Reduction of Cr(VI) by a Bacillus sp.

A Bacillus sp. RE was resistant to chromium and reduced Cr(VI) without accumulating chromium inside the cell. When Cr(VI) was 10 and 40 μg ml⁻¹, >95% of the total Cr(VI) was reduced in 24 and 72 h of growth, respectively, whereas at 80 μg Cr(VI) ml⁻¹ only 50% of Cr(VI) was reduced. However growth was not affected; the cell mass was 0.7–0.8 mg ml⁻¹ in all cases. The cell-free extract showed Cr(VI) reducing enzyme activity which was enhanced (>5 fold) by NADH and NADPH. Like whole cells the enzyme also reduced Cr(VI) with decreasing efficiency on increasing Cr(VI) concentration. The enzyme activity was optimal at pH 6.0 and 30 °C. The enzyme was stable up to 30 °C and from pH 5.5 to 8, but from pH 4 to 5 the enzyme was severely destabilized. Its K_m and V_max were 14 μm and 3.8 nmol min⁻¹ mg⁻¹ respectively. The enzyme activity was enhanced by Cu²⁺ and Ni²⁺ and inhibited by Hg²⁺. Received 21 September 2005; Revisions requested 5 October 2005; Revisions received 16 November 2005; Accepted 16 November 2005     

A novel isolate of Desulfovibrio sp. with enhanced ability to reduce Cr(VI)

Kinetic analysis of the reduction of Cr(VI) by resting cell suspensions of Desulfovibrio vulgaris ATCC 29579 and a new isolate, Desulfovibrio sp. (`Oz7') was studied using lactate as the electron donor at 30 °C. The apparent K <sub>m</sub> (K <sub>m app</sub>) and V <sub>max</sub> with respect to Cr(VI) reduction was compared for both strains. Desulfovibio sp. `Oz7' had a K _{m app} of 90 M (threefold lower than that of D. vulgaris ATCC 29579) and a V _max of 120 nmol h^–1 mg^–1 biomass dry wt (approx. 30% lower than for the reference strain). The potential of the new isolate for bioremediation of Cr(VI) wastewaters is discussed.     

Effect of complexing agents on reduction of Cr(VI) by Desulfovibrio vulgaris ATCC 29579

The reduction of Cr(VI) at the expense of molecular hydrogen was studied using resting cells of Desulfovibrio vulgaris ATCC 29579 in anaerobic resting cell suspensions in MOPS buffer. Bioreduction occurred only in the presence of ligands or chelating agents (CO32-, citrate, NTA, EDTA, DTPA). The stimulatory effect of these ligands on the rate of Cr(VI) reduction was correlated (r = 0.988) with the strength of the ligand/chelate complex of Cr(III). The data are examined with respect to likely solution and redox equilibria in the ionic matrix of the carrier solution, and with respect to the potential for bioremediation of Cr(VI).     

Reduction of Cr(VI) by immobilized cells of Desulfovibrio vulgaris NCIMB 8303 and Microbacterium sp. NCIMB 13776

Hexavalent chromium, a carcinogen and mutagen, can be reduced to Cr(III) by Desulfovibrio vulgaris NCIMB 8303 and Microbacterium sp. NCIMB 13776. This study examined Cr(VI) reduction by immobilized cells of the two strains in a common solution matrix using various entrapment matrices. Chitosan and PVA-borate beads did not retain integrity and supported low or no reduction of Cr(VI) by the cells. A commercial preparation (Lentikats) was stable but also did not support Cr(VI) reduction. K-carrageenan beads were stable in batch suspensions but gel integrity was lost after only 5 h in a flow-through system in the presence of 100 microM Cr(VI). The best immobilization matrices were agar and agarose, where the initial rates of reduction of Cr(VI) (from 500 microM solution) for D. vulgaris NCIMB 8303 and Microbacterium sp. NCIMB 13776 were 127 (agar) and 130 (agarose), and 15 (agar) and 12 (agarose) nmol h(-1) mg dry cell wt(-1), respectively. The higher removal of Cr(VI) by D. vulgaris was also seen in 14-mL packed-bed flow-through columns, where, at a flow rate of 2.4 mL h(-1), the percentage removal of Cr(VI) was approximately 95% and 60% for D. vulgaris and Microbacterium sp., respectively (agar-immobilized cells). The Cr(VI) reducing activities of D. vulgaris and Microbacterium sp. were lost after 159 and 140 h, respectively. Examination of the beads for structural integrity within the columns in situ using magnetic resonance imaging after 24 and 100 h of continuous operation against Cr(VI) (with negligible Cr retained within the columns) showed that agar beads were more stable with time. The most appropriate system for development of a continuous bioprocess is thus the use of D. vulgaris NCIMB 8303 immobilized in an agar gel matrix.     

Cr(Ⅵ)还原菌Microbacterium sp.BD6的分离鉴定及还原特性

【目的】从电镀厂下水道的淤泥中分离筛选Cr(Ⅵ)高效还原菌,并对其生长和还原特性进行研究,以期为Cr(Ⅵ)污染的生物修复提供优质的菌种资源和应用参考。【方法】采用富集培养法从淤泥中分离、筛选出Cr(Ⅵ)还原菌,通过生理生化及16S rRNA基因序列分析进行初步鉴定。采用单因素实验确定菌株的最佳培养条件和抵抗胁迫环境的能力,利用外加电子供体改善菌株的Cr(Ⅵ)还原能力,筛选出最佳电子供体研究对菌株还原的影响。【结果】经分离筛选得到1株Cr(Ⅵ)耐受还原菌,初步鉴定为微杆菌属(Microbacterium sp.),命名为BD6。菌株BD6适宜在中温、偏碱性的环境条件下生长,能耐受50.0 g/L NaCl的高盐环境。Mn^2+对菌种的生长表现出较高的抑制,Ni^2+、Zn^2+、Cd^2+的抑制作用较小,Cu^2)产生了一定的促进作用。Cr(Ⅵ)对BD6的最低抑菌浓度为1700 mg/L。添加甘油、果糖、乳糖、葡萄糖、丙酮酸钠作为电子供体促进了菌株对Cr(Ⅵ)的还原。选择甘油作为菌株还原Cr(Ⅵ)的最佳电子供体,无电子供体添加时菌株96 h内对100 mg/L Cr(Ⅵ)的还原率仅为69.63%,添加2 g/L的甘油菌株在36 h内的还原率达到了100%。通过加大甘油的添加量可以促进菌株对初始浓度较高Cr(Ⅵ)的还原,但要受到Cr(Ⅵ)的毒性限制。菌株的最适还原条件和最适生长条件吻合,在50.0 g/L NaCl的高盐条件和50 mg/L Cd^2+的毒性环境中,添加2 g/L的甘油,菌株对100 mg/L Cr(Ⅵ)的还原率分别为72 h 96.79%、54 h 99.86%。【结论】分离筛选得到的Microbacterium sp.BD6是一株潜在的可用于Cr(Ⅵ)污染生物还原修复的候选菌株。     

Chromate reduction by Microbacterium liquefaciens immobilised in polyvinyl alcohol

A polyvinyl alcohol-based immobilisation technique has been utilised for entrapping the newly-isolated chromate-reducing bacterium, Microbacterium liquefaciens MP30. Three immobilisation methods were evaluated: PVA-nitrate, PVA-borate and PVA-alginate. Chromate reduction was studied in batch and continuous-flow bioreactors, where the beads maintained integrity during continuous operation. PVA-borate and PVA-alginate cell beads showed a higher rate and extent of chromate reduction than PVA-nitrate cell beads in batch experiments. With the former 100 M Cr(VI) was removed within 4 days, while only 40 M Cr(VI) was removed using the latter, and with no increase in Cr(VI) removal subsequently. Cell activity was maintained during immobilisation but the rate of Cr(VI) removal by immobilised cells was only half that of an equivalent mass of free cells. Using PVA-alginate cell beads in a continuous-flow system, chromate removal was maintained at 90–95% from a 50 M solution over 20 days without signs of bead breakdown.     

Reduction of Cr(VI) by "palladized" biomass of Desulfovibrio desulfuricans ATCC 29577

A novel catalytic activity of palladium [Pd(0)]-coated cells of Desulfovibrio desulfuricans ATCC 29577 ["bio-Pd(0)"] is demonstrated. Reduction of 700 microM Cr(VI) occurred within 24 h using formate (25 mM) or hydrogen (1 atm) as the electron donor, under conditions whereby cells lacking bound Pd(0), or palladium metal manufactured via chemical reduction of soluble Pd(II), did not reduce Cr(VI). The biomass-bound Pd(0) also functioned in the continuous removal of 400 microM Cr(VI) from a 1 mM solution under H(2) (flow residence time approximately 5 h), where chemically prepared Pd(0) was ineffective. This demonstrates a new type of active bioinorganic catalysis, whereby the presence of biomass bound to Pd(0) confers a novel catalytic capability not seen with Pd base metal or biomass alone.     

Aerobic Cr(VI) reduction by Thermus scotoductus strain SA-01

AIM: To evaluate Thermus scotoductus SA-01's ability to reduce Cr(VI) aerobically. METHODS AND RESULTS: T. scotoductus SA-01 is able to reduce Cr(VI) aerobically when grown in a complex organic medium containing Cr(VI) concentrations up to 0.5 mmol l(-1). Suspension of T. scotoductus SA-01 cells also reduced Cr(VI) aerobically under nongrowth conditions using a variety of electron donors as well as in the absence of an exogenous electron donor. The optimum temperature and pH for Cr(VI) reduction under nongrowth conditions were found to be 80 degrees C and 7, respectively. It was also found that the Cr(VI) reduction was catalysed by a cytoplasmic, constitutively expressed enzyme. CONCLUSIONS: Apart from SA-01's ability to reduce Cr(VI) through a strictly anaerobic membrane-bound mechanism (unpublished data), it also has a second enzyme localized in the cytoplasm that can reduce Cr(VI) aerobically. As this enzyme is constitutively expressed and not induced by Cr(VI), it remains to be determined whether it has any other physiological functions. SIGNIFICANCE AND IMPACT OF THE Study: This is the first report of a Thermus species able to reduce Cr(VI) aerobically and extends the knowledge of parameters associated with Cr(VI) reduction. Employing thermophiles in bioremediation using industrial bioreactors would cancel the need for expensive cooling systems.     

Reduction of chromate by microorganisms isolated from metal contaminated sites of Karachi, Pakistan

Three bacterial strains, two identified as Pseudomonas stutzeri and one as a strain of cucurbit yellow vine disease bacterium, isolated from a foundry soil and a tannery, respectively, in Pakistan, were resistant to up to 1 mM chromate and anaerobically reduced Cr(VI) up to 100 M. The highest removal was by P. stutzeri CMG463: 88 mol l^–1 (88% of that supplied; specific rate was 3.0 nmol mg^–1 protein h^–1), while 58 and 76 mol l^–1 (58% and 76%) were removed by P. stutzeri CMG462 and cucurbit yellow vine disease bacterium CMG480, respectively. These isolates were compared to strains isolated from an uncontaminated coastal site in the UK and designated as K2 (Pseudomonas synxantha) K3 (Bacillus sp.), and J3 (unidentified Gram-positive strain). Strain K3 was Cr-sensitive, partially lysed by Cr(VI), but had the highest removal of chromate anaerobically: 92 mol l^–1 (92% of that supplied) at a specific rate of 71 nmol mg^–1 protein h^–1. Analysis of cell sections using transmission electron microscopy with energy dispersive X-ray analysis showed intracellular chromium in P. stutzeri but the cucurbit yellow vine disease bacterium and the Bacillus sp. precipitated chromium extracellularly. The isolates from the Cr-contaminated sites did not remove more Cr(VI), overall, than Cr-unstressed bacteria, but their tolerance to Cr(VI) is potentially useful for bioremediation, particularly since other studies have shown that the two P. stutzeri strains can bioaccumulate Cu²⁺.     

Concurrent Removal of Mn(II) and Cr(VI) by Achromobacter sp. TY3-4

In this study, we report a bacterium, Achromobacter sp. TY3-4, capable of concurrently removing Mn (II) and Cr (VI) under oxic condition. TY3-4 reduced as much as 2.31?mM of Cr (VI) to Cr (III) in 70?h, and oxidized as much as 20?mM of Mn(II) to Mn oxides in 80?h. When 0.58?mM Cr (VI) and 10?mM Mn(II) were present together, both Cr(VI) and Mn(II) were completely removed by TY3-4 and the generated precipitates are Mn^IIIOOH, Mn^III,IV₃O₄, Mn^IVO₂ and Cr^III(OH)₃. Experiments also show that both biosroption and bioreduction of Mn(II) are the driving forces for Mn(II) removal, whereas bioreduction of Cr(VI) is the driving force for Cr(VI) removal. On the basis of these results, a possible reaction was proposed that TY3-4 concurrently reduces Cr(VI) and oxidizes Mn(II). This study is fundamental for Mn and Cr cycles. The strain shows potential for practical application.     

Cr(VI) reduction by Enterobacter sp. DU17 isolated from the tannery waste dump site and characterization of the bacterium and the Cr(VI) reductase

Out of nineteen bacteria screened from the tannery waste dump site, the most effective isolate, strain DU17 was selected for Cr(VI) reduction process among the non-pathogenic once. Based on 16S rRNA gene sequence analysis, the bacterium was identified as Enterobacter sp. DU17. Its amplified Cr(VI) reductase gene showed maximum homology with flavoprotein of Enterobacter cloacae. Enterobacter sp. DU17 reduced Cr(VI) maximally at 37 °C and pH 7.0. Various co-metals, electron (e⁻) donors and inhibitors were tested to study their effect on Cr(VI) reduction. In presence (0.2% each) of glucose and fructose, Enterobacter sp. DU17 reduced Cr(VI) completely after 16 and 20 h, respectively. Since the concentration of total Cr was invariable after remediation as detected through AAS analysis, this experiment disclosed that responsible operation was associated with extracellular Cr(VI) reduction process rather than uptake mechanism. Multiple antibiotic resistance index of 0.08 for this bacterium was very low as compared to standard risk assessment value of 0.20. With high Cr(VI) reducing capability, non-pathogenicity and antibiotic sensitivity, Enterobacter sp. DU17 is found to be very efficient in removing Cr(VI) toxicity from the environment.     

Permeable reactive biobarriers for in situ Cr(VI) reduction: bench scale tests using Cellulomonas sp. strain ES6

Chromate (Cr(VI)) reduction studies were performed in bench scale flow columns using the fermentative subsurface isolate Cellulomonas sp. strain ES6. In these tests, columns packed with either quartz sand or hydrous ferric oxide (HFO)-coated quartz sand, were inoculated with strain ES6 and fed nutrients to stimulate growth before nutrient-free Cr(VI) solutions were injected. Results show that in columns containing quartz sand, a continuous inflow of 2 mg/L Cr(VI) was reduced to below detection limits in the effluent for durations of up to 5.7 residence times after nutrient injection was discontinued proving the ability of strain ES6 to reduce chromate in the absence of an external electron donor. In the HFO-containing columns, Cr(VI) reduction was significantly prolonged and effluent Cr(VI) concentrations remained below detectable levels for periods of up to 66 residence times after nutrient injection was discontinued. Fe was detected in the effluent of the HFO-containing columns throughout the period of Cr(VI) removal indicating that the insoluble Fe(III) bearing solids were being continuously reduced to form soluble Fe(II) resulting in prolonged abiotic Cr(VI) reduction. Thus, growth of Cellulomonas within the soil columns resulted in formation of permeable reactive barriers that could reduce Cr(VI) and Fe(III) for extended periods even in the absence of external electron donors. Other bioremediation systems employing Fe(II)-mediated reactions require a continuous presence of external nutrients to regenerate Fe(II). After depletion of nutrients, contaminant removal within these systems occurs by reaction with surface-associated Fe(II) that can rapidly become inaccessible due to formation of crystalline Fe-minerals or other precipitates. The ability of fermentative organisms like Cellulomonas to reduce metals without continuous nutrient supply in the subsurface offers a viable and economical alternative technology for in situ remediation of Cr(VI)-contaminated groundwater through formation of permeable reactive biobarriers (PRBB).     

Continuous removal of Cr(VI) from aqueous solution catalysed by palladised biomass of Desulfovibrio vulgaris

Growth-decoupled cells of Desulfovibrio vulgaris NCIMB 8303 can be used to reduce Pd(II) to cell-bound Pd(0) (Bio-Pd⁰), a bioinorganic catalyst capable of reducing hexavalent chromium to less toxic Cr(III), using formate as the electron donor. Magnetic resonance imaging showed that Bio-Pd⁰, immobilized in chitosan and agar beads, is distinguishable from the surrounding gel and is evenly dispersed within the immobilization matrix. Agar-immobilized Bio-Pd⁰ and `chemical Pd⁰' were packed into continuous-flow reactors, and challenged with a solution containing 100 m Cr(VI) (pH 7) at a flow rate of 2.4 ml h^–1. Agar-immobilized chemical Pd⁰ columns lost Cr(VI) reducing ability by 160 h, whereas columns containing immobilized Bio-Pd⁰ maintained 90% reduction until 680 h, after which reduction efficiency was gradually lost.     

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.     

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.     

Molecular cloning, expression and characterization of a chitosanase from Microbacterium sp.

A gene encoding a chitosanase (mschito) was cloned from Microbacterium sp. OU01. The ORF consists of 801 bp which encoded a polypeptide of 266 amino acid residues. The deduced amino acid sequence shows 98% identity to that of the chitosanase reported in Pseudomonas sp. A-01. In addition, the fusion protein containing MSCHITO was expressed in E. coli and purified using Ni-NTA affinity chromatography. The purified rMSCHITO protein degraded the chitosan (the degree of deacetylation of 99%) and produced a mixture of chitooligosaccharides. The MSCHITO is thus an endo-chitosanase.     

Comparative Study of Cr(VI) Removal by Exiguobacterium sp. in Free and Immobilized Forms

Cr(VI) is a toxic environmental pollutant. To determine the potential role of microbes towards chromate bioremediation, two bacterial strains, E1 and E4, that could tolerate Cr(VI) at levels up to 2250 μg ml^?1 were isolated from the soil of a tannery. They were identified as Exiguobacterium sp. To estimate the removal of Cr(VI) using immobilized bacterial cells, 2% sodium alginate and 2.5% agar were used as immobilizing matrices. In the case of sodium alginate, 89% and 93% of Cr(VI) removal by E1 and E4, respectively, were observed. When agar beads were used as an immobilizing matrix, removal was recorded as 39% and 48% for E1 and E4, respectively. Removal of Cr(VI) was also estimated in sterile and nonsterile tannery effluent. More Cr(VI) removal was noted in the nonsterile effluent than in the sterile effluent. The maximum uptake of Cr(VI) of bound cells of E1 and E4 was found to be 17.54 and 20.04 μg ml^?1, respectively. Fourier transform infrared (FTIR) spectra of cells of E4 with Cr(VI), without Cr(VI), and immobilized cells depicted several absorption peaks, mainly for P?OH group, C?H bending, C?O bond, and amide II groups, reflecting the complex nature of the bacterial cells and the contribution of these functional groups to the Cr(VI) binding process.     

Bioremediation Potential of Cr(VI)-Contaminated Soil Using Indigenous Microorganisms

Amendment of Cr(VI)-contaminated soil (approx. 200 mg/kg) with various treatments resulted in greater CO₂ evolution and Cr(VI) reduction with organic amendments relative to controls receiving no organics, indicating bacterial reduction of Cr(VI) under anaerobic conditions. Isolation of Cr(VI)-reducing, indigenous bacteria, representative of the dominant soil population, further indicated Cr(VI) reduction by indigenous bacteria. Although desorption of Cr(VI) was evident with some treatments, its reduction was not affected.     

Comparative study of Cr(VI) uptake and reduction in industrial effluent by Ochrobactrum intermedium and Brevibacterium sp.

Two chromium-resistant bacterial strains, CrT-1 and CrT-13, tolerant up to 40mg K₂CrO₄ ml^–1 on nutrient agar, 25mgml^–1 in nutrient broth, and up to 10mgml^–1 in acetate-minimal media, were identified as Ochrobactrum intermedium and Brevibacterium sp., respectively, on the basis of 16S rRNA gene sequencing. Uptake of chromate was greater in living cells than in heat-killed on dried cells. CrT-1 reduced 82%, 28% and 16% of Cr(VI) at 100, 500, and 1000gml^–1 after 24h while CrT-13 reduced 41%, 14% and 9%. Other heavy metals at low concentrations did not affect these reductions. At 150 and 300gml^–1 in an industrial effluent sample Cr(VI) was reduced by 87% and 71%, respectively, with CrT-1 and by 68% and 47% with CrT-13.Revisions requested 17 May 2004; Revisions received 2 July 2004