Anaerobic co-reduction of chromate and nitrate by bacterial cultures of Staphylococcus epidermidis L-02
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- Vatsouria, A., Vainshtein, M., Kuschk, P. et al. J IND MICROBIOL BIOTECHNOL (2005) 32: 409. doi:10.1007/s10295-005-0020-0
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Industrial wastewater is often polluted by Cr(VI) compounds, presenting a serious environmental problem. This study addresses the removal of toxic, mutagenic Cr(VI) by means of microbial reduction to Cr(III), which can then be precipitated as oxides or hydroxides and extracted from the aquatic system. A strain of Staphylococcus epidermidis L-02 was isolated from a bacterial consortium used for the remediation of a chromate-contaminated constructed wetland system. This strain reduced Cr(VI) by using pyruvate as an electron donor under anaerobic conditions. The aims of the present study were to investigate the specific rate of Cr(VI) reduction by the strain L-02, the effects of chromate and nitrate (available as electron acceptors) on the strain, and the interference of chromate and nitrate reduction processes. The presence of Cr(VI) decreased the growth rate of the bacterium. Chromate and nitrate reduction did not occur under sterile conditions but was observed during tests with the strain L-02. The presence of nitrate increased both the specific Cr(VI) reduction rate and the cell number. Under denitrifying conditions, Cr(VI) reduction was not inhibited by nitrite, which was produced during nitrate reduction. The average specific rate of chromate reduction reached 4.4 μmol Cr 1010 cells−1 h−1, but was only 2.0 μmol Cr 1010 cells−1 h−1 at 20 °C. The maximum specific rate was as high as 8.8–9.8 μmol Cr 1010 cells−1 h−1. The role of nitrate in chromate reduction is discussed.
KeywordsChromate reductionNitrate reductionBacteriaStaphylococcus epidermidis
Chromium is a transition metal which is able to exist in several oxidation states. The most stable and common forms are the trivalent Cr(III) and hexavalent Cr(VI) species [6, 31]. Cr(VI) is considered to be the most toxic and carcinogenic form of Cr, and is usually associated with oxygen as chromate (CrO42−) or dichromate (Cr2O72−) ions. By contrast, Cr(III) forms insoluble oxides and hydroxides above pH 5 , is much less mobile, and mostly exists bound to organic matter in soil and aquatic environments . The use of chromium in leather-tanning, electroplating, paint pigment and dye production, automobile manufacturing, the steel industry and other industries has led to Cr(VI) being discharged into natural ecosystems. Since high levels of Cr(VI) may overcome the reducing capacity of the environment, it persists as a pollutant. Problems of incremental chromate pollution and the relatively low cost of biological methods of heavy metal recovery have encouraged interest in both Cr(VI)-reducing microorganisms and chromate-resistant bacteria. A wide variety of bacteria belonging to various systematic and physiological groups are involved in Cr(VI) reduction under anaerobic conditions [1, 3, 8, 10, 16, 20, 21, 23, 24, 32]. However, the reduction of CrO42−—a terminal electron acceptor during anaerobic respiration—may not produce enough energy to enable bacterial growth [14, 15]. Consequently, the distribution of chromate reduction in the bacterial world is not specific and can be affiliated to various physiological processes, such as simultaneous reductive processes with alternative electron acceptors (nitrate, nitrite, sulphate). These compounds are present in contaminated systems and often vary spatiotemporally. Cr(VI) reduction by bacterial consortiums has been shown to be related to sulphate and nitrate reduction . It has also been suggested that the microbial reduction of nitrate, Cr(VI) and sulphate takes place consecutively [4, 13].
As far as bioremediation applications are concerned, it is important to know how various electron acceptors affect Cr(VI) reduction, and in turn how Cr(VI) affects other terminal electron-accepting processes. Hence, the goal of this work was to investigate the interaction of chromate and nitrate reduction by Staphylococcus epidermidis in model experiments.
Materials and methods
The pure bacterial culture, strain L-02, was isolated by the authors from the active chromium-reducing bacterial consortium obtained from the Grosskayna experimental wetland station in Merseburg, Germany . The culture was identified at the German collection of microorganisms and cells (DSMZ). Its 100% similarity with the type strain of Staphylococcus epidermidis was determined by 16SrRNA sequencing [17, 26].
The basic medium for culturing S. epidermidis L-02 contained (g/L): KH2PO4, 0.9; Na2HPO4·2 H2O, 1.2; NH4Cl, 0.5; yeast extract, 0.9; Na pyruvate, 2.0; NaHCO3, 0.2; MgSO4·7 H2O, 0.5 (pH 7). The high concentration of phosphates resulted in no change of pH during experiments. The experiments were performed in 55 ml Wheaton glass serum bottles (Sigma) with 25 ml of the medium. The bottles were purged with N2, sealed with Wheaton butyl stoppers (Sigma), and sterilized in an autoclave at 105 °C for 20 min. Stock solutions of K2Cr2O4 (10 g Cr(VI)/L) and KNO3 (100 g/L) were sterilized separately and then added to the base medium either individually or combined to reach the final concentrations. All the supplements (inoculum, Cr (VI) and nitrate) were added by injection with sterile syringes. The ability of the isolated strain to reduce Cr(VI) was examined in cultures with 0.3 mmol (standard concentration) or 0.6 mmol (double concentration) Cr(VI) in the medium. The final working nitrate concentration was 3.2 mmol. Despite the strain’s optimal temperature of 30–37 °C, our experiments were carried out at 20 °C to expand the process dynamics over time.
Chromium (VI) concentration was estimated with diphenylcarbazide reagent (1% in acetone) and the resulting colour was measured at 540 nm using the standard method . Nitrate and nitrite concentrations were analysed by ion chromatography using a Dionex 100 (AS4A-SC column/AG4A-SC column) with UV detection at 215 nm (NO3−/NO2−) . Cell numbers were counted by direct microscopy in a counting chamber . All the bottles were set up in duplicate.
Specific reduction rates were described as the relationship between the substrate concentration decrease per time unit and the number of bacterial cells. These specific rates can be expressed mathematically as: Vsp = dC/N*t, where Vsp is the specific reduction rate in μmol Cr 1010 cells−1 h−1, dC the difference in the reduced substrate concentrations in micromole, N the cell concentration in 1010/L, and t the time in hours. Analyses of parallel samples showed that variations never exceeded 5–8%.
Results and discussion
A representative of the Staphylococcus genus, namely S. cohnii, has already been reported to reduce chromate . We found that our isolated S. epidermidis strain L-02 can also reduce chromate. Resistance to Cr(VI) has been shown for several species of the Staphylococcus genus including S. epidermidis . Nevertheless, whether all the members of this genus possess chromate-reducing ability is yet unknown. Information on the reductive activity of Staphylococci suggests that one of the typical genus features is nitrate reduction. We tested the combined and separate effects of chromate and nitrate on the strain L-02 under anaerobic conditions in the presence of pyruvate, which provided an alternative possibility of growth with a fermentative process. In addition, pyruvate could be used as an electron donor in processes of nitrate or chromate reduction.
Values of maximum specific Cr(VI) reduction as reported, or calculated from published data
Maximum specific reduction rate, Vmax (mmol Cr(VI) cells−1 h−1)
Escherichia coli ATCC 33456 
Desulfovibrio vulgaris ATCC 29579 
Enterobacter cloaceae HO1 
Shewanella oneidensis MR-1 
Staphylococcus epidermidis L-02 [This study]
Pantoea agglomerans SP1 
The physiological features of the culture during the process of chromate reduction are characterized by the specific rate of reduction and its change in the experiments. The study on the specific rate of Cr(VI) reduction revealed a few stages of the process: (1) adaptation during the first 3 days (initial growth, enzyme induction), (2) a peak of activity on the fourth day (initial phase of active growth and the cell multiplication related to fast reductase formation as a protective mechanism), (3) a stable phase (5–13 days for the variant with no nitrate and 5–10 days for the variant supplemented with nitrate) of the reduction processes, (4) the appearance of a new peak of the specific nitrate reduction activity when chromate was nearly exhausted but nitrate was still present in the medium (only in the variant supplemented with nitrate). In the medium supplemented with nitrate, the biomass increase (Fig. 1) did not cause a corresponding increase in the specific Cr reduction rate. The same situation was observed in the medium supplemented with chromate only. (Fig. 2). Nevertheless, the specific rate of Cr reduction was fivefold higher in the absence of nitrate. This fact testifies to the fact of the nitrate competition as an alternative final electron acceptor to chromate. The chromate and nitrate reductions appear to be provided with the same reductase complex.
Judged by the specific rate, the nitrate reduction process also showed different stages (Fig. 3). There was an increase at the beginning of growth and at the end of the stable phase when chromate was more or less exhausted (Fig. 2). The specific rate of nitrate reduction at log-phase was about 3.5 times higher in the absence of chromate (Fig. 3).
This paper describes the pattern of chromate reduction by S. epidermidis. The average specific reduction rate was 4.4 μmol Cr 1010 cells−1 h−1 at 30 °C. The specific chromate reduction rate at 20 °C was generally only 2.0 μmol Cr 1010 cells−1 h−1, although at the maximum stages it reached 8.8 μmol Cr 1010 cells−1 h−1 without nitrate supplements and 9.8 μmol Cr 1010 cells−1 h−1 in the presence of nitrate. Nitrate also stimulated Cr(VI) reduction by S. epidermidis L-02 by increasing the cell numbers, too. The nitrite produced did not affect the process of Cr(VI) reduction under the experimental conditions. The mutual negative effect of nitrate and chromate on the specific reduction rate can be explained as the alternative use of the oxidizers by joint enzymes.
The research was kindly supported by the Linkage NATO grant EST-CLG-978918.