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.     

Hexavalent chromium reduction by an actinomycete, Arthrobacter crystallopoietes ES 32

Environmental contamination by hexavalent chromium, Cr(VI), presents a serious public health problem. This study assessed the reduction of Cr(VI) by intact cells and a cell-free extract (CFE) of an actinomycete, Arthrobacter crystallopoietes (strain ES 32), isolated from soil contaminated with dichromate. Both intact cells and CFE of A. crystallopoietes, displayed substantial reduction of Cr(VI). Intact cells reduced about 90% of the Cr(VI) added within 12 h and Cr(VI) was almost completely reduced after 24 h. The K _M and V _max of Cr(VI) bioreduction by intact cells were 2.61 μM and 0.0142 μmol/min/mg protein, respectively. Cell-free chromate reductase of the A. crystallopoietes (ES 32) reduced hexavalent chromium at a K _M of 1.78 μM and a V _max of 0.096 μmol/min/mg protein. The rate constant (k) of chromate reduction was inversely related to Cr(VI) concentration and the half-life (t _1/2) of Cr(VI) reduction increased with increasing concentration. A. crystallopoietes produced a periplasmic chromate reductase that was stimulated by NADH. Results indicate that A. crystallopoietes ES 32 can be used to detoxify Cr(VI) in polluted sites, particularly in stressed environments.     

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.     

Reduction and precipitation of chromate by mixed culture sulphate-reducing bacterial biofilms

The ability of sulphate-reducing bacterial biofilms to reduce hexavalent chromium (Cr(VI)) to insoluble Cr(III), a process of environmental and biotechnological significance, was investigated. The reduction of chromate to insoluble form has been quantified and the effects of chromate on the carbon source utilization and sulphate-reducing activity of the bacterial biofilms evaluated. Using lactate as the carbon/energy source and in the presence of sulphate, reduction of 500 micromol l-1 Cr(VI) was monitored over a 48-h period where 88% of the total chromium was removed from solution. Mass balance calculations showed that ca 80% of the total chromium was precipitated out of solution with the bacterial biofilm retaining less than 10% of the chromium. Only ca 12% of the chromate added was not reduced to insoluble form. Although Cr(VI) did not have a significant effect on C source utilization, sulphate reduction was severely inhibited by 500 micromol-1 Cr(VI) and only ca 10% of the sulphate reducing activity detected in control biofilms occurred in the presence of Cr(VI). Low levels of sulphide were also produced in the presence of chromate, with control biofilms producing over 10-times more sulphide than Cr(VI)-exposed biofilms. Sulphide- or other chemically-mediated Cr(VI) reduction was not detected. The biological mechanism of Cr(VI) reduction is likely to be similar to that found in other sulphate-reducing bacteria.     

Reduction of hexavalent chromium by Pseudomonas fluorescens LB300 in batch and continuous cultures

Batch and continuous cultures of Pseudomonas fluorescens LB300 were shown to reduce hexavalent chromium, Cr(VI), aerobically at neutral pH (pH 7.0) with citrate as carbon and energy source. The product of Cr(VI) reduction was previously shown and confirmed in this work to be trivalent chromium, Cr(III), by quantitative reoxidation to Cr(VI) with KMnO₄. In separate batch cultures (100 ml) containing initial Cr(VI) concentrations of 314.0, 200.0 and 112.5 mg Cr(VI) L^–1, the organism reduced 61%, 69% and 99.7% of the Cr(VI), respectively. In a comparison of stationary and shaken cultures, the organism reduced 81% of Cr(VI) in 147 h in stationary culture and 80% in 122 h in shaken culture. In continuous culture, the organism lowered the influent Cr(VI) concentration by 28% with an 11.7-h residence time, by 39% with a 20.8-h residence time and by 57% with a 38.5-h residence time. A mass balance of chromium in a continuous culture at steady state showed an insignificant uptake of chromium by cells of P. fluorescens LB300. Correspondence to: P. C. DeLeo     

Hexavalent chromium removal in vitro and from industrial wastes, using chromate-resistant strains of filamentous fungi indigenous to contaminated wastes

Two chromate-resistant filamentous fungi, strains H13 and Ed8, were selected from seven independent fungal isolates indigenous to Cr(VI)-contaminated soil because of their ability to decrease hexavalent chromium levels in the growth medium. Morphophysiological studies identified strain H13 as a Penicillium sp. isolate and Ed8 as an Aspergillus sp. isolate. When incubated in minimal medium with glucose as a carbon source and in the presence of 50 microg/mL Cr(VI), these strains caused complete disappearance of Cr(VI) in the growth medium after about 72 h of incubation. Total chromium concentration in growth medium was constant during culture growth, and no accumulation of chromium in fungal biomass was observed. Quantitative determinations of oxidized and reduced chromium species during the reduction process revealed stoichiometric conversion of Cr(VI) to Cr(III). A decrease in Cr(VI) levels from industrial wastes was also induced by Ed8 or H13 biomass. These results indicate that chromate-resistant filamentous fungi with Cr(VI)-reducing capability could be useful for the removal of Cr(VI) contamination.     

Removal of Hexavalent Chromium by Aspergillus niger Through Reduction and Accumulation

Abstract

Industrial activities discharge a large amount of wastes containing hexavalent chromium [Cr(VI)] into the environment, which poses a threat to human health. Microorganisms can be used as efficient tools for Cr(VI) remediation. In this study, the Cr(VI) removal capacity of Aspergillus niger was evaluated. A. niger could tolerate and reduce Cr(VI) by nearly 100% at concentrations ranging from 10 to 50?mg/L. Overall, almost 97% of the Cr(VI) removal was caused by extracellular reduction whereas 3% was caused by accumulation. Extracellular reduction was mediated by non-enzymatic cell secretions, whereas extracellular accumulated Cr formed precipitates on the hyphal surfaces and was partially absorbed on the cell wall. Cr(VI) also entered the cell and was reduced by the strong chromate reductase activity in cell-free extracts and then accumulated within the cell. These data suggest that A. niger, which has the capacity to remove Cr(VI) by reduction and accumulation, can be a useful tool for Cr(VI) remediation.     

铬还原菌的分离筛选及其在微生物燃料电池生物阴极中的应用

【目的】水溶性的Cr(Ⅵ)对环境及人类造成的危害是社会亟待解决的问题。Cr(Ⅵ)还原菌株的分离筛选、还原特性的分析和在微生物燃料电池中的应用为六价铬污染水体的微生物修复提供科学依据和新的方法。【方法】从黄河兰州段排污口采集样本,用平板法分离筛选获得具有Cr(Ⅵ)还原能力的菌株,并将Cr(Ⅵ)还原能力最强的LZU-26菌株应用到微生物燃料电池中,检测其产电能力和Cr(Ⅵ)还原特性。【结果】共分离得到21株具有Cr(Ⅵ)还原能力的菌株,其中LZU-26菌株Cr(Ⅵ)还原能力最强,属于Cellulosimicrobium cellilans。0.4 mmol/L初始Cr(Ⅵ)在LZU-26的作用下24 h铬还原率可达到95.89%,在48 h后达99.97%。将LZU-26运用在微生物燃料电池生物阴极,所获得的最大电压和最大功率密度分别为68 mV和6.8 W/cm~2。生物阴极Cr(Ⅵ)还原率(68.9%)也远高于化学阴极(14.7%)和对照组(2.7%)。【结论】利用Cr(Ⅵ)还原菌作为微生物燃料电池生物阴极处理含铬废水,将会是一种高效、节能和环境友好的方法。     

Efficient removal of hexavalent chromium by a tolerant Streptomyces sp. affected by the toxic effect of metal exposure

AIMS: To isolate and analyse chromium-resistant micro-organisms suitable for bioremediation. METHODS AND RESULTS: Strain CG252, with a minimal inhibitory concentration of 500 microg ml(-1), was isolated from contaminated soils and identified as a Streptomyces sp. by 16S rDNA sequence analysis. Assays carried out at various Cr(VI) concentrations indicated that chromium removal was more efficient at lower concentrations and that this activity resulted in accumulation of Cr(III). Atomic adsorption analysis indicated that the chromium removed was not associated with cell mass and activity assays showed that the capacity to reduce Cr(VI) was most probably due to a soluble cytosolic enzyme. Cells grown as biofilms showed enhanced removal of Cr(VI) with respect to planktonic cells, while analysis of growth and colony morphology indicated that Cr(VI) had a toxic effect on this strain. CONCLUSIONS: Streptomyces sp. CG252 tolerated heavy metals and elevated levels of chromium, despite its negative effect on growth and development, and was efficient at removing Cr(VI) by promoting reduction to Cr(III). SIGNIFICANCE AND IMPACT OF THE STUDY: Strain CG252's capacity to tolerate heavy metals and to reduce Cr(VI) to the less toxic Cr(III), especially when forming biofilms, makes it a promising candidate for detoxification of sites containing this heavy metal.     

Bacterial removal of chromium (VI) and (III) in a continuous system

The capacity of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans to reduce different concentrations of hexavalent chromium in shake flask cultures has been investigated. A. ferrooxidans reduces 100% of chromium (VI) at concentrations of 1, 2.5 and 5 ppm, but in the presence of 10 ppm only 42.9% of chromium (VI) was reduced after 11 days of incubation. A. thiooxidans showed a lower capacity to reduce this ion and total reduction of chromium (VI) was only obtained for concentrations of 1 and 2.5 ppm, whereas 64.7% and 30.5% was reached for 5 and 10 ppm, respectively, after 11 days. A continuous flow mode system was subsequently investigated, in which A. thiooxidans was immobilized on elemental sulphur and the acidic medium obtained was employed to solubilize chromium (III) and to reduce chromium (VI) present in a real electroplating waste [30% of chromium (III) and 0.1% of chromium (VI)]. The system enabled the reduction of 92.7% of hexavalent chromium and represents a promising way to treat this type of waste in the industry.     

Chromium(VI) induced alterations in mouse spleen cells: a short-term assay

Cr(VI), the highest oxidation state for chromium, is a carcinogenic and mutagenic agent. In vivo and in vitro Cr(VI) toxic effects are related to its intracellular fate. Once inside the cell it is reduced to stable Cr(III) by cysteine, glutathione and ascorbic acid. Additionally, as Cr(V) and/or Cr(IV) intermediates have been reported in Cr(VI) reactions with biological reductants, chromium damage is thought to originate from these chemical species. This work investigated the morphology of splenic cells after short-term exposure to Cr(VI). A dose of 30 mg of K2CrO4/kg body weight was administered to mice and the effects were studied 24 and 48 h after the injections. Histological results revealed a time-dependency effect of Cr(VI) on splenic cells. Changes included enlargement of the capsule and depletion of the red pulp cells, accompanied by an increase in macrophages, 24 h after injection. Partial restoration of red pulp was noted after 48 h.     

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.     

Hexavalent Chromium Reduction by Immobilized Cells and the Cell-Free Extract of Bacillus sp. ES 29

Bacillus sp. ES 29 (ATCC: BAA-696) is an efficient chromate reducing bacterium. We evaluated hexavalent chromium (Cr[VI]) reduction by immobilized intact cells and the cell-free enzyme extracts of Bacillus sp. ES 29 in a bioreactor system. Influences of different flow rates (3 to 14 mL h-1), Cr(VI) concentration (2 to 8 mg L-1), and immobilization support materials (Celite, amberlite, and Ca-alginate) on Cr(VI) reduction were examined. Both immobilized intact cells and the cell-free extract of Bacillus sp. ES 29 displayed substantial Cr(VI) reduction. Increasing flow rates from 3 to 6 mL h-1 did not affect the rate of Cr(VI) reduction, but above 6 mL h-1, the Cr(VI) reducing capacity of the immobilized intact cells and cell-free extract of Bacillus sp. ES 29 decreased. With both intact cells and the cell-free extracts, the rate of Cr(VI) reduction was inversely related to the concentration. Intact cells immobilized to Celite displayed the highest rate (k = 0.443 at 3 mL h-1) of Cr(VI) reduction. For the immobilized cell-free extract, maximal reduction (k = 0.689 at 3 mL h-1) was observed with Ca-alginate. Using initial Cr(VI) concentrations of 2 to 8 mg L-1 at flow rates of 3 to 6 mL h-1 both immobilized intact cells and the cell-free extracts reduced 84 to 98% of the influent Cr(VI). Results indicate that immobilized cells and the cell-free extracts of Bacillus sp. ES 29 could be used for large-scale removal of Cr(VI) from contaminated water and waste streams in containment systems.     

Hexavalent Chromium Reduction by Immobilized Cells and the Cell-Free Extract of Bacillus sp. ES 29

Bacillus sp. ES 29 (ATCC: BAA-696) is an efficient chromate reducing bacterium. We evaluated hexavalent chromium (Cr[VI]) reduction by immobilized intact cells and the cell-free enzyme extracts of Bacillus sp. ES 29 in a bioreactor system. Influences of different flow rates (3 to 14 mL h?1), Cr(VI) concentration (2 to 8 mg L?1), and immobilization support materials (Celite, amberlite, and Ca-alginate) on Cr(VI) reduction were examined. Both immobilized intact cells and the cell-free extract of Bacillus sp. ES 29 displayed substantial Cr(VI) reduction. Increasing flow rates from 3 to 6 mL h?1 did not affect the rate of Cr(VI) reduction, but above 6 mL h?1, the Cr(VI) reducing capacity of the immobilized intact cells and cell-free extract of Bacillus sp. ES 29 decreased. With both intact cells and the cell-free extracts, the rate of Cr(VI) reduction was inversely related to the concentration. Intact cells immobilized to Celite displayed the highest rate (k = 0.443 at 3 mL h?1) of Cr(VI) reduction. For the immobilized cell-free extract, maximal reduction (k = 0.689 at 3 mL h?1) was observed with Ca-alginate. Using initial Cr(VI) concentrations of 2 to 8 mg L?1 at flow rates of 3 to 6 mL h?1 both immobilized intact cells and the cell-free extracts reduced 84 to 98% of the influent Cr(VI). Results indicate that immobilized cells and the cell-free extracts of Bacillus sp. ES 29 could be used for large-scale removal of Cr(VI) from contaminated water and waste streams in containment systems.     

Modeling hexavalent chromium reduction in Escherichia coli 33456

A model based on te analysis of the mechanism of enzymatic reactions was developed to characterize the rate and extent of microbial reduction of hexavalent chromium in Escherichia coli 33456. A finite reduction capacity (R(c)) was proposed and incorporated into the enzymatic model to regulate the toxicity effect on cells due to the oxidizing power of Cr(VI). The parameter values were determined by nonlinear least-square analysis using experimental data of anaerobic cultures. The obtained parameters were then used to predict Cr(VI) reduction in aerobic cultures along with a modification term of uncompetitive inhibition from molecular oxygen. The applicability of the developed model was demonstrated through excellent prediction of the results of batch studies conducted over range of initial Cr(VI) concentrations, initial cell densities, and DO levels. A sensitivity analysis revealed that the parameters obtained using the experimental data were unique, and neither change in K(c), the half-velocity constant, at high initial Cr(VI) concentrations nor change in R(c), the reduction capacity, at low initial Cr(VI) concentrations was sensitive to model prediction. (c) 1994 John Wiley & Sons, Inc.     

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.     

Reduction of high concentrations of chromate by Leucobacter sp. CRB1 isolated from Changsha, China

In recent years, more and more attentions are put on the remediation of Cr(VI) contamination with chromate resistant bacteria. Leucobacter sp. CRB1 was a novel chromate reducing bacteria isolated from the soil of chromite ore processing residue (COPR) disposal site in Changsha, China. The objectives of this study were to evaluate the Cr(VI) tolerance of Leucobacter sp. CRB1 as well as its tolerant mechanism, and Cr(VI) reduction ability. The results showed that Leucobacter sp. CRB1 was able to tolerate 4,000 mg/l of hexavalent chromium with 34.5% reduction efficiency. At the optimum pH 9.0, the maximum concentration of chromate be reduced completely was 1,818 mg/l in growing cells and 2,100 mg/l in resting cells. Scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDAX) showed that extracellular Cr(VI) reduction of Leucobacter sp. CRB1 contributed to its high tolerance and high reduction ability. With repeating spiking, 2,490 mg/l hexavalent chromium was reduced totally within 17 h. The results suggest Leucobacter sp. CRB1 has potential application for remediation of high concentration of Cr(VI) contamination.     

Differential effect of chromium compounds on the immune response of the African mouth breeder Oreochromis mossambicus (Peters)

The objective of this study was to investigate the effect of trivalent (chromic chloride) and hexavalent (potassium dichromate) forms of chromium in the African mouth breeder Oreochromis mossambicus (Peters), with reference to the humoral immune response and lymphoid cells/organs. The 96 h LD50 for hexavalent and trivalent chromium was found to be 75 and 1,000microg fish(-1), respectively. Groups of fishes were injected intraperitoneally with 10, 1, 0.1 and 0.01% LD50 hexavalent and trivalent forms of chromium and subsequently immunised with bovine serum albumin (5 mg in 0.2 ml physiological saline). Both forms of chromium suppressed the antibody response, with hexavalent chromium being more suppressive than trivalent chromium. Reduction in spleen weight, splenocyte number and the percentage of blood lymphocytes was observed following administration of both forms of chromium. The possible immunological mechanisms behind the differential suppression of the antibody response and the reduction in spleen weight, splenocyte and lymphocyte counts are discussed.     

Bioremediation of Cr(VI) from Chromium-Contaminated Wastewater by Free and Immobilized Cells of Cellulosimicrobium cellulans KUCr3

In this report, possible utilization of a chromium-reducing bacterial strain Cellulosimicrobium cellulans KUCr3 for effective bioremediation of hexavalent chromium (Cr(VI))-containing wastewater fed with tannery effluents has been discussed. Cr(VI) reduction and bioremediation were found to be related to the growth supportive conditions in wastewater, which is indicative of cell mass dependency for Cr(VI) reduction. Cr(VI) reduction was determined by measuring the residual Cr(VI) in the cell-free supernatant using colorimetric reagent S-diphenylcarbazide. Nutrient availability and initial cell density showed a positive relation with Cr(VI) reduction, but it was inhibited with increasing concentration of Cr(VI) under laboratory condition. The optimum temperature and pH for effective Cr(VI) reduction in wastewater were found to be 35°C and 7.5, respectively. The viable cells of KUCr3 were successfully entrapped in an agarose bead that was used in continuous column and batch culture for assaying Cr(VI) reduction. In packed bed column (continuous flow) experiment, approximately 25% Cr(VI) reduction occurred after 144 h. Cr(VI) was almost 75% and 52% reduced at concentrations of 0.5 mM and 2 mM Cr(VI), respectively, after 96 h in batch culture experiment in peptone-yeast extract-glucose medium, whereas it could decrease the Cr(VI) content up to 40% from the water containing tannery waste. This study suggests that KUCr3 could be used as a candidate for possible environmental clean up operation with respect to Cr(VI) bioremediation.