of Wastewater and Polluted Soil

of tight binding and its potential as a biosorption material [68].

of this emerging class of compound.

Use of diverse biomass material as a biosorption candidate has been infrequently examined. Free biomass, such as microbial cells suffers from a number of disadvantages, including low mechanical strength, the small size of individual microbial cells and the difficulty of separating cells once they have been utilised to adsorb metals in liquid effluents. Several processes using biomass immobilisation have been investigated to overcome these disadvantages. Immobili‐ sation of biomass in bio-towers, trickle filters, airlift reactors or rotating systems where microbial biofilms play a key role have been examined [76]. As the immobilised biomass grows and its size increases, there is natural expansion and leakage of the biomass, which can then be collected as a microbial sludge. Provided the metals in the wastewater do not have a deleterious effect on the biofilm or other co- habiting organisms, this system can work well. The advantage of rotating immobilised systems, in the case of cyanobacteria, would be that they can still be exposed to light, as opposed to bio-tower systems. Moving sand bed reactors have also been used [77] to develop consortia to treat mixed metal pollutant effluents, which could also provide enough light for cyanobacterial consortia. Technologies and processes for metal recovery are reviewed in [78].

Dried or dead cells may absorb more metals than live cells and for this reason encapsulation of biomass may be advantageous [79], which would mean the utilisation of different process configurations. Although dead cells or biomass can be used, there is little data on the relative merits when compared to live cells. Generally, in addition to metallic pollution, natural waste materials may contain other substances that need remediation, and thus having live biomass may, on occasion, be more advantageous. It is envisaged however that should biosoption be employed at scale then some form of continuous flow through system would need to be employed. Many variables need to be considered; including biomass concentration, pollutant metal concentration, pH of the system, and flow rate. As such studies have been carried out at small laboratory scale there is little data available on large scale systems particularly with cyanobacteria.

Metals absorbed by EPS or biomass are often required to undergo elution in subsequent processes. The nature of such elution processes is dependent on whether the biomass needs to be reused or recycled. Acid or alkali desorption can generally be used for elution [1]. For particular cases, such as precious metal recovery, selective desorption may be used. In the case of radionucleotide recovery, this can occur via combustion and ash removal. In other cases simple liquid extraction may be used on occasion with a variety of solvents. The desorption procedures utilised are thus dependent on the metal, its value and whether the biomass will be reused.
