Membrane-associated chromate reductase activity from Enterobacter cloacae.

Washed cells of Enterobacter cloacae HO1 reduced hexavalent chromium (chromate: CrO4(2-) anaerobically. Chromate reductase activity was preferentially associated with the membrane fraction of the cells. Right-side-out membrane vesicles prepared from E. cloacae cells showed high chromate reductase activities when ascorbate-reduced phenazine methosulfate was added as an electron donor.     

Isolation and Characterization of an Enterobacter cloacae Strain That Reduces Hexavalent Chromium under Anaerobic Conditions

An Enterobacter cloacae strain (HO1) capable of reducing hexavalent chromium (chromate) was isolated from activated sludge. This bacterium was resistant to chromate under both aerobic and anaerobic conditions. Only the anaerobic culture of the E. cloacae isolate showed chromate reduction. In the anaerobic culture, yellow turned white with chromate and the turbidity increased as the reduction proceeded, suggesting that insoluble chromium hydroxide was formed. E. cloacae is likely to utilize toxic chromate as an electron acceptor anaerobically because (i) the anaerobic growth of E. cloacae HO1 accompanied the decrease of toxic chromate in culture medium, (ii) the chromate-reducing activity was rapidly inhibited by oxygen, and (iii) the reduction occurred more rapidly in glycerol- or acetate-grown cells than in glucose-grown cells. The chromate reduction in E. cloacae HO1 was observed at pH 6.0 to 8.5 (optimum pH, 7.0) and at 10 to 40°C (optimum, 30°C).     

A method for removal of toxic chromium using dialysis-sac cultures of a chromate-reducing strain of Enterobacter cloacae

Summary A method for removal of toxic hexavalent chromium (chlomate: CrO _inf4 ^sup2– ) was developed by use of dialysis-sac cultures of a chromate-reducing strain of Enterobacter cloacae (HO1). E. cloacae strain HO1 cells were put in dialysis (semipermeable membrane) sacs, and the sacs were submerged in water containing toxic CrO _inf4 ^sup2– . The dialysis sacs allowed CrO _inf4 ^sup2– to diffuse into the culture, and CrO _inf4 ^sup2– was reduced anaerobically in the dialysis sacs by the E. cloacae cells. Because reduced chromium readily formed insoluble chromium hydroxides in the dialysis sacs, the greater part of reduced chromium was unable to diffuse out through the semipermeable membrane. Thus the dialysis culture of E. cloacae strain HO1 could successfully remove toxic chromium from the surrounding water. If the initial concentration of CrO _inf4 ^sup2– was less than 4mM (208 ppm as chromium), about 90% of the total chromium could be removed from water by the described method. Offprint requests to: H. Ohtake     

Kinetics and modeling of hexavalent chromium reduction in Enterobacter cloacae

Kinetics of bacterial reduction of toxic hexavalent chromium (chromate: CrO(4) (2-)) was investigated using batch and fedbatch cultures of Enterobacter cloacae strain HO1. In fedbatch cultures, the CrO(4) (2-) feed was controlled on the basis of the rate of pH change. This control strategy has proven to be useful for avoiding toxic CrO(4) (2-) overload. A simple mathematical model was developed to describe the bacterial process of CrO(4) (2-) reduction. In this model, two types of bacterial cells were considered: induced, CrO(4) (2-)-resistant cells and uninduced, sensitive ones. Only resistant cells were assumed to be able to reduce CrO(4) (2-). These fundamental ideas were supported by the model predictions which well approximated all experimental data. In a simulation study, the model was also used to optimize fed-batch cultures, instead of lengthy and expensive laboratory experiments. (c) 1993 John Wiley & Sons, Inc.     

Chromate reduction and 16S rRNA identification of bacteria isolated from a Cr(VI)-contaminated site

A gram-positive, hexavalent chromium [chromate: Cr(VI)]-tolerant bacterium, isolated from tannery waste from Pakistan, was identified as a Microbacterium sp. by 16S rRNA gene sequence homology. The strain (designated as MP30) reduced toxic Cr(VI) only under anaerobic conditions at the expense of acetate as the electron donor. The bacterium was able to grow aerobically in L-broth supplemented with 15 mM CrO4(2-) but then did not reduce Cr(VI). At a concentration of 2.4x10(9) cells/ml, 100 microM sodium chromate was reduced within 30 h; however, the maximum specific reduction rate was obtained at lower initial cell concentrations.     

Effects of oxygen stress on chromate reduction in Enterobacter cloacae strain HO1

Enterobacter cloacae strain HO1 was able to reduce toxic hexavalent chromium (chromate) anaerobically. The reduction of chromate by E. cloacae cells was sensitive to oxygen stress. Cultures under continuous aeration showed no chromate reduction. However, when released from the oxygen stress, the cultures readily resumed chromate reduction.     

Microsomal metabolism of hexavalent chromium. Inhibitory effect of oxygen and involvement of cytochrome P-450

The reduction of hexavalent chromium (Cr(VI] by rat liver microsomes was studied. With 15-120 microM Na2CrO4 microsomes (0.5 mg protein/ml) effectively reduced Cr(VI) in the presence of NADPH provided anaerobic conditions. Phenobarbital (PB) and Aroclor 1254 (PCB) pretreatment increased microsomal Cr(VI) reduction while CoCl2 reduced the rate. The rates with 30 microM Na2CrO4 were: 6.4 +/- 0.1, 7.8 +/- 0.7, 13.4 +/- 0.5, 2.95 +/- 0.09 nmol Cr.mg prot.-1 min-1 for control, PB, PCB and cobalt pretreated microsomes respectively. Kinetic studies gave a Michaeli-Menten like first-order kinetics with increases both in Km and Vmax values after pretreatment with PB or PCB. CO partly inhibited the microsomal Cr(VI) reduction. The CO-sensitive reduction rate was directly correlated to the cyt. P-450 content of the different microsomal preparations. Substituting NADH for NADPH gave approximately 27% lower activity with 30 microM Na2CrO4. This activity was neither inducible by cyt. P-450 inducers nor influenced by CO. Oxygen 1.0% and 0.10% gave approximately 100% and 30% inhibition of Cr(VI) reduction (30 microM Na2CrO4) respectively, and an uncompetitive like inhibitory pattern was found. No redox cycling of Cr(VI) was seen. 51Cr binding to the microsomes was approximately 10% after complete reduction of 30 microM Na2CrO4. Externally added FMN, Fe3+-ADP and nitrobenzen stimulated microsomal Cr(VI) reduction. A 60% higher reduction rate of Cr(VI) by isolated hepatocytes was found during anaerobic in comparison with aerobic conditions.     

Bacterial Reduction of Hexavalent Chromium: Kinetic Aspects of Chromate Reduction by Enterobacter cloacae HO1

Kinetic aspects of the bacterial reduction of hexavalent chromium (chromate: CrO^2-₄) were investigated using Enterobacter cloacae strain HO1. E. cloacae strain HO1 could reduce hexavalent chromium to the trivalent form (Cr³⁺) anaerobically. High concentrations of CrO^2-₄ inhibited the reduction, and a substrate inhibition model gave a good fit to the observed data. The rate of chromate reduction was proportional to cell density. The effect of temperature on the reduction rate followed the Arrhenius equation. The rate of chromate reduction was also dependent on pH and the concentrations of carbon and energy sources in the culutre medium. Amino acids including asparagine, methionine, serine and threonine were utilized effectively as carbon and energy sources for chromate reduction.     

微生物铬转化和抗性机制与生物修复研究进展

铬(Chromium,Cr)是过渡金属元素,在自然界中以六价[CrO_4~(2-),Cr_2O_7~(2-),Cr(Ⅵ)]和三价[Cr(OH)_3,Cr(Ⅲ)]为主。很多微生物在长期铬胁迫的条件下,进化出了一系列铬转化和抗性机制。微生物对铬的转化包括Cr(Ⅵ)的还原和Cr(Ⅲ)的氧化。微生物的Cr(Ⅵ)还原可以将毒性强的六价铬转化为毒性弱或无毒的三价铬,这类微生物有较强的土壤和水体铬污染治理潜力。Cr(Ⅲ)的氧化也在铬的生物地球化学循环过程中起着至关重要的作用。除了Cr(Ⅵ)的还原,微生物对铬的抗性机制还有:(1)减少摄入;(2)外排;(3)清除胞内氧化压力;(4)DNA修复。本文主要介绍微生物的铬转化和抗性机制,以及其在铬污染生物修复中应用的最新研究进展。     

Bacterial Reduction of Hexavalent Chromium: Kinetic Aspects of Chromate Reduction by Enterobacter cloacae HO1

Kinetic aspects of the bacterial reduction of hexavalent chromium (chromate: CrO^2-₄) were investigated using Enterobacter cloacae strain HO1. E. cloacae strain HO1 could reduce hexavalent chromium to the trivalent form (Cr³⁺) anaerobically. High concentrations of CrO^2-₄ inhibited the reduction, and a substrate inhibition model gave a good fit to the observed data. The rate of chromate reduction was proportional to cell density. The effect of temperature on the reduction rate followed the Arrhenius equation. The rate of chromate reduction was also dependent on pH and the concentrations of carbon and energy sources in the culutre medium. Amino acids including asparagine, methionine, serine and threonine were utilized effectively as carbon and energy sources for chromate reduction.     

Hexavalent-chromium reduction by a chromate-resistantBacillus sp. strain

Bacillus strain QC1-2, isolated from a chromium-polluted zone, was selected by its high ability to both tolerate and reduce hexavalent chromium [Cr(VI)] to less-toxic trivalent chromium [Cr(III)]. Cell suspensions of strain QC1-2 rapidly reduced Cr(VI), in both aerobic and anaerobic conditions, to Cr(III) which remained in the supernatant. Cr(VI) reduction was dependent on the addition of glucose but sulfate, an inhibitor of chromate transport, had no effect. Studies with permeabilized cells and cell extracts showed that the Cr(VI) reductase of strain QC1-2 is a soluble NADH-dependent enzyme.     

Bio-reduction of Cr(VI) by exopolysaccharides (EPS) from indigenous bacterial species of Sukinda chromite mine, India

Chrome mining activity has contributed intensively towards pollution of hexavalent chromium around Sukinda Valley, Orissa, India. In an attempt to study the specific contribution of exopolysaccharides (EPS) extracted from indigenous isolates towards Cr(VI) reduction, three chromium (VI) tolerant strains were isolated from the effluent mining sludge. Based on the tolerance towards Cr(VI) and EPS production capacity, one of them was selected for further work. The taxonomic identity of the selected strain was confirmed to be Enterobacter cloacae (showing 98% similarity in BLAST search to E. cloacae) through 16S rRNA analysis. The EPS production was observed to increase with increasing Cr(VI) concentration in the growth medium, highest being 0.078 at 100?mg/l Cr(VI). The extracted EPS from Enterobacter cloacae SUKCr1D was able to reduce 31.7% of Cr(VI) at 10?mg/l concentration, which was relevant to the prevailing natural concentrations at Sukinda mine effluent sludge. The FT-IR spectral studies confirmed the surface chemical interactions of hexavalent chromium with EPS.     

Type I nitroreductases in soil enterobacteria reduce TNT (2,4,6,-trinitrotoluene) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)

Many enteric bacteria express a type I oxygen-insensitive nitroreductase, which reduces nitro groups on many different nitroaromatic compounds under aerobic conditions. Enzymatic reduction of nitramines was also documented in enteric bacteria under anaerobic conditions. This study indicates that nitramine reduction in enteric bacteria is carried out by the type I, or oxygen-insensitive nitroreductase, rather than a type II enzyme. The enteric bacterium Morganella morganii strain B2 with documented hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) nitroreductase activity, and Enterobacter cloacae strain 96-3 with documented 2,4,6-trinitrotoluene (TNT) nitroreductase activity, were used here to show that the explosives TNT and RDX were both reduced by a type I nitroreductase. Morganella morganii and E. cloacae exhibited RDX and TNT nitroreductase activities in whole cell assays. Type I nitroreductase, purified from E. cloacae, oxidized NADPH with TNT or RDX as substrate. When expression of the E. cloacae type I nitroreductase gene was induced in an Escherichia coli strain carrying a plasmid, a simultaneous increase in TNT and RDX nitroreductase activities was observed. In addition, neither TNT nor RDX nitroreductase activity was detected in nitrofurazone-resistant mutants of M. morganii. We conclude that a type I nitroreductase present in these two enteric bacteria was responsible for the nitroreduction of both types of explosive.     

Addition of DNA to Cr(VI) and cytochrome b5 containing proteoliposomes leads to generation of DNA strand breaks and Cr(III) complexes

Chromium (Cr) is a cytotoxic metal that can be associated with a variety of types of DNA damage, including Cr-DNA adducts and strand breaks. Prior studies with purified human cytochrome b(5) and NADPH:P450 reductase in reconstituted proteoliposomes (PLs) demonstrated rapid reduction of Cr(VI) (hexavalent chromium, as CrO(4)(2-), and the generation of Cr(V), superoxide (O(2)(*-)), and hydroxyl radical (HO(*)). Studies reported here examined the potential for the species produced by this system to interact with DNA. Strand breaks of purified plasmid DNA increased over time aerobically, but were not observed in the absence of O(2). Cr(V) is formed under both conditions, so the breaks are not mediated directly by Cr(V). The aerobic strand breaks were significantly prevented by catalase and EtOH, but not by the metal chelator diethylenetriaminepentaacetic acid (DTPA), suggesting that they are largely due to HO(*) from Cr-mediated redox cycling. EPR was used to assess the formation of Cr-DNA complexes. Following a 10-min incubation of PLs, CrO(4)(2-), and plasmid DNA, intense EPR signals at g=5.7 and g=5.0 were observed. These signals are attributed to specific Cr(III) complexes with large zero field splitting (ZFS). Without DNA, the signals in the g=5 region were weak. The large ZFS signals were not seen, when Cr(III)Cl(3) was incubated with DNA, suggesting that the Cr(III)-DNA interactions are different when generated by the PLs. After 24 h, a broad signal at g=2 is attributed to Cr(III) complexes with a small ZFS. This g=2 signal was observed without DNA, but it was different from that seen with plasmid. It is concluded that EPR can detect specific Cr(III) complexes that depend on the presence of plasmid DNA and the manner in which the Cr(III) is formed.     

Insolubilization of hexavalent chromium in highly alkaline cement sludge by anaerobic bioremediation

Chemical treatment using reducing agents such as ferrous sulfate is the most popular method for remediation of hexavalent chromium–contaminated sludge. However, the use of such chemical agents poses the risk of secondary pollution through the elution of other heavy metals. Therefore, a bioremediation method was developed to remediate high-alkali cement sludge containing hexavalent chromium. When a biomediator mainly composed of lignocellulose and lignoarabinoxylan was added to hexavalent chromium–contaminated sludge under anaerobic conditions, the amount of hexavalent chromium eluted from the treated sludge decreased significantly to below the level of the environmental standard value in Japan and its pH was reduced to 8. Moreover, the oxidation-reduction potential of the treated sludge decreased and its microbiota changed. These results indicate that anaerobic microbes can facilitate the change of hexavalent chromium to an immobilized form of trivalent chromium. Nucleotide sequence analysis suggested that anaerobic microbes activated in the sludge were Exiguobacterium aurantiacum, which are known to tolerate high pH environments and produce organic acids, even in the cement sludge. Finally, treated sludge did not elute hexavalent chromium during shaking in acid or alkaline solution.     

Isolation and characterization of a chromium-reducing bacterium from a chromated copper arsenate-contaminated site

A Gram-negative bacterium (CRB5) was isolated from a chromium-contaminated site that was capable of reducing hexavalent chromium to an insoluble precipitate, thereby removing this toxic chromium species from solution. Analysis of the 16S rRNA from the isolate revealed that it was a pseudomonad with high similarity to Pseudomaonas synxantha . CRB5 was tolerant to high concentrations of chromate (500 mg l⁻¹) and can reduce Cr(VI) under aerobic and anaerobic conditions. It also exhibited a broad range of reduction efficiencies under minimal nutrient conditions at temperatures between 4°C and 37°C and at pH levels from 4 to 9. As reduction increased, so did total cellular protein, indicating that cell growth was a requirement for reduction. Under low nutrient conditions with CRB5 or when using non-sterile contaminated groundwater from the site, reduction of Cr(VI) was followed by a increase in solution turbidity as a result of the formation of fine-grained Cr(III) precipitates, most probably chromium hydroxide mineral phases such as Cr(OH)₃. Chromium adsorption and precipitation, as observed by transmission electron microscopy coupled with energy dispersive X-ray spectroscopy (TEM/EDS), revealed that the surfaces of the cells were uniformly stained with bound Cr(III) and amorphous precipitates (as determined by selected area electron diffraction; SAED). A mass balance of chromium in a batch bioreactor revealed that up to 30% of the total Cr was as settable precipitates or bound to cells.     

Hexavalent chromium reduction by bacteria from tannery effluent

Chromium is generated from several industrial processes. It occurs in different oxidation states, but Cr(III) and Cr(VI) are the most common ones. Cr(VI) is a toxic, soluble environmental contaminant. Some bacteria are able to reduce hexavalent chromium to the insoluble and less toxic Cr(III), and thus chromate bioremediation is of considerable interest. An indigenous chromium-reducing bacterial strain, Rb-2, isolated from a tannery water sample, was identified as Ochrobactrum intermedium, on the basis of 16S rRNA gene sequencing. The influence of factors like temperature of incubation, initial concentration of Cr, mobility of bacteria, and different carbon sources were studied to test the ability of the bacterium to reduce Cr(VI) under variable environmental conditions. The ability of the bacterial strain to reduce hexavalent chromium in artificial and industrial sewage water was evaluated. It was observed that the mechanism of resistance to metal was not due to the change in the permeability barrier of the cell membrane, and the enzyme activity was found to be inductive. Intracellular reduction of Cr(VI) was proven by reductase assay using cell-free extract. Scanning electron microscopy revealed chromium precipitates on bacterial cell surfaces, and transmission electron microscopy showed the outer as well as inner distribution of Cr(VI). This bacterial strain can be useful for Cr(VI) detoxification under a wide range of environmental conditions.     

The cytotoxicity and genotoxicity of hexavalent chromium in Steller sea lion lung fibroblasts compared to human lung fibroblasts

In this study we directly compared soluble and particulate chromate cytotoxicity and genotoxicity in human (Homo sapiens) and sea lion (Eumetopias jubatus) lung fibroblasts. Our results show that hexavalent chromium induces increased cell death and chromosome damage in both human and sea lion cells with increasing intracellular chromium ion levels. The data further indicate that both sodium chromate and lead chromate are less cytotoxic and genotoxic to sea lion cells than human cells, based on an administered dose. Differences in chromium ion uptake explained some but not all of the reduced amounts of sodium chromate-induced cell death. By contrast, uptake differences could explain the differences in sodium chromate-induced chromosome damage and particulate chromate-induced toxicity. Altogether they indicate that while hexavalent chromium induces similar toxic effects in sea lion and human cells, there are different mechanisms underlying the toxic outcomes.     

Competition between different strains of Streptococcus cremoris

Abstract Streptococcus cremoris strain HP was found to grow poorly on agar plates under aerobic conditions in comparison to several other strains of S. cremoris (Wg2, ML1, AM1, E8). This made it possible to determine the numbers of strain HP in mixed cultures with other strains under different culture conditions. None of the mixtures was stable in batch cultures as a result of differences in the maximum specific growth rate. In continuous culture under lactose limitation strain HP outcompeted strains E8 and ML1 at low dilution rates, but at high dilution rates and in batch culture the reverse was observed. This represents another example of crossing μ-s curves in anaerobic bacteria.