Extra-cellular chromate-reducing activity of the yeast cultures

This paper reports on the experimental data supporting an essential role of extra-cellular reduction in chromate detoxification by baker’s and non-conventional yeasts. A decrease of chromate content in the yeast culture coincides with an increase of Cr(III) content in extra-cellular liquid. At these conditions, cell-bound chromium level was insignificant and a dominant part of extra-cellular Cr(III) species was detected in the reaction with chromazurol S only after mineralization of the cell-free samples. This phenomenon of chromium “disappearance” can be explained by the formation of Cr(III) stable complexes with extra-cellular yeast-secreted components which are “inaccessible” in the reaction with chromazurol S without mineralization. It was shown that increasing sucrose concentration in a growth medium resulted in an increase of chromate reduction. A strong inhibition of chromate reduction by 0.25 mM sodium azide, a respiration inhibitor and a protonophore, testifies that extra-cellular chromate detoxification depends on energetic status of the yeast cells. It was shown that Cr(III)-biochelates produced in extra-cellular medium are of a different chemical nature and can be separated into at least two components by ion-exchange chromatography on anionit Dowex 1x10. A total yield of the isolated Cr(III)-biocomplexes is approximately 65 % (from initial level of chromate) with a relative molar ratio 8:5.     

Chromate-resistant mutants of the yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis>: Selection and properties

Chromate-resistant mutants of the non-conventional yeast Pichia guilliermondii L2 were selected by different methods. The isolated mutants were capable of better growth and higher biomass yield at toxic (1.8–2.4 mM) chromate concentrations than the parent strain. The capacity of the mutants for extracellular chromate reduction and chelation of Cr(III) in the culture liquid was demonstrated. The effectiveness of these processes was shown to correlate with the resistance of P. guilliermondii strains to chromate. Extracellular metabolites of the yeast cells cultivated without chromate were shown to be capable of reducing chromate and forming stable soluble Cr(III)-biocomplexes.     

Chromate-reducing properties of soluble flavoproteins from Pseudomonas putida and Escherichia coli

Cr(VI) (chromate) is a toxic, soluble environmental contaminant. Bacteria can reduce chromate to the insoluble and less toxic Cr(III), and thus chromate bioremediation is of interest. Genetic and protein engineering of suitable enzymes can improve bacterial bioremediation. Many bacterial enzymes catalyze one-electron reduction of chromate, generating Cr(V), which redox cycles, generating excessive reactive oxygen species (ROS). Such enzymes are not appropriate for bioremediation, as they harm the bacteria and their primary end product is not Cr(III). In this work, the chromate reductase activities of two electrophoretically pure soluble bacterial flavoproteins--ChrR (from Pseudomonas putida) and YieF (from Escherichia coli)-were examined. Both are dimers and reduce chromate efficiently to Cr(III) (kcat/Km = approximately 2 x 10(4) M(-1) x s(-1)). The ChrR dimer generated a flavin semiquinone during chromate reduction and transferred >25% of the NADH electrons to ROS. However, the semiquinone was formed transiently and ROS diminished with time. Thus, ChrR probably generates Cr(V), but only transiently. Studies with mutants showed that ChrR protects against chromate toxicity; this is possibly because it preempts chromate reduction by the cellular one-electron reducers, thereby minimizing ROS generation. ChrR is thus a suitable enzyme for further studies. During chromate reduction by YieF, no flavin semiquinone was generated and only 25% of the NADH electrons were transferred to ROS. The YieF dimer may therefore be an obligatory four-electron chromate reducer which in one step transfers three electrons to chromate and one to molecular oxygen. As a mutant lacking this enzyme could not be obtained, the role of YieF in chromate protection could not be directly explored. The results nevertheless suggest that YieF may be an even more suitable candidate for further studies than ChrR.     

Chromate-Reducing Properties of Soluble Flavoproteins from Pseudomonas putida and Escherichia coli

Cr(VI) (chromate) is a toxic, soluble environmental contaminant. Bacteria can reduce chromate to the insoluble and less toxic Cr(III), and thus chromate bioremediation is of interest. Genetic and protein engineering of suitable enzymes can improve bacterial bioremediation. Many bacterial enzymes catalyze one-electron reduction of chromate, generating Cr(V), which redox cycles, generating excessive reactive oxygen species (ROS). Such enzymes are not appropriate for bioremediation, as they harm the bacteria and their primary end product is not Cr(III). In this work, the chromate reductase activities of two electrophoretically pure soluble bacterial flavoproteins—ChrR (from Pseudomonas putida) and YieF (from Escherichia coli)—were examined. Both are dimers and reduce chromate efficiently to Cr(III) (k_cat/K_m = ~2 × 10⁴ M⁻¹·s⁻¹). The ChrR dimer generated a flavin semiquinone during chromate reduction and transferred >25% of the NADH electrons to ROS. However, the semiquinone was formed transiently and ROS diminished with time. Thus, ChrR probably generates Cr(V), but only transiently. Studies with mutants showed that ChrR protects against chromate toxicity; this is possibly because it preempts chromate reduction by the cellular one-electron reducers, thereby minimizing ROS generation. ChrR is thus a suitable enzyme for further studies. During chromate reduction by YieF, no flavin semiquinone was generated and only 25% of the NADH electrons were transferred to ROS. The YieF dimer may therefore be an obligatory four-electron chromate reducer which in one step transfers three electrons to chromate and one to molecular oxygen. As a mutant lacking this enzyme could not be obtained, the role of YieF in chromate protection could not be directly explored. The results nevertheless suggest that YieF may be an even more suitable candidate for further studies than ChrR.     

Exogenously applied sulphate as a tool to investigate transport and reduction of chromate in the duckweed Spirodela polyrhiza

The accumulation of chromium in Spirodela polyrhiza was investigated in the presence and absence of exogenously applied sulphate. Precultivation (10 d) at minimum sulphate concentration (0.013 m m versus 1 m m in controls) enhanced the rate of chromium accumulation. This effect was caused by the increased number of sulphate transporters which transport chromate into cells. Chromate and sulphate compete for the available sulphate transporters. The kinetics of reduction Cr(VI)→Cr(V) was investigated by l -band electron paramagnetic resonance (EPR) spectroscopy. The kinetic model developed previously (Appenroth et al., Journal of Inorganic Biochemistry 78, 235–242, 2000) was refined and extended to include chromate transport and reduction in the presence of competing ions. The following conclusions were drawn from the fitting procedure: without simultaneously applied sulphate, the rate constant of Cr(VI) transport from apoplast into plant cells and the rate constant of Cr(VI) to Cr(V) reduction within the apoplast are comparable (7.0 versus 5.7 h⁻¹) demonstrating that these two processes are competing. Moreover, the rate constant of reduction Cr(V)→Cr(III) is much lower within cells than in apoplast (0.39 versus 7.0 h⁻¹) showing that Cr(V) is stabilized in the symplast. The rate of transport of Cr(VI) into plant cells is at least one order of magnitude higher than that of Cr(V) or Cr(III). The treatment with sulphate (10 m m ) decreases the rate constant of the transport of Cr(VI) into cells (2.0 h⁻¹) confirming the competition of chromate and sulphate for the same transporters. Simultaneously, the rate constant of Cr(V)→Cr(III) reduction is increased in the apoplast (by the factor of 3) and decreased in the symplast (by the factor of 5). Treatment with higher sulphate concentrations (100 m m ) increases the accumulation of chromium by enhancing the rate constant of Cr(VI) transport into cells leaving other processes essentially unchanged. We suggest that 100 m m sulphate opens a new pathway for chromate transport into cells.     

Intracellular chromium reduction

Two steps are involved in the uptake of Cr(VI): (1) the diffusion of the anion CrO4(2-) through a facilitated transport system, presumably the non-specific anion carrier and (2) the intracellular reduction of Cr(VI) to Cr(III). The intracellular reduction of Cr(VI), keeping the cytoplasmic concentration of Cr(VI) low, facilitates accumulation of chromate from extracellular medium into the cell. In the present paper, a direct demonstration of intracellular chromium reduction is provided by means of electron paramagnetic (spin) resonance (EPR) spectroscopy. Incubation of metabolically active rat thymocytes with chromate originates a signal which can be attributed to a paramagnetic species of chromium, Cr(V) or Cr(III). The EPR signal is originated by intracellular reduction of chromium since: (1) it is observed only when cells are incubated with chromate, (2) it is present even after extensive washings of the cells in a chromium-free medium; (3) it is abolished when cells are incubated with drugs able to reduce the glutathione pool, i.e., diethylmaleate or phorone; and (4) it is abolished when cells are incubated in the presence of a specific inhibitor of the anion carrier, 4-acetamido-4'-isothiocyanatostilbene-2-2'-disulfonic acid.     

Kinetics of chromium(V) formation and reduction in fronds of the duckweed Spirodela polyrhiza--a low frequency EPR study

The uptake of chromate by the duckweed Spirodela polyrhiza was investigated with atomic absorption spectroscopy and the reduction of Cr(VI) to Cr(V) was measured using low frequency EPR spectroscopy. The biphasic kinetics of the uptake was fitted to parameters of a proposed kinetic model. Another model was developed to simulate chromate reduction. The first step of chromate reduction was found to be much faster than the uptake of Cr(VI) from the free space. Most probably, this step occurs already in the cell wall or on the cell membrane surface. Further reduction of Cr(V) to Cr(III) was estimated to be slower. The disappearance of the Cr(V) signal, following transfer of the plants into a Cr-free solution, lasted several tens of hours; the kinetics was mono- or biexponential depending on the length of Cr loading. The rate constants for Cr reduction in living plants were determined for the first time.     

Mechanisms of bacterial resistance to chromium compounds

Chromium is a non-essential and well-known toxic metal for microorganisms and plants. The widespread industrial use of this heavy metal has caused it to be considered as a serious environmental pollutant. Chromium exists in nature as two main species, the trivalent form, Cr(III), which is relatively innocuous, and the hexavalent form, Cr(VI), considered a more toxic species. At the intracellular level, however, Cr(III) seems to be responsible for most toxic effects of chromium. Cr(VI) is usually present as the oxyanion chromate. Inhibition of sulfate membrane transport and oxidative damage to biomolecules are associated with the toxic effects of chromate in bacteria. Several bacterial mechanisms of resistance to chromate have been reported. The best characterized mechanisms comprise efflux of chromate ions from the cell cytoplasm and reduction of Cr(VI) to Cr(III). Chromate efflux by the ChrA transporter has been established in Pseudomonas aeruginosa and Cupriavidus metallidurans (formerly Alcaligenes eutrophus) and consists of an energy-dependent process driven by the membrane potential. The CHR protein family, which includes putative ChrA orthologs, currently contains about 135 sequences from all three domains of life. Chromate reduction is carried out by chromate reductases from diverse bacterial species generating Cr(III) that may be detoxified by other mechanisms. Most characterized enzymes belong to the widespread NAD(P)H-dependent flavoprotein family of reductases. Several examples of bacterial systems protecting from the oxidative stress caused by chromate have been described. Other mechanisms of bacterial resistance to chromate involve the expression of components of the machinery for repair of DNA damage, and systems related to the homeostasis of iron and sulfur.     

Reduction of Hexavalent Chromium by Immobilized Viable Cells of Arthrobacter sp. SUK 1201

Arthrobacter sp. SUK 1201, a potent isolate reported from chromite mine overburden of Orissa, India, has been evaluated for Cr(VI) reduction with immobilized whole cells. For whole-cell immobilization, Ba-alginate was found to be most effective, and the Cr(VI) reduction potential was maximum in minimal salts (MS) medium with cells immobilized in 2% alginate. Fourier transform infrared spectra of depolymerized cells has failed to detect any sign of complexation of Cr(VI) or its reduced products with the cell mass. Reduction efficiency of the beads increased with increase in cell load, but decreased with increase in Cr(VI) concentration in the medium. Glycerol was the most potent electron donor for chromate reduction, followed by glucose and peptone. Optimum pH for Cr(VI) reduction was 7.0, and the process was inhibited by metal ions such as Ni(II), Co(II), Cd(II), Zn(II), and Mn(II) but not by Cu(II) and Fe(III). Similarly, CCCP (carbonyl cyanide-m-chlorophenylhydrazone), DCC (N,N,-dicyclohexylcarbodiimide), sodium azide, and sodium fluoride were inhibitory in nature, whereas chromate reduction was unaffected in the presence of DNP (2,4-dinitrophenol). Moreover, immobilized cells of SUK 1201 remained biologically active for four consecutive cycles, accompanied with an initial increase in cell number in the beads, although a decline in chromate reduction was recorded from the second cycle onward. Immobilized cells of Arthrobacter sp. SUK 1201, therefore, could be a potential tool for long-term uses in chromium detoxification.     

Reduction of chromate,selenite, tellurite,and iron (III) by the moderately thermophilic bacterium <Emphasis Type="Italic">Bacillus thermoamylovorans</Emphasis> SKC1

A moderately thermophilic, facultatively anaerobic bacterium capable of reducing Cr(VI) (strain SKC1) was isolated from municipal sewage. Based on the analysis of the 16S rRNA gene nucleotide sequence and DNA-DNA hybridization data, strain SKC1 was identified as a representative of the species Bacillus thermoamylovorans. B. thermoamylovorans SKC1 is capable of reducing chromate with L-arabinose as an electron donor with an optimum at 50°C and neutral pH. The culture is able to reduce Cr(VI) at its initial concentration in the medium of up to 150 mg/l. In addition to chromate, strain SKC1 is capable of reducing selenite and tellurite, as well as soluble forms of Fe(III). It was shown that Cr(VI), Te(IV), and Se(IV) exert a bacteriostatic effect on strain SKC1, and the reduction of these anions performs the detoxification function. This is the first communication on the reduction of chromate, selenite, tellurite, and soluble Fe(III) species by a culture of thermophilic bacilli.     

Characterization and genomic analysis of chromate resistant and reducing <Emphasis Type="Italic">Bacillus cereus</Emphasis> strain SJ1

Background  

Chromium is a toxic heavy metal, which primarily exists in two inorganic forms, Cr(VI) and Cr(III). Chromate [Cr(VI)] is carcinogenic, mutational, and teratogenic due to its strong oxidizing nature. Biotransformation of Cr(VI) to less-toxic Cr(III) by chromate-resistant and reducing bacteria has offered an ecological and economical option for chromate detoxification and bioremediation. However, knowledge of the genetic determinants for chromate resistance and reduction has been limited so far. Our main aim was to investigate chromate resistance and reduction by Bacillus cereus SJ1, and to further study the underlying mechanisms at the molecular level using the obtained genome sequence.     

A bacterial flavin reductase system reduces chromate to a soluble chromium(III)-NAD(+) complex

Biological reduction of carcinogenic chromate has been extensively studied in eukaryotic cells partly because the reduction produces stable chromium(III)-DNA adducts, which are mutagenic. Microbial reduction of chromate has been studied for bioremediation purposes, but little is known about the reduction mechanism. In eukaryotic cells chromate is mainly reduced non-enzymatically by ascorbate, which is usually absent in bacterial cells. We have characterized the reduction of chromate by a flavin reductase (Fre) from Escherichia coli with flavins. The Fre-flavin system rapidly reduced chromate, whereas chemical reduction by NADH and glutathione was very slow. Thus, enzymatic chromate reduction is likely the dominant mechanism in bacterial cells. Furthermore, the end-product was a soluble and stable Cr(III)-NAD(+) complex, instead of Cr(III) precipitate. Since intracellularly generated Cr(III) forms adducts with DNA, protein, glutathione, and ascorbate in eukaryotic cells, we suggest that the produced Cr(III) is primarily complexed to NAD(+), DNA, and other cellular components inside bacteria.     

Isolation and characterization of an NAD+-degrading bacterium PTX1 and its role in chromium biogeochemical cycle

Microorganisms can reduce toxic chromate to less toxic trivalent chromium [Cr(III)]. Besides Cr(OH)₃ precipitates, some soluble organo-Cr(III) complexes are readily formed upon microbial, enzymatic, and chemical reduction of chromate. However, the biotransformation of the organo-Cr(III) complexes has not been characterized. We have previously reported the formation of a nicotinamide adenine dinucleotide (NAD⁺)-Cr(III) complex after enzymatic reduction of chromate. Although the NAD⁺-Cr(III) complex was stable under sterile conditions, microbial cells were identified as precipitates in a non-sterile NAD⁺-Cr(III) solution after extended incubation. The most dominant bacterium PTX1 was isolated and assigned to Leifsonia genus by phylogenetic analysis of 16S rRNA gene sequence. PTX1 grew slowly on NAD⁺ with a doubling time of 17 h, and even more slowly on the NAD⁺-Cr(III) complex with an estimated doubling time of 35 days. The slow growth suggests that PTX1 passively grew on trace NAD⁺ dissociated from the NAD⁺-Cr(III) complex, facilitating further dissociation of the complex and formation of Cr(III) precipitates. Thus, organo-Cr(III) complexes might be an intrinsic link of the chromium biogeochemical cycle; they can be produced during chromate reduction and then further mineralized by microorganisms.     

Reduction of chromate by bacteria isolated from the cooling water of an electricity generating station

Summary Chromate-reducing bacteria were isolated from the cooling water of an electricity generating station where reduction of chromate had caused blockage of pipes by precipitation of chromium(III) oxide. Isolates identified included the generaAlcaligenes, Vibrio, Bacillus, Micrococcus, Staphylococcus andCorynebacterium. Isolate VMC-2 with the highest chromate-reducing activity was tentatively identified asComanonas testosteroni. The concentration of added chromate (K₂CrO₄, 20 M)_decreased by 95% during 45 min incubation with whole cells of VMC-2. In comparison, two Fe(III)-reducing isolates,Vibrio metschnikovii andAeromonas hydrophila, from lake sediments, showed similarly high chromate-reducing activities, and were able to reduce 99% of added chromate (20 M) in 45 min. Moderate Cr(VI)-reducers included strains ofBacillus, Vibrio andCorynebacterium. Micrococcus andStaphylococcus did not reduce Cr(VI). Sulfate (0.5 and 1.0 mM) inhibited the reduction of chromate by VMC-2 suggesting competition between the two oxyanions. Chromate-reducing activity was located in the soluble fraction of this isolate. The intermediacy of Cr(V)_in the reduction of chromate was confirmed by EPR spectroscopy. The bactericidal activity of hypochlorite towards isolate VMC-2 was determined.     

Reduction of chromate, selenite, tellurite, and iron(III) by the moderately thermophilic bacterium Bacillus thermoamylovorans SKC1

A moderately thermophilic, facultatively anaerobic bacterium capable of reducing Cr(VI) (strain SKC1) was isolated from municipal sewage. Based on the analysis of the 16S rRNA gene nucleotide sequence and DNA-DNA hybridization data, strain SKC1 was identified as a representative of the species Bacillus thermoamylovorans. B. thermoamylovorans SKC1 is capable of reducing chromate with L-arabinose as an electron donor with an optimum at 50 degrees C and neutral pH. The culture is able to reduce Cr(VI) at its initial concentration in the medium of up to 150 mg/l. In addition to chromate, strain SKC1 is capable of reducing selenite and tellurite, as well as soluble forms of Fe(III). It was shown that Cr(VI), Te(IV), and Se(IV) exert a bacteriostatic effect on strain SKC1, and the reduction of these anions performs the detoxification function. This is the first communication on the reduction of chromate, selenite, tellurite, and soluble Fe(III) species by a culture of thermophilic bacilli.     

Chromate reduction by a chromate-resistant bacterium, <Emphasis Type="Italic">Microbacterium</Emphasis> sp.

Heavy-metal chromium [Cr(VI)] is a ubiquitous environmental pollutant. Comparing with chemical reduction, microbiological reduction is considered to be a friendly and cheaper way to decrease the damage caused by chromate. A bacterial strain, CR-07, which is resistant to and capable of reducing chromate was isolated from a mud sample of iron ore and identified as a Microbacterium sp. The bacterium had a high degree of tolerance to chromate, and could grow in LB medium containing 4.08 mM of K₂Cr₂O₇. It also had a degree of resistance to other heavy metals, e.g. Cd²⁺, Pb²⁺, Zn²⁺, Cu²⁺, Co²⁺, Hg²⁺ and Ag⁺. The bacterium could remove 1.02 mM of Cr(VI) from LB medium within 36 h of incubation. Chromate removal was achieved in the supernatant from the bacterial cultures, and corresponded to chromate reduction. The activity of chromate reduction by the bacterium was not related to enzymes or reducing sugars, while fluorometric assay suggested that glutathione, a chromate-reducing substance which was produced by the bacterium, was one of the factors that contributed to the reduction of Cr(VI).     

Purification to homogeneity and characterization of a novel Pseudomonas putida chromate reductase

Cr(VI) (chromate) is a widespread environmental contaminant. Bacterial chromate reductases can convert soluble and toxic chromate to the insoluble and less toxic Cr(III). Bioremediation can therefore be effective in removing chromate from the environment, especially if the bacterial propensity for such removal is enhanced by genetic and biochemical engineering. To clone the chromate reductase-encoding gene, we purified to homogeneity (>600-fold purification) and characterized a novel soluble chromate reductase from Pseudomonas putida, using ammonium sulfate precipitation (55 to 70%), anion-exchange chromatography (DEAE Sepharose CL-6B), chromatofocusing (Polybuffer exchanger 94), and gel filtration (Superose 12 HR 10/30). The enzyme activity was dependent on NADH or NADPH; the temperature and pH optima for chromate reduction were 80 degrees C and 5, respectively; and the K(m) was 374 microM, with a V(max) of 1.72 micromol/min/mg of protein. Sulfate inhibited the enzyme activity noncompetitively. The reductase activity remained virtually unaltered after 30 min of exposure to 50 degrees C; even exposure to higher temperatures did not immediately inactivate the enzyme. X-ray absorption near-edge-structure spectra showed quantitative conversion of chromate to Cr(III) during the enzyme reaction.     

Generation of PM2 DNA breaks in the course of reduction of chromium(VI) by glutathione

The carcinogen chromate is efficiently taken up and reduced to chromium(III) compounds by various biological systems. To test the possible DNA damage induced in the course of chromium(VI) reduction, we used a combination of chromate with the reductant glutathione (GSH) as well as a green complex of chromium(V), which is formed in the reaction of chromate with GSH. The combination of chromate and glutathione was found to cause single-strand breaks in supercoiled circular DNA of the bacteriophage PM2. The green chromium(V) complex Na4(GSH)4Cr(V).8H2O, prepared from chromate and glutathione, also cleaved supercoiled PM2 DNA. No DNA-degrading effects were observed with either chromate or the final product of the reaction with GSH, a purple anionic chromium(III) GSH complex. The nature of the buffering agents revealed a strong influence on the extent of DNA strand breaks produced by chromate and GSH. A variation of the GSH concentration in the reaction with chromate and PM2 DNA, performed in sodium phosphate-buffered solutions showed an initial increase in the number of strand breaks at GSH concentrations up to 1 mM followed by a decline at higher GSH concentrations. Since neither chromate, when administered individually, nor the final product of chromium(VI) reduction, the purple chromium(III) GSH complex, produced any detectable DNA cleavage, the critical steps leading to DNA strand breaks occur in the course of the conversion of chromium(VI) to chromium(III) by GSH, the most abundant intracellular low molecular thiol. Moreover, the demonstration that DNA cleavage is induced in the presence of the chromium(V) complex identifies chromium(V) as the oxidation state of the metal, which is involved in the steps leading to DNA-damaging effects of chromate.     

Chromium(VI)-reducing <Emphasis Type="Italic">Chlorella</Emphasis> spp. isolated from disposal sites of paper-pulp and electroplating industry

We isolated four cultures of chromate resistant, unicellular, non-motile green algae from disposal sites of the paper-pulp and electroplating industries. These algae were maintained in Tris-acetate-glycerophosphate medium containing 30 μM K₂Cr₂O₇. The morphological features as well as analysis of the 500-bp fragment of 18S rDNA (NS 12 region) showed that these isolates belong to Chlorella spp. These isolates showed EC₅₀ values for chromate ranging from 60 to 125 μM. Uptake studies with radioactive ⁵¹Cr(VI) showed that 10–19% of total radioactivity was intracellular, and 1–2% was bound to the cell wall. The rest of the activity remained in the medium, suggesting that resistance was not related to accumulation of Cr(VI) in the cells. Interestingly, when these isolates were grown in the presence of 30 μM of K₂Cr₂O₇, a decrease in the Cr(VI) concentration in the medium was observed. Only live cells could deplete Cr(VI) from the supernatant, suggesting the presence of chromium reduction activity in these Chlorella isolates. Cr(VI) reduction activity of the cells of Chlorella was stimulated by light as well as by acetate and glycerophosphate. Treatment of Chlorella cells with 3-(3,4 dichlorophenyl),1,1dimethyl urea (DCMU) did not affect the Cr(VI) reduction. However, if the cells were treated with sodium azide, Cr(VI) reduction was severely affected. Though chromate resistance has been well documented in algae, the information on chromate reduction by algae is scant. This paper discusses the Cr(VI) reduction by Cr(VI) resistant Chlorella, which may find a use in the effective bioremediation of Cr(VI).     

Chromate resistance and reduction in Pseudomonas fluorescens strain LB300

Pseudomonas fluorescens LB300 is a chromateresistant strain isolated from chromium-contaminated river sediment. Chromate resistance is conferred by the plasmid pLHB1. Strain LB300 grew in minimal salts medium with as much as 1000 g of K₂CrO₄ ml^–1, and actively reduced chromate to Cr(III) while growing aerobically on a variety of substrates. Chromate was also reduced during anaerobic growth on acetate, the chromate serving as terminal electron acceptor. P. fluorescens LB303, a plasmidless, chromatesensitive variant of P. fluorescens LB300, did not grow in minimal salts medium with more than 10 g of K₂CrO₄ ml^–1. However, resting cells of strain LB303 grown without chromate reduced chromate as well as strain LB300 cells grown under the same conditions. Furthermore, resting cells of chromate-sensitive Pseudomonas putida strain AC10, also catalyzed chromate reduction. Evidently chromate resistance and chromate reduction in these organisms are unrelated. Comparison of the rates of chromate reduction by chromate grown cells and cells grown without chromate indicated that the chromate reductase activity is constitutive. Studies with cell-free extracts show that the reductase is membrane-associated and can mediate the transfer of electrons from NADH to chromate.