Hexavalent Chromium Reduction and Accumulation by <Emphasis Type="Italic">Acinetobacter</Emphasis> AB1 Isolated from Fez Tanneries in Morocco

Hexavalent chromium reduction and accumulation by Acinetobacter AB1 isolated from Fez tanneries effluents were tested. The effects of some environmental factors such as pH, temperature, and exposure time on Cr(VI) reduction and resistance were investigated. We found that this strain was able to resist to concentrations as high as 400 mg/l of Cr(VI). Moreover, pH 10 and the temperature 30°C constitute favourable conditions to the growth and reduction of Acinetobacter AB1. Complete reduction of Cr(VI) was observed at low initial Cr(VI) concentrations of 50 mg/l after 72 h of incubation. Furthermore, Transmission electron microscope (TEM) analysis showed morphological changes in AB1 strain due 48H exposure to 100 mg/l chromate concentration and revealed circular electron dense (dark black point) inclusion within the cell cytoplasm suggesting chromium deposition within the cells.     

Isolation and characterization of a chromium-resistant bacterium Serratia sp. Cr-10 from a chromate-contaminated site

A novel bacterium, Cr-10, was isolated from a chromium-contaminated site and capable of removing toxic chromium species from solution by reducing hexavalent chromium to an insoluble precipitate. Sequence analysis of 16S rRNA gene of strain Cr-10 showed that it was most closely related to Serratia rubidaea JCM 1240^T (97.68%). Physiological and chemotaxonomic data also supported that strain Cr-10 was identified as Serratia sp., a genus which was never specially reported chromate-resistant before. Serratia sp., Cr-10 was tolerant to a concentration of 1,500 mg Cr(VI) L⁻¹, which was the highest level reported until now. The optimum pH and temperature for reduction of Cr(VI) by Serratia sp. Cr-10 were found to be 7.0 and 37 °C, respectively. The Cr(VI) reduction was significantly influenced by additional carbon sources, and among them fructose and lactose offered maximum reduction, with a rate of 0.28 and 0.25 mg Cr(VI) L⁻¹ h⁻¹, respectively. The cell-free extracts and filtrate of the culture were able to reduce Cr(VI) while concentration of total chromium remained stable in the process, indicating that the enzyme-catalyzed mechanism was applied in Cr(VI) reduction by the isolate. Additionally, it was found that there was hardly any chromium on the cell surface of the strain, further supporting that reduction, rather than bioadsorption, plays a major role in the Cr(VI) removal.     

Low temperature reduction of hexavalent chromium by a microbial enrichment consortium and a novel strain of Arthrobacter aurescens

Background  

Chromium is a transition metal most commonly found in the environment in its trivalent [Cr(III)] and hexavalent [Cr(VI)] forms. The EPA maximum total chromium contaminant level for drinking water is 0.1 mg/l (0.1 ppm). Many water sources, especially underground sources, are at low temperatures (less than or equal to 15 Centigrade) year round. It is important to evaluate the possibility of microbial remediation of Cr(VI) contamination using microorganisms adapted to these low temperatures (psychrophiles).     

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.     

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.     

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.     

Bioremoval of hexavalent chromium from water by a salt tolerant bacterium, Exiguobacterium sp. GS1

Pollution of terrestrial surfaces and aquatic systems by hexavalent chromium, Cr(VI), is a worldwide public health problem. A chromium resistant bacterial isolate identified as Exiguobacterium sp. GS1 by 16S rRNA gene sequencing displayed high rate of removal of Cr(VI) from water. Exiguobacterium sp. GS1 is 99% identical to Exiguobacterium acetylicum. The isolate significantly removed Cr(VI) at both high and low concentrations (1–200 μg mL⁻¹) within 12 h. The Michaelis–Menten K _m and V _max for Cr(VI) bioremoval were calculated to be 141.92 μg mL⁻¹ and 13.22 μg mL⁻¹ h⁻¹, respectively. Growth of Exiguobacterium sp. GS1 was indifferent at 1–75 μg mL⁻¹ Cr(VI) in 12 h. At initial concentration of 8,000 μg L⁻¹, Exiguobacterium sp. GS1 displayed rapid bioremoval of Cr(VI) with over 50% bioremoval in 3 h and 91% bioremoval in 8 h. Kinetic analysis of Cr(VI) bioremoval rate revealed zero-order in 8 h. Exiguobacterium sp. GS1 grew and significantly reduced Cr(VI) in cultures containing 1–9% salt indicating high salt tolerance. Similarly the isolate substantially reduced Cr(VI) over a wide range of temperature (18–45  °C) and initial pH (6.0–9.0). The T _opt and initial pH_opt were 35–40  °C and 7–8, respectively. Exiguobacterium sp. GS1 displayed a great potential for bioremediation of Cr(VI) in diverse complex environments.     

Chromium resistance strategies and toxicity: what makes <Emphasis Type="Italic">Ochrobactrum tritici</Emphasis> 5bvl1 a strain highly resistant

Large-scale industrial use of chromium (Cr) resulted in widespread environmental contamination with hexavalent chromium (Cr(VI)). The ability of microorganisms to survive in these environments and detoxify chromate requires the presence of specific resistance systems. Several Cr(VI) resistant species, belonging to a variety of genera, have been isolated in recent years. Ochrobactrum tritici strain 5bvl1 is a model for a highly Cr(VI)-resistant and reducing microorganism, with different strategies to cope with chromium. The strain contains the transposon-located (TnOtChr) chromate resistance genes chrB, chrA, chrC, chrF. The chrB and chrA genes were found to be essential for the establishment of high resistance but not chrC or chrF genes. Other mechanisms involved in chromium resistance in this strain were related to strategies such as specific or unspecific Cr(VI) reduction, free-radical detoxifying activities, and repairing DNA damage. Expression of the chrB, chrC or chrF genes was related to increased resistance to superoxide-generating agents. Genetic analyses also showed that, the ruvB gene is related to chromium resistance in O. tritici 5bvl1. The RuvABC complex probably does not form when ruvB gene is interrupted, and the repair of DNA damage induced by chromium is prevented. Aerobic or anaerobic chromate reductase activity and other unspecific mechanisms for chromium reduction have been identified in different bacteria. In the strain O. tritici 5bvl1, several unspecific mechanisms were found. Dichromate and chromate have different effects on the physiology of the chromium resistant strains and dichromate seems to be more toxic. Toxicity of Cr(VI) was evaluated by following growth, reduction, respiration, glucose uptake assays and by comparing cell morphology.     

Bioreduction of Hexavalent Chromium from Soil Column Leachate by Pseudomonas stutzeri

Bioreduction of Cr(VI) to less toxic Cr(III) by chromate-reducing bacteria has offered an ecological and economical option for chromate detoxification. The present study reports isolation of chromate-resistant bacterial strain Cr8 from chromium slag, identified as Pseudomonas stutzeri, based on 16S rRNA gene sequencing and their potential use in Cr(VI) reduction. The reduced product associated with bacterial cell was characterized by scanning electron microscopy–energy-dispersive x-ray spectroscopy (SEM-EDS) and x-ray diffraction (XRD) analyses. At initial concentrations of 100 and 200 mg L^?1 Cr(VI), P. stutzeri Cr8 reduced Cr(VI) completely within 24 h, whereas it reduced almost 1000 mg L^?1 Cr(VI) at the end of 120 h. Further, soil column leaching experiments were performed and found that bacterial cells reduced Cr(VI) leachate at faster rate that almost disappeared at the end of 168 h. The leachate precipitates also revealed efficient chromate bioreduction. The remediation process utilizing P. stutzeri could be considered as a viable alternative to reduce Cr(VI) contamination, especially emanating from the overburden dumps of chromite ores and mine drainage.     

Toxicity of Hexavalent Chromium and Its Reduction by Bacteria Isolated from Soil Contaminated with Tannery Waste

An Arthrobacter sp. and a Bacillus sp., isolated from a long-term tannery waste contaminated soil, were examined for their tolerance to hexavalent chromium [Cr(VI)] and their ability to reduce Cr(VI) to Cr(III), a detoxification process in cell suspensions and cell extracts. Both bacteria tolerated Cr(VI) at 100 mg/ml on a minimal salts agar medium supplemented with 0.5% glucose, but only Arthrobacter could grow in liquid medium at this concentration. Arthrobacter sp. could reduce Cr(VI) up to 50 μg/ml, while Bacillus sp. was not able to reduce Cr(VI) beyond 20 μg/ml. Arthrobacter sp. was distinctly superior to the Bacillus sp. in terms of their Cr(VI)-reducing ability and resistance to Cr(VI). Assays with permeabilized (treated with toluene or Triton X 100) cells and crude extracts demonstrated that the Cr(VI) reduction was mainly associated with the soluble protein fraction of the cell. Arthrobacter sp. has a great potential for bioremediation of Cr(VI)-containing waste. Received: 13 June 2002 / Accepted: 13 September 2002     

Modulation of tolerance to Cr(VI) and Cr(VI) reduction by sulfate ion in a <Emphasis Type="Italic">Candida</Emphasis> yeast strain isolated from tannery wastewater

The main aim of this study was to investigate the influence of the sulfate ion on the tolerance to Cr(VI) and the Cr(VI) reduction in a yeast strain isolated from tannery wastewater and identified as Candida sp. FGSFEP by the D1/D2 domain sequence of the 26S rRNA gene. The Candida sp. FGSFEP strain was grown in culture media with sulfate concentrations ranging from 0 to 23.92 mM, in absence and presence of Cr(VI) [1.7 and 3.3 mM]. In absence of Cr(VI), the yeast specific growth rate was practically the same in every sulfate concentration tested, which suggests that sulfate had no stimulating or inhibiting effect on the yeast cell growth. In contrast, at the two initial Cr(VI) concentrations assayed, the specific growth rate of Candida sp. FGSFEP rose when sulfate concentration increased. Likewise, the greater efficiencies and volumetric rates of Cr(VI) reduction exhibited by Candida sp. FGSFEP were obtained at high sulfate concentrations. Yeast was capable of reducing 100% of 1.7 mM Cr(VI) and 84% of 3.3 mM Cr(VI), with rates of 0.98 and 0.44 mg Cr(VI)/L h, with 10 and 23.92 mM sulfate concentrations, respectively. These results indicate that sulfate plays an important role in the tolerance to Cr(VI) and Cr(VI) reduction in Candida sp. FGSFEP. These findings may have significant implications in the biological treatment of Cr(VI)-laden wastewaters.     

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).     

Evaluation of biosorption potency of <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. for removal of hexavalent chromium from tannery effluent

Two bacterial consortia were developed by continuous enrichment of microbial population of tannery and pulp and paper mill effluent contained Serratia mercascens, Pseudomonas fluorescence, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter sp. identified by 16S rDNA method. The consortia evaluated for removal of chromate [(Cr(VI)] in shake flask culture indicated pulp and paper mill consortium had more potential for removal of chromate. Acinetobacter sp. isolated from pulp and paper mill consortium removed higher amount of chromate [Cr(VI)] under aerobic conditions. Parameters optimized in different carbon, nitrogen sources, and pH, indicated maximum removal of chromate in sodium acetate (0.2%), sodium nitrate (0.1%) and pH 7 by Acinetobacter sp. Bacteria was applied in 2-l bioreactor significantly removed chromate after 3 days. The results of the study indicated removal of more than 75% chromium by Acinetobacter sp. determined by diphenylcarbazide colorimetric assay and atomic absorption spectrophotometer after 7 days. Study of microbial [Cr(VI)] removal and identification of reduction intermediates has been hindered by the lack of analytical techniques. Therefore, removal of chromium was further substantiated by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) which indicated bioaccumulation of chromium in the bacterial cells.     

In vitro reduction of hexavalent chromium by a cell-free extract of Bacillus sp. ES 29 stimulated by Cu2+

Chromium-resistant bacteria (CRB) isolated from soils can be used to reduce toxic Cr(VI) from contaminated environments. This study assessed in vitro reduction of hexavalent Cr using a cell-free extract (CFE) of CRB isolated from soil contaminated with dichromate. One isolate, ES 29, that substantially reduced Cr(VI) was identified as a Bacillus species by 16S rRNA gene-sequence homology. The isolate reduced Cr(VI) under aerobic conditions, using NADH as an electron donor and produced a soluble Cr(VI)-reducing enzyme stimulated by copper (Cu2+). The CFE of the bacterial isolate reduced 50% of Cr(VI) in 6 h. The Cr(VI)-reduction activity of the CFE had a Km of 7.09 microM and a Vmax of 0.171 micromol min(-1) mg(-1) protein. Mercury inhibited the enzyme, but not competitively, with a Vmax of 0.143 micromol min(-1) mg(-1) protein, a Km of 7.07 microM and a Ki of 1.58 microM. This study characterizes the enzymatic reduction of Cr(VI) by Bacillus sp. ES 29 which can be used for the bioremediation of chromate.     

Chromate Reduction by a Pseudomonad Isolated from a Site Contaminated with Chromated Copper Arsenate

A pseudomonad (CRB5) isolated from a decommissioned wood preservation site reduced toxic chromate [Cr(VI)] to an insoluble Cr(III) precipitate under aerobic and anaerobic conditions. CRB5 tolerated up to 520 mg of Cr(VI) liter⁻¹ and reduced chromate in the presence of copper and arsenate. Under anaerobic conditions it also reduced Co(III) and U(VI), partially internalizing each metal. Metal precipitates were also found on the surface of the outer membrane and (sometimes) on a capsule. The results showed that chromate reduction by CRB5 was mediated by a soluble enzyme that was largely contained in the cytoplasm but also found outside of the cells. The crude reductase activity in the soluble fraction showed a K_m of 23 mg liter⁻¹ (437 μM) and a V_max of 0.98 mg of Cr h⁻¹ mg of protein⁻¹ (317 nmol min⁻¹ mg of protein⁻¹). Minor membrane-associated Cr(VI) reduction under anaerobiosis may account for anaerobic reduction of chromate under nongrowth conditions with an organic electron donor present. Chromate reduction under both aerobic and anaerobic conditions may be a detoxification strategy for the bacterium which could be exploited to bioremediate chromate-contaminated or other toxic heavy metal-contaminated environments.     

Removal of toxic chromate using free and immobilized Cr(VI)-reducing bacterial cells of Intrasporangium sp. Q5-1

Chromate-reducing microorganisms with the ability of reducing toxic chromate [Cr(VI)] into insoluble trivalent chromium [Cr(III)] are very useful in treatment of Cr(VI)-contaminated water. In this study, a novel chromate-reducing bacterium was isolated from Mn/Cr-contaminated soil. Based on morphological, physiological/biochemical characteristics and 16S rRNA gene sequence analyses, this strain was identified as Intrasporangium sp. strain Q5-1. This bacterium has high Cr(VI) resistance with a MIC of 17 mmol l⁻¹ and is able to reduce Cr(VI) aerobically. The best condition of Cr(VI) reduction for Q5-1 is pH 8.0 at 37°C. Strain Q5-1 is also able to reduce Cr(VI) in resting (non-growth) conditions using a variety of carbon sources as well as in the absence of a carbon source. Acetate (1 mmol l⁻¹) is the most efficient carbon source for stimulating Cr(VI) reduction. In order to apply strain Q5-1 to remove Cr(VI) from wastewater, the bacterial cells were immobilized with different matrices. Q5-1 cells embedded with compounding beads containing 4% PVA, 3% sodium alginate, 1.5% active carbon and 3% diatomite showed a similar Cr(VI) reduction rates to that of free cells. In addition, the immobilized Q5-1 cells have the advantages over free cells in being more stable, easier to re-use and minimal clogging in continuous systems. This study provides potential applications of a novel immobilized chromate-reducing bacterium for Cr(VI) bioremediation.     

Studies on microbial chromate reduction by Pseudomonas Sp. In aerobic continuous suspended growth cultures

Reduction of hexavalent chromium was studied in three bench-scale continuous stirred tank reactors. The inoculum was a culture of Pseudomonas sp., capable of giving 83% to 87% chromate reduction in 72-h batch assays with 60 mg Cr(VI) L(-1) in synthetic medium. The continuous culture studies were conducted for about 100 days using synthetic feed containing different levels of chromate (5 to 124 mg L(-1)) at 28 degrees to 30 degrees C and pH 6.8. The feed rate was varied over the range 0.5 to 1 L d(-1) to obtain hydraulic retention time of 36 to 72 h. Chromate reduction efficiency was 81% to 91% and 100% for influent Cr(VI) concentrations of 15 to 124 and 5 mg L(-1), respectively, with a hydraulic retention time of 72 h. (c) 1994 John Wiley & Sons, Inc.     

The Reduction of Cr(VI) by Bacteria of the Genus Pseudomonas

Non-nitrate-reducing collection bacteria from the genus Pseudomonas were found to be able to use hexavalent chromium as a terminal electron acceptor. The reduction of Cr(VI) was accompanied by an increase in the cell biomass. At Cr(VI) concentrations in the medium lower than 15 mg/l, the non-nitrate-reducing pseudomonads reduced Cr(VI) less efficiently than did denitrifying pseudomonads. In contrast, at Cr(VI) concentrations higher than 30 mg/l, Cr(VI) was reduced more efficiently by the non-nitrate-reducing pseudomonads than by the denitrifying pseudomonads.     

Evaluation of chromium(VI) removal behaviour by two isolates of Synechocystis sp. in terms of exopolysaccharide (EPS) production and monomer composition

Chromium(VI) removal and its association with exopolysaccharide (EPS) production in cyanobacteria were investigated. Synechocystis sp. BASO670 produced higher EPS (548 mg L⁻¹) than Synechocystis sp. BASO672 (356 mg L⁻¹). While the EC₅₀ of the Cr(VI) for Synechocystis sp. BASO670 and Synechocystis sp. BASO672 were determined as 11.5 mg L⁻¹, and 2.0 mg L⁻¹, respectively, there was no relation between Cr(VI) removal and EPS production. Synechocystis sp. BASO672, which has higher EPS value, removed (33%) more Cr(VI) than Synechocystis sp. BASO670. Monomer compositions of EPS of each of the isolates were determined differently. Synechocystis sp. BASO672 which removed higher Cr(VI), had higher values of uronic acid and glucuronic acid (192 μg/mg and 89%, respectively). Our results showed that EPS might play a role in Cr(VI) tolerance. Monomer composition, especially uronic acid and glucuronic acid content of EPS may have enhanced Cr(VI) removal.     

Anaerobic co-reduction of chromate and nitrate by bacterial cultures of Staphylococcus epidermidis L-02

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.