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

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 × 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 × 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 × 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.

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.

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.