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Applied and Environmental Microbiology, May 2008, p. 2797-2804, Vol. 74, No. 9
0099-2240/08/$08.00+0     doi:10.1128/AEM.02212-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Sheathless Mutant of Cyanobacterium Gloeothece sp. Strain PCC 6909 with Increased Capacity To Remove Copper Ions from Aqueous Solutions

Ernesto Micheletti,¹ Sara Pereira,^2,3 Francesca Mannelli,¹ Pedro Moradas-Ferreira,^2,4 Paula Tamagnini,^2,3 and Roberto De Philippis^1*

Department of Agricultural Biotechnology, University of Florence, Piazzale delle Cascine 24, I-50144, Florence, Italy,¹ IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal,² Departamento de Botânica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre 1191, 4150-181 Porto, Portugal,³ Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Largo Abel Salazar 2, 4099-003 Porto, Portugal⁴

Received 28 September 2007/ Accepted 27 February 2008

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The cyanobacterium Gloeothece sp. strain PCC 6909 and its sheathless mutant were tested for their abilities to remove copper ions from aqueous solutions, with the aim of defining the role of the various outermost polysaccharidic investments in the removal of the metal ions. Microscopy studies and chemical analyses revealed that, although the mutant does not possess a sheath, it releases large amounts of polysaccharidic material (released exocellular polysaccharides [RPS]) into the culture medium. The RPS of the wild type and the mutant are composed of the same 11 sugars, although they are present in different amounts, and the RPS of the mutant possesses a larger amount of acidic sugars and a smaller amount of deoxysugars than the wild type. Unexpectedly, whole cultures of the mutant were more effective in the removal of the heavy metal than the wild type (46.3 ± 3.1 and 26.7 ± 1.5 mg of Cu²⁺ removed per g of dry weight, respectively). Moreover, we demonstrated that the contribution of the sheath to the metal-removal capacity of the wild type is scarce and that the RPS of the mutant is more efficient in removing copper. This suggests that the metal ions are preferably bound to the cell wall and to RPS functional groups rather than to the sheath. Therefore, the increased copper binding efficiency observed with the sheathless mutant can be attributed to the release of a polysaccharide containing larger amounts and/or more accessible functional groups (e.g., carboxyl and amide groups).

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Cyanobacteria are a large and widespread group of photoautotrophic microorganisms that combine the ability to perform an oxygenic plant-like photosynthesis with typical prokaryotic features (40). Outside their atypical gram-negative cell walls (15), many strains possess outermost structures, mainly of a polysaccharidic nature, that differ in thickness and consistency. These structures can be referred to as sheaths, capsules, and slimes (4). The term sheath is applied to the usually thin and electron-dense layer surrounding the cells or the cell groups; the capsule refers to the thick and gelatinous layer intimately associated with the cell surface and presenting sharp outlines, whereas the term slime is used to designate the mucilaginous material that is dispersed around the organism but does not reflect the shape of the cells. During cell growth, aliquots of these polysaccharides can be released into the surrounding medium (referred to as released exocellular polysaccharides [RPS]), causing a progressive increase in its viscosity (4). The cyanobacterial exopolysaccharides (EPSs; a general term which includes both the polysaccharidic structures that remain intimately associated with the cell, i.e., the sheath and capsule, and those released into the culture medium, i.e., the RPS) exhibit some typical characteristics that distinguish them from the polymers synthesized by other bacteria: they frequently contain two different uronic acids, they build a polymer particularly rich in negatively charged groups, they possess a larger number of different monosaccharides, which increases the number of possible conformations of the polymer, and they contain sulfate groups, a unique feature among prokaryotes and one that is shared by the EPSs produced by Archaea (5, 31).

At present, heavy metals are one of the most widespread causes of pollution, and their continuous accumulation in water bodies and soils constitutes a serious hazard to both the environment and human health (11, 20). The use of EPS-producing microorganisms (or isolated EPSs) is a valid alternative to conventional chemical and physicochemical methods to remove metallic cations from polluted waters (35). This new technology presents advantages such as the use of natural and renewable sources, reduced costs, rapid kinetics of metal removal, the ability to remove metallic ions present at low concentrations, the possibility to treat contaminated waters simultaneously with several different metal ions, and the possibility of recovering valuable metals from the biosorbent (6, 18). In this context, EPS-producing cyanobacteria appear to be promising candidates due to the unique characteristics of their polysaccharidic envelopes (see above). The efficiency of cyanobacterial EPS in the removal of metal ions has been discussed previously, with an emphasis on the monosaccharidic composition of the polymer, the isolation of EPS, and the subsequent utilization with removal assays (5, 8, 21). Although the chemical composition of the sheaths of several cyanobacterial strains has been determined (14, 15, 32, 33, 38, 39) and the importance of the capsules and RPS in the metal-removal process has been recognized (6), information about the exact contribution of each type of EPSs and/or functional group to the biosorption of the metal is still very limited.

This work aimed to understand the role of the various outermost polysaccharidic investments in the process of copper removal by the unicellular N₂-fixing cyanobacterium Gloeothece sp. strain PCC 6909. This strain is characterized by a well-defined laminated sheath that encloses cells and cell groups, maintaining a firm colonial structure. In this study, both the wild-type and a sheathless mutant, previously obtained by chemical mutagenesis, were used to elucidate the contribution of each type of EPS to the metal-removal process. Moreover, chemical and physical analyses were performed to identify the major sites responsible for the metal binding.

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Organisms and growth conditions.
The unicellular cyanobacterium Gloeothece sp. strain PCC 6909 wild type (obtained from the Pasteur Culture Collection, Paris, France) and its sheathless mutant strain PCC 6909/1 (obtained by chemical mutagenesis using nitrosoguanidine, as described by S. Shestakov, Moscow, Russia, and provided by L. Stal, Nederlands Instituut voor Ecologie [NIOO-KNAW]), The Netherlands) were grown in BG11₀ (28) supplemented with 0.375 g liter^–1 NaNO₃, in an Orbital incubator (Gallenkamp, Loughborough, United Kingdom) at 100 revolutions min^–1, at 30 ± 1°C, and under continuous illumination provided by cool white fluorescent tubes (100 µmol photon m^–2 s^–1; photosynthetic active radiation). The 16S rRNA gene sequence of the Gloeothece sp. strain PCC 6909/1 sheathless mutant is available from GenBank under the accession number EU499305.

Light microscopy.
Cells were observed with a Reichert-Jung Polyvar photomicroscope (Vienna, Austria) using Nomarski differential interference contrast before and after they were negatively stained with India ink or after being stained with Alcian blue (in 3% acetic acid [pH 2.5]) (27).

TEM.
Cells were fixed in 2% glutaraldehyde in 50 mM sodium cacodylate buffer (pH 7.2) and 2% osmium tetroxide in the same buffer, dehydrated in ethanol (25% to 100%), and embedded in Epon. Sections were contrasted with uranyl acetate and lead citrate and visualized by transmission electron microscopy (TEM) with a Zeiss EM C10 (Gottingen, Germany).

SEM and EDS.
Culture samples were placed into dialysis tubes (12 to 14 kDa of molecular mass cutoff; Medicell International Ltd., London, United Kingdom) and dialyzed against water at pH 5.0 for a minimum of 16 h. The culture confined in the dialysis tubes was then transferred into a 30-mg liter^–1 copper solution, and after 24 h an aliquot was transferred onto an aluminum support and dried in a desiccator at room temperature. A graphite layer was applied on the sample surface by thermal dispersion in the chamber of a JEE-4B vacuum apparatus (Jeol Electron Optics Laboratory Co., Ltd., Tokyo, Japan). The elemental composition of the samples was determined by scanning electron microscopy (SEM) (Philips 515; Eindhoven, The Netherlands) coupled to an energy-dispersive spectrometry (EDS) machine (EDAX Inc., Mahwah, NJ). The concentration of the elements in the samples was measured by using electron probe microanalysis performed at 25 kV accelerating voltage and with an accumulation of X radiation for 40 s.

Copper removal assays.
Aliquots of 250 ml of both cultures, the Gloeothece sp. strain PCC 6909 wild type and the sheathless mutant, were placed into dialysis tubes (12 to 14 kDa of molecular mass cutoff; Medicell International Ltd., London, United Kingdom) and dialyzed against water at pH 5.0 for at least 16 h. For the experiments to determine the effects of the acid pretreatment, the dialysis tubes containing the cultures were first dipped into a 0.1 M HCl solution for 40 min, before cultures underwent dialysis against water. Afterward, 50 ml of each dialyzed culture (biomass plus RPS) was suspended in 490 ml of a 10-mg liter^–1 copper solution (pH 4.5 to 5.5) and incubated at 30 ± 1°C with continuous stirring. The pH of the system (biosorbent plus copper solution) was controlled and, when necessary, adjusted to pH 5.0 by using 0.1 M HCl. Five-milliliter samples were withdrawn at known intervals. At the end of the experiment, the biomass was separated from the metal solution by centrifugation at 3,000 x g for 7 min, followed by vacuum filtration through a 0.7-µm-pore-size membrane filter (Whatman International, Ltd., Maidstone, England). The final copper content in the supernatant was determined with an Atomic Absorption spectrophotometer (SpectrAA 10 plus; Varian, Inc., CA) operating at a wavelength of 232 nm. The amount of metal removed from the solution was calculated by the differences in the metal concentration before and after the contact with cyanobacterial cultures, as determined by the Atomic Absorption spectrophotometer, and compared with a blank obtained by adding 50 ml of distilled water to 490 ml of a 10-mg liter^–1 copper solution (pH 4.5 to 5.5). All the experiments were done at least in triplicate, and the data are reported as the means ± standard deviations. Specific metal removal, q, expressed as the amount, mg, of metal removed per g of dry weight, was calculated as q (mg g^–1) = V (C_i – C_t) m^–1, where V is the sample volume (in liters), C_i and C_t are the initial and final metal concentrations (in mg liter^–1), respectively, and m is the amount of dry weight (in g) (36). To determine the fraction of the culture responsible for the removal of copper ions, bioremoval assays were performed with the dialyzed whole cultures (biomass plus RPS), with the isolated biomass suspended in deionized water at pH 5.0 and pure EPS solutions.

All bioremoval assays were performed maintaining the pH in the range 4.5 to 5.5, taking into account previous indications that copper binds more efficiently to the cyanobacterial exopolysaccharides at these pH values (7).

Analytical analysis.
Protein, carbohydrate, and uronic acid contents of the cells and of the EPS were determined using the Lowry (22), phenol-sulfuric acid (10), and carbazole (12) colorimetric assays, respectively. The dry weight (g liter^–1) was determined by vacuum filtration of the dialyzed cultures, followed by the desiccation of the filter at 100°C, until a constant weight was reached.

Isolation of the EPSs (RPS and sheath).
Cells were separated from the culture medium by centrifugation at 3,000 x g for 7 min, and 2 volumes of 97% ethanol were added to the supernatant. The precipitated RPS were collected with a sterile forceps, dried in a desiccator, and then solubilized in 50 ml of deionized water. The sheath of the wild-type strain was obtained according to the method described previously by Del Gallo and Haegi (3). The cells were washed at least three times with deionized water to remove the RPS, suspended in deionized water, stirred in an incubator at 80°C for 1 h, and centrifuged at 8,000 x g for 1 h. The sheath was precipitated from the supernatant with 2 volumes of ethanol.

Determination of the monosaccharidic composition of the EPS.
EPS samples (5 mg) were hydrolyzed with a 2 M solution of trifluoroacetic acid at 120°C for an appropriate period of time (45 min for the wild type, 45 and 120 min for the mutant), cooled on ice, and dried in a rotary evaporator. The samples were washed twice with MilliQ-grade water and then analyzed by ion exchange chromatography using a Dionex ICS-2500 ion chromatograph (Sunnyvale, CA) with an ED50 pulsed amperometric detector using a gold working electrode (Dionex, Sunnyvale, CA). A Carbopac PA1 4 mm by 250 mm column (Dionex, Sunnyvale, CA) was used. The eluants used were MilliQ-grade water (solution A), 0.185 M sodium hydroxide solution (solution B), and 0.488 M sodium acetate solution (solution C). A gradient elution was used consisting of a first stage (injection time to the 7th min) with an eluant constituted by 84% solution A, 15% solution B, and 1% solution C; a second stage (injection time from the 7th to 13th min) with 50% solution B and 50% solution C; and a final stage (injection time from the 13th to the 30th min) with 84% solution A, 15% solution B, and 1% solution C. The flow rate was 1 ml min^–1.

Determination of the sulfate and phosphate contents of the EPS.
Lyophilized EPS samples (5 mg) were hydrolyzed with 2 M HCl at 100°C for 2 h, and the solution, after cooling at room temperature, was analyzed by ion-exchange chromatography. The analysis was performed using a Dionex ICS-2500 system chromatograph equipped with a continuously regenerated anion-trap column (Sunnyvale, CA), a continuous anionic self-regenerating suppressor, a conductivity detector (ED50), an Ion Pac PA11 4 mm by 250 mm column (Dionex, Sunnyvale, CA), and a reagent-free Dionex system producing high-purity 50 mM KOH at a flow rate of 2 ml min^–1. Sulfate and phosphate solutions (1 to 10 mg liter^–1; Fluka, Buchs, Switzerland) were used as standards.

Chemical modifications of functional groups present in the RPS.
Solutions of the RPS of both the wild type and the sheathless mutant were confined in dialysis tubes and pretreated in 0.1 M HCl for 40 min before being dialyzed against water at pH 5.0 for 16 h. To determine the role of the carboxylic groups in the copper removal process, the RPS solutions (60 mg liter^–1) were lyophilized, and the powder obtained was dipped for 16 h in a 1:100 (vol/vol) solution of hydrochloric acid-anhydrous methanol, according to the method described in reference 13. The amide groups were blocked by dipping the RPS solutions, confined in dialysis tubes, in a 1:2 (vol/vol) formaldehyde-formic acid solution, according to the method described in reference 16. Both treatments were applied for 24 h with continuous stirring. Subsequently, the RPS solutions, confined in dialysis tubes, were dialyzed against water at pH 5.0 for 16 h, dipped in a 10-mg liter^–1 copper solution (pH 4.5 to 5.5), and maintained in contact with the metal for 24 h with continuous stirring at 30 ± 1°C. Determination of the final amount of copper removed was performed as described above. RPS solutions without any chemical treatment were used as controls.

Potentiometric titration.
RPS and sheath solutions were titrated by adding 0.1 M sodium hydroxide, and the pH was measured with a pH meter (pH 300; Hanna Instruments, Québec, Canada).

DRIFT spectrometry.
RPS were freeze dried after 2 h of contact with a 30-mg liter^–1 copper solution. The lyophilized samples were mixed with desiccated spectroscopic-grade potassium bromide, 1:10 (wt/wt), and subjected to diffuse reflectance infrared Fourier transform (DRIFT) spectrometry analysis with a 1710 Fourier transform infrared spectrometer (Perkin-Elmer, Inc., Wellesley, MA), operating in the range of 4,000 to 400 cm^–1. Samples not exposed to copper and prepared with the same procedure were used as controls for determining the spectra of RPS not exposed to the metal.

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Morphology and composition of the EPS produced by Gloeothece wild type and its sheathless mutant.
Optical and electron microscopy observations showed that the Gloeothece wild type is characterized by the presence of a firm sheath surrounding the cells (Fig. 1a and c), and in several optical micrographs, an amorphous layer outside the sheath, resembling slime, was also observed (Fig. 1a). On the other hand, in the mutant, neither the sheath nor the surrounding slime was observed (Fig. 1b and d). When the cultures were stained with Alcian blue, a specific dye for negatively charged polysaccharides, it was observed that both the wild type and the mutant released polysaccharides into the culture medium (Fig. 1e and f).

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FIG. 1. Light microscopy micrographs of the Gloeothece sp. strain PCC 6909 wild type (a) and its sheathless mutant (b) stained with India ink; the TEM images of the wild type (c) and of the mutant (d); light microscopy micrographs of the wild type (e) and the mutant (f) stained with Alcian blue. The sheath (arrows) and the amorphous layer (arrowhead) are highlighted. Bars, 10 µm (light micrographs) and 1 µm (TEM).

The monosaccharidic composition of the exopolysaccharides produced by the wild type (sheath and RPS) and by the mutant (RPS) was determined by ion exchange chromatography. On the whole, 11 different monosaccharides were found in the RPS, synthesized by the wild type and by the mutant: the hexoses glucose, galactose, mannose, and fructose; the pentoses arabinose, xylose, and ribose; the deoxyhexoses fucose and rhamnose; and the acidic hexoses glucuronic and galacturonic acids (Table 1). However, the RPS of the wild type and the mutant differed in the amounts of specific sugar residues, and especially in the total content of uronic acids (glucuronic plus galacturonic acid), which were larger in the RPS of the mutant than in the wild type, and in the total amounts of the deoxysugars (fucose and rhamnose), which were larger in the wild type. Interestingly, the sheath of the wild type contained a smaller number of different sugar residues compared to that of the RPS. Although the sheath possesses glucosamine, absent in RPS, it lacks fucose, arabinose, xylose, fructose, and galacturonic acid (Table 1). Furthermore, the RPS of both the wild type and the mutant contained similar amounts of sulfate groups, about 10% (wt/wt) of the RPS dry weight, in contrast to the sheath of the wild type, which contained an amount of sulfate that was 10-fold smaller (Table 1). Small amounts of phosphate (about 3.6% of the polysaccharide dry weight) were detected in the sheath of the wild type but not in the RPS of both the wild type and the mutant (Table 1). Analysis of the crude composition of the RPS showed a protein content of 7.3% ± 0.8% and 3.8% ± 0.2% and a total carbohydrate content of 13.9% ± 0.9% and 11.4% ± 0.4% of RPS dry weight for the polysaccharide produced by the mutant and the wild type, respectively.

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TABLE 1. Monosaccharidic composition and sulfate and phosphate content of the exopolysaccharides produced by the Gloeothece sp. strain PCC 6909 wild type and its sheathless mutanta

Copper removal assays.
Dialyzed whole cultures (biomass plus RPS) of the Gloeothece sp. strain PCC 6909 wild type and its sheathless mutant were tested to determine their capacities to remove copper ions from aqueous solutions. Unexpectedly, the mutant strain proved to be more efficient in the metal removal, reaching values of specific copper removal (q) approximately two times higher than those of the wild type (46.3 ± 3.1 and 26.7 ± 1.5 mg of copper removed per g of dry weight, respectively; Fig. 2). When dialyzed whole cultures were subjected to a pretreatment with 0.1 M HCl for 40 min, their q values increased considerably. However, even with the pretreatment, the highest specific copper removal was obtained with the mutant; 102.8 ± 5.8 mg g^–1 compared to 61.1 ± 1.4 mg g^–1 for the wild type. Under the conditions tested, the maximum copper removal capacity of the cultures was attained by 30 to 60 min after contact with the metal solution (Fig. 2). In order to further investigate the unexpected higher efficiency of copper removal exhibited by whole cultures of the sheathless mutant, the metal-removal capacities of both suspended biomasses (without RPS) and solutions of RPS of the wild-type and mutant strains were determined. The q values obtained for the biomasses of the wild type and the mutant were 25.8 ± 1.8 and 20.2 ± 1.7 mg Cu²⁺ removed per g of dry weight, respectively (Fig. 3), pointing out that in the absence of the RPS, the wild type has a slightly higher metal-removal capacity in comparison with that of the sheathless mutant. In contrast, when the metal-removal capacities of the pure RPS solutions were compared, the q value obtained with the polysaccharide released by the mutant was 52.4% higher than that of the wild type (149.2 ± 4.0 and 97.9 ± 5.2 mg Cu²⁺ removed per g of RPS dry weight, respectively). In addition, the cultures of the mutant released a 27% larger amount of polysaccharide into the culture medium than the wild type; indeed, the ratio between the released carbohydrates and dry weights was 0.14 and 0.11 for the mutant and the wild type, respectively.

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FIG. 2. Time course of specific copper removal (q), expressed as mg of copper removed per gram of biomass dry weight by the Gloeothece sp. strain PCC 6909 wild type (wt, ) and its sheathless mutant (mt, ). Data are the mean values of at least three independent experiments, and bars represent the standard deviations.

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FIG. 3. Specific copper removal (q) expressed as mg of copper removed per gram of biomass (dry weight) using the biomass only or the whole cultures (biomass plus RPS) of the Gloeothece sp. strain PCC 6909 wild type (wt) and its sheathless mutant (mt). Data are the mean values of at least three independent experiments, and bars represent the standard deviations.

Metal-binding groups in the EPS.
SEM studies, performed in order to localize the copper ions on the external layers of the cells, pointed to large amounts of metal ions adsorbed in a confined area of the sheath of the wild type (Fig. 4a and b), a result that was supported by the elemental analysis carried out with SEM/EDS (Fig. 4I). On the other hand, copper ions appeared to be more evenly distributed on the surfaces of the unsheathed cells of the mutant strain (Fig. 4c and d), even if in smaller quantities per surface area (Fig. 4II) in comparison with the wild type.

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FIG. 4. SEM images of cultures of the Gloeothece sp. strain PCC 6909 wild type (wt) (a and b) and its sheathless mutant (mt) (c and d) exposed to a copper solution (30 mg liter–1). Secondary electron images show the cell surfaces (a and c), and backscattered electron images (b and d) reveal the presence and the position of metals (white halos). Elemental compositions of the wild type (I) and mutant (II) samples, as determined by SEM/EDS analyses and pointing out the concentration of the metals in selected areas, are shown. Note that the high content in Al is due to the aluminum support grid.

In order to characterize the reactivity of all available functional groups, potentiometric titrations of aqueous solutions of the sheath and RPS produced by Gloeothece wild type and of the RPS produced by the mutant were carried out. The curves of both fractions of the wild type were characterized by the presence of only one inflection point, located within the pH range 5.5 to 6.9, while the curve obtained for the RPS of the mutant exhibited an additional inflection point located at pH 7.9 (Fig. 5), suggesting the presence of another functional group on the RPS of the mutant that is able to participate in the metal ions’ binding capacity.

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FIG. 5. Potentiometric titration of the exopolysaccharides of the Gloeothece sp. strain PCC 6909 wild type (wt) (, sheath; , RPS) and its sheathless mutant (mt) (, RPS).

The DRIFT spectra of the RPS of both the Gloeothece wild type and the mutant strains, performed before and after contact between the polysaccharides and the copper ions (Fig. 6), showed significant variations in the fingerprint region. The major modifications in the spectra of the RPS exposed to the metal were observed for the bands at 1,700 and 1,400 cm^–1, which are due to the carboxylates, at 1,264 cm^–1, which is related to the sulfate groups, and at 1,646 and 1,548 cm^–1, which are due to the amide groups (Fig. 6).

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FIG. 6. DRIFT spectra of the released polysaccharides of the Gloeothece sp. strain PCC 6909 wild type (wt) (a) and its sheathless mutant (mt) (b), exposed (dashed lines) or not exposed (solid lines) to a 30-mg liter–1 copper solution. Arrows indicate the major band shifts.

In order to corroborate the indications coming from the DRIFT spectra regarding the main functional groups involved in the metal-binding process, a number of copper removal assays were carried out with the RPS chemically treated to specifically block the putative functional groups. When methylation of the amide groups was performed, the q values decreased to 47% and 35% of the q values obtained with the nonmethylated RPS of the wild type and the mutant, respectively. An even more drastic decrease of the q values was observed with the esterification of the carboxyl groups, in which the q values dropped to 19% and 21% of the q values reached with the RPS of the wild type and the mutant, respectively.

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The fast kinetics of copper removal exhibited by the cultures of both the Gloeothece sp. strain PCC 6909 wild type and the mutant strongly suggest that the metal-binding process is essentially due to an ion-exchange mechanism, in accordance with the hypothesis previously formulated for the metal-removal process carried out by other cyanobacteria (6, 30). The positively charged copper ions most probably interacted with the negative charges distributed on the polysaccharidic layer on the cell surface of the wild type or on the released polysaccharide of both the wild type and the mutant (the hypothesis is supported by the SEM images), even if some metabolic activity cannot be completely excluded (1, 23, 25, 26, 34, 37, 41). This assumption is also supported by the significant increase of the q values observed when the cultures were pretreated with HCl before the contact with the metal. Indeed, it was previously shown that HCl is a very effective desorbing agent, capable of removing, by an ion-exchange mechanism, the cations derived from the nutrients of the culture medium and still bound to the negatively charged polysaccharides in spite of the preliminary dialysis (24). Subsequently, the protons of the HCl solution will be easily exchanged for the metal ions.

The copper-removal-specific values (q) obtained with whole cultures (biomass plus RPS) of the Gloeothece strain PCC 6909 wild type and its mutant revealed that unexpectedly, the cultures of the mutant removed copper about two times more efficiently than did those of the wild type, in spite of the absence of the sheath outside the cells. On the other hand, when the experiments were carried out with biomasses only, the wild type showed slightly higher q values than the mutant, suggesting a role for the sheath, although not very significant, in the metal-removal process. This poor contribution of the sheath might be related to a differential distribution of the metal binding sites between the sheath and the cell wall, with these sites predominantly located on the cell walls, as was previously reported for a Calothrix strain (26, 41).

On the other hand, the higher metal removal observed with the cultures of the mutant can be explained by the larger amounts of RPS released into the culture medium than with the wild type, as clearly shown by the microscopic observations carried out with the Alcian blue, as well as by the determination of the amount of soluble carbohydrates in the culture medium. These results also imply that the random mutagenesis did not block the synthesis and release of the carbohydrates but only their mobilization for the formation of a structured sheath. In addition, the metal-removal capacity of the RPS solution of the mutant was considerably higher than that of the wild type. Taking into account these results, it is possible to conclude that the higher copper removal capacity observed for the whole cultures of the mutant is due to the release of large quantities of polysaccharidic material, together with better metal-binding properties of its polymer. In agreement, an important contribution of RPS to the metal-removal process was previously demonstrated for two capsulated cyanobacteria, Cyanospira capsulata and Nostoc sp. strain PCC 7936. Indeed, it was experimentally shown that the RPS of the two cyanobacteria contributed to the metal-removal capacity. This was confirmed by the observation that the differences of q_max values between those of the whole cultures and those of cultures lacking RPS correspond to the q_max values obtained with pure RPS in solution (6). The determination of the chemical composition of the RPS synthesized by the wild type and the mutant pointed out that the two polymers are composed by the same 11 monomers. However, some significant quantitative differences emerged, in particular with regard to the uronic acids and the deoxysugars. Indeed, the acidic sugars are 35% more abundant in the RPS of the mutant, thus conferring a higher affinity for the positively charged copper ions, while the deoxysugars fucose and rhamnose were 28% more abundant in the RPS of the wild type, providing this polymer with a higher degree of hydrophobicity (5) and reducing its solubility and, as a consequence, its accessibility for copper ions. In the Gloeothece wild type, the compositions of the sheath and the released polysaccharide were significantly different, indicating that the RPS is not merely due to the solubilization of the external layer(s) of the sheath, as has been previously suggested for this organism (33), but that it probably arose from another biosynthetic process. It is also worth mentioning that the monosaccharidic composition of the sheath of the Gloeothece strain PCC 6909 wild type reported here (Table 1) differs qualitatively and quantitatively from that reported previously, performed with cells grown with different amounts of nitrate (or no nitrate at all) and at different temperatures (32, 33), confirming that the growth conditions may play a significant role in determining the sheath composition, as was previously suggested (32).

The titration curves obtained for the sheath and the RPS of the wild type showed that only one functional group, with a pK_a in the range of 5.5 to 6.9, and putatively assigned to carboxyl or phosphate groups (1, 2, 26), plays a significant role in their ion exchange properties toward cations. On the other hand, in the case of the RPS of the mutant, an additional functional group, with a pK_a of 7.9 and putatively assigned to the amide groups (26), is most probably involved in its ion exchange properties. Further investigations with DRIFT spectrometry confirmed the presence of more than one functional group capable of interacting with copper ions. Indeed, the spectra of RPS of both the wild type and the mutant showed the presence of major band shifts after the contact with Cu²⁺ for the signals at (i) 1,700 and 1,400 cm^–1 due to the carbonyl stretching of un-ionized and ionized carboxyl groups, respectively (9, 16, 17, 19, 41); (ii) at 1,264 cm^–1 due to the S=O stretching of sulfate groups (29); and (iii) at 1,646 and 1,548 cm^–1, due to the amide I and amide II peptide bond vibrations, respectively (41). These results, together with the data from the potentiometric titrations, suggest that the carboxyl and the amide groups are the most important sites for the metal binding process. The absence of a second inflection point, related to the amide groups, in the exopolysaccharides of the wild type may be due to a lower accessibility and/or fewer numbers of these groups compared to the ones present in the RPS of the mutant. This hypothesis is supported by the fact that the protein fraction of the RPS of the mutant is about two times higher than that of the RPS of the wild type (7.3% ± 0.8% and 3.8 ± 0.2% of RPS dry weight, respectively). The relevance of the amide groups present in the RPS of the mutant compared to the ones present in the wild type is corroborated by the sharper decrease in the q value observed when the amide groups were methylated with formaldehyde.

In conclusion, from the results described above, it is possible to infer that in the Gloeothece strain PCC 6909 wild type, the role of the sheath in the removal of copper ions is limited, while the increased copper binding efficiency of the sheathless mutant can be ascribed to the release of a polysaccharide bearing three functional groups (the carboxyl, amide, and sulfate groups), two of which (the carboxyl and amide groups) are present in higher number than in the wild type and are probably more accessible for the metal ions. These results emphasize the idea that the performance of a given EPS-producing cyanobacterium in metal-removal processes depends not only on the amount of EPS produced but also on its quality and structure.

 
This work was supported by Fundação Calouste Gulbenkian: Programa Ambiente e Saúde; Proc. 76910, FCT (SFRH/BD/22733/2005), POCI 2010 (III Quadro Comunitário de Apoio), and by Acordos de Cooperação Científica e Tecnológica GRICES/CNR: Proc. 4.1.1., 2005 and 2007.

We thank Lucas Stal for providing the Gloeothece sheathless mutant, Rui Seabra and Arlete Santos (IBMC) for TEM, Mario Paolieri (Interdipartimental Center for Electronic Microscopy and Microanalysis [MEMA], University of Florence) for SEM, and Luca Calamai (Department of Soil Science and Plant Nutrition, University of Florence) for his technical assistance with the DRIFT spectrometry.

 
* Corresponding author. Mailing address: Department of Agricultural Biotechnology, University of Florence, Piazzale delle Cascine 24, I-50144, Florence, Italy. Phone: 39-0553288284. Fax: 39-0553288272. E-mail: roberto.dephilippis{at}unifi.it

Published ahead of print on 7 March 2008.

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Applied and Environmental Microbiology, May 2008, p. 2797-2804, Vol. 74, No. 9
0099-2240/08/$08.00+0     doi:10.1128/AEM.02212-07
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