1 Background, aim, and scope

Chromium is a heavy metal used extensively in industry, and high levels contaminate both terrestrial and aquatic habitats. Toxic effects of chromium on bacteria and algae have been reviewed by Wong and Trevors (1988). Many microorganisms are capable of secreting high molecular-mass polymers that can either be released into the surrounding environment [extracellular polysaccharides (EPS), exopolysaccharides] or remain attached to the cell surface (capsular polysaccharides). These polysaccharides are believed to protect bacterial cells from desiccation, heavy metals, or other environmental stresses by producing biofilms, thus enhancing the cells chances of colonizing special ecological habitats (Kazy et al. 2002). EPSs have found a wide range of applications in industries. Generally, they may replace polysaccharides used in the food industry as thickeners, stabilizers, emulsifiers, bodying agents, foam enhancers, and gelling agents (Ruas-Madeido et al. 2002). Cyanobacteria are important organisms in water that have a cell surface consisting of polysaccharides, proteins, and lipids that may act as a basic binding site of heavy metals. Studies on exopolysaccharides from cyanobacteria have dealt largely with chemical composition and their protective role (Panof et al. 1988). This study was designed to determine the correlation between metal resistance and EPS concentration of some cyanobacterial isolates isolated from different freshwaters of Turkey and to investigate the effect of Cr(VI) concentrations on EPS concentration, and in particular, on monosaccharide composition of EPS.

2 Materials and methods

Samples were isolated from different freshwaters of Turkey and isolates were maintained in BG-11 medium [NaNO3, 15; K2HPO4, 0.4; MgSO4·7H2O, 0.75; CaCl2·2H2O, 0.36; citric acid, 0.06; iron(III) ammonium citrate, 0.06; Na2–EDTA, 0.01; Na2CO3, 0.2 g/L, 1 mL; trace elements solution, (H3BO3, 61; MnSO4·H2O, 169; ZnSO4·7H2O, 287; CuSO4·5H2O, 2.5; (NH4)6Mo7O24·4H2O, 12.5 mg/L) pH 6.8; Rippka et al. 1979] for Cr(VI) resistance and for EPS concentration. Cultures were incubated at 22°C to 25°C with light/dark cycle of 12/12 h by using an incubator shaker (MINITRON) for 12 days (light intensity period: 12,000 μmol m−2 s−1). Cr(VI) resistance of cyanobacterial cultures was investigated by determining chlorophyll-a (Hirschberg and Chamovitz 1994) every 48 h, during 12 days. Five, 15, and 35 ppm Cr(VI) concentrations were prepared by dissolving K2Cr2O7 salt (Merck) in distilled water. Also, flasks containing medium lacking Cr(VI) were inoculated in the same manner to serve as controls. The EC50 was determined by probit analysis (Finney 1971). EPS was extracted by the modified procedure of Cérantola et al. (2000). Total carbohydrate contents of the EPS samples were determined by the method of Dubois et al. (1956) using glucose as a reference standard. The monosaccharide composition of freeze-dried exopolysaccharides samples was determined with high-performance liquid chromatography (HPLC; VARIAN ProStar) by using Metacarb 87H column (300 × 7.8 mm, Cat No. 5210). Monomer analyses of EPS were carried out by Middle East Technical University, Central Laboratory, Molecular Biology and Biotechnology R&D Center. To determine the effect of Cr(VI) on EPS concentration, Chroococcus sp. H4 and Synechocystis sp. S63 were incubated with 15 and 35 ppm Cr(VI) concentrations for 5 days, and final biomass of the isolates were equaled by determining chlorophyll-a. EPS was isolated as described earlier. Results of each representative experiment were analyzed by ANOVA. P values smaller than 0.05 are considered significant.

3 Results

Ten cyanobacterial isolates from different freshwaters of Turkey were obtained. The isolates, original habitats, EC50, and EPS values are listed in Table 1. The EC50 values of the ten strains ranged from 1.5 to 10.7 ppm. Chroococcus sp. H4 (EC50 of 10.7 ppm) was the isolate most resistant to Cr(VI). Microcystis sp. S80 (EC50 of 1.5 ppm) was the most sensitive to Cr(VI). EPS concentration of the ten strains ranged from 427 to 108 mg/L. Chroococcus sp. H4 (427 mg/L) and Synechocystis sp. S63 (418 mg/L) were high EPS-producing isolates. Microcystis sp. S80 (108 mg/L) was low EPS-producing isolate. The sugar monomer make-up was characterized of the EPSs produced by Chroococcus sp. H4 with and without exposure to Cr(VI) and quantified the monomer content by HPLC. In the absence of Cr(VI) exposure, the EPSs were composed mainly of glucose (99%) and a very small amount of galacturonic acid (1%). Following Cr(VI) exposure, EPSs were composed mainly of xylose (75%) and small amounts of glucose (9%), rhamnose (14%), and galacturonic acid (2%). Mannose, galactose, arabinose, ribose, and glucuronic acid were not detected in the EPSs produced by Chroococcus sp. H4 with or without Cr(VI) exposure. Final biomasses of isolates (H4 and S63) were equaled before determining the effect of Cr(VI) on EPS concentration. A significant and regular increase in the concentration of EPS by both Chroococcus sp. H4 (ANOVA; F 2,3 = 25.92; P = 0.013) and Synechocystis sp. S63 (ANOVA; F 2,3 = 22.58; P = 0.016) was observed. EPS concentrations of Chroococcus sp. H4 at control, 15, and 35 ppm Cr(VI) were determined as 477 ± 2, 482 ± 2, and 510 ± 6 mg/L respectively and 457 ± 6, 497 ± 8, 525 ± 5 mg/L for Synechocystis sp. S63.

Table 1 Geographical origins EPS concentrations and EC50 values of the isolates
Full size table

4 Discussion

In this study, Cr(VI)-resistant isolates produced high amounts of EPS, and sensitive isolates produced low amounts of EPS. Kazy et al. (2002) reported that EPS production by a copper-resistant isolate of Pseudomonas aeruginosa was considerably higher than its copper-sensitive counterpart. The monosaccharide most frequently found in the cyanobacterial EPSs is glucose (in more than 90% of the polymers; Bar-Or and Shilo 1987). Glucose was the most common monosaccharide detected in this study, but the ratio of glucose to other monosaccharides decreased following cyanobacteria exposure to Cr(VI), Chroococcus sp. H4 in particular. Conversely, xylose was a major sugar in Chroococcus sp. H4 treated with Cr(VI), but xylose was not detected in the controls. Additionally, only one acidic sugar was found in both control and Cr(VI)-treated Chroococcus sp. H4. Priester et al. (2006) also detected differences in the monosaccharide composition of EPS in Cr(VI)-exposed Pseudomonas putida. Differences in the monosaccharide composition and values of EPS may promote heavy-metal resistance in these microorganisms. The EPS concentration from both isolates (H4 and S63) was considerably higher than control and was observed to have a good correlation between Cr(VI) exposure and EPS concentration in both isolates. A similar correlation was detected by Fang et al. (2002). In sulfate-reducing bacterial biofilms, exposure to trivalent Cr resulted in a nearly 82% increase in extracellular carbohydrate. Many different environmental stresses increase production of extracellular carbohydrates (Priester et al. 2006). Here, it was shown that Cr(VI) is an important stress factor that increases EPS concentration in cyanobacteria.

5 Conclusions

Very little comparative information about the effect of Cr(VI) on EPS concentration and composition is available, especially in case of practical applications, indicating the need for more research in this area. Ultimately, our results suggest a potential to exploit EPS-producing cyanobacteria for the production of a wide range of biopolymers suited to various industrial and environmental applications such as gelling agents and heavy-metal biosorption.