Aerobic chromate reduction by Bacillus subtilis

We have studied the reduction of hexavalent chromium (chromate) to the less toxic trivalent form by using cell suspensions and cell-free extracts from the common soil bacterium, Bacillus subtilis. B. subtilis was able to grow and reduce chromate at concentrations ranging from 0.1 to 1 mM K₂CrO₄. Chromate reduction was not affected by a 20-fold excess of nitrate-compound that serves as alternate electron acceptor and antagonizes chromate reduction by anaerobic bacteria. Metabolic poisons including sodium azide and sodium cyanide inhibited chromate reduction. Reduction was effected by a constitutive system associated with the soluble protein fraction and not with the membrane fraction. The reducing activity was heat labile and showed a K_m of 188 m CrO₄ ^2-. The reductase can mediate the transfer of electrons from NAD(P)H to chromate. The results suggest that chromate is reduced via a detoxification system rather than dissimilatory electron transport.     

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.     

Chromate reduction by Rhodobacter sphaeroides

Rhodobacter sphaeroides grew in the presence of up to 43 μM chromate and reduced hexavalent chromium to the trivalent form under both aerobic and anaerobic conditions. Reduced chromium remained in the external medium. Reductase activity was present in cells of R. sphaeroides independent of whether chromate was present or not in the growth medium. The reducing activity was found in the cytoplasmic cell fraction and was dependent on NADH. The chromate-reducing enzyme was purified by anion exchange, hydroxyapatite and hydrophobic interaction chromatography, and gel filtration. The molecular weight of the enzyme was 42 kDa as determined by gel filtration. The optimum of the reaction is at pH 7.0 and 30°C. The enzyme activity showed a hyperbolic dependence on the concentrations of both substrates, NADH and chromate, with a maximum velocity at 0.15 mM NADH. A K _m of 15±1.3 μM CrO₄ ²⁻ and a V _max of 420±50 μmol min⁻¹ mg protein⁻¹ was determined for the enzyme isolated from anaerobically grown cells and 29±6.4 μM CrO₄ ²⁻ and 100±9.6 μmol CrO₄ ²⁻ min⁻¹ mg protein⁻¹ for the one from aerobically grown ones. Journal of Industrial Microbiology & Biotechnology (2000) 25, 198–203. Received 05 January 2000/ Accepted in revised form 27 May 2000     

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.     

Bacterial reduction of toxic hexavalent chromium using a fed-batch culture of Enterobacter cloacae strain HOl

Bacterial reduction of hexavalent chromium (chromate: CrO₄²⁻) was investigated using fed-batch cultures of Enterobacter cloacae strain HOl. In the fed-batch cultures, toxic CrO₄²⁻ was continuously added in small doses to minimize the toxic effect. The fed-batch process was proved to be an effective biological method for detoxifying CrO₄²⁻ in aqueous solutions.     

Effects of vitamin E and vitamin B2 on chromate-induced DNA lesions

The induction of DNA single strand breaks by carcinogenic chromate compounds has been found to be altered by vitamin E and vitamin B₂. Pretreatment with vitamin E for 24 h prior to exposure to Na₂CrO₄ resulted in a decrease of chromate-induced DNA single strand breaks, whereas similar treatment with vitamin B₂ enhanced levels of breaks induced by chromate. In contrast, levels of DNA protein crosslinks, the other major lesion induced, were not affected by vitamin E or vitamin B₂. The uptake of Na₂CrO₄ was not affected by pretreatment with these vitamins. The role of vitamins in chromate-induced DNA damages is discussed.     

The contribution of endophytic bacteria to Albizia lebbeck-mediated phytoremediation of tannery effluent contaminated soil

Toxicity of chromium often impairs the remediation capacity of plants used in phytoremediation of polluted soils. In this study, we have identified Albizia lebbeck as a prospective chromium hyperaccumulator and examined cultivable diversity of endophytes present in chromium-treated and control saplings. High numbers (22–100%) of endophytic bacteria, isolated from root, stem, and leaf tissues, could tolerate elevated (1–3 mM) concentrations of K₂CrO₇. 16S rRNA gene sequence-based phylogenetic analysis showed that the 118 isolates obtained comprised of 17 operational taxonomic units affiliated with the proteobacterial genera Rhizobium (18%), Marinomonas (1%), Pseudomonas (16%), and Xanthomonas (7%) but also with members of Firmicutes genera, such as Bacillus (35%) and Salinococcus (3%). The novel isolates belonging to Salinococcus and Bacillus could tolerate high K₂CrO₇ concentrations (3 mM) and also showed elevated activity of chromate reductase. In addition, majority (%) of the endophytic isolates also showed production of indole-3-acetic acid. Taken together, our results indicate that the innate endophytic bacterial community assists plants in reducing heavy metal toxicity.     

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.     

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.     

Bacterial Cr(VI) Reduction Concurrently Improves Sunflower (<Emphasis Type="Italic">Helianthus Annuus</Emphasis> L.) Growth

Four Cr(VI)-reducing bacterial strains (Ochrobactrum intermedium, CrT-2, CrT-3 and CrT-4) previously isolated from chromium-contaminated sites were inoculated on to seeds of sunflower (Helianthus annuus var SF-187), which were germinated and grown along with non-inoculated controls with chromate salts (300 μg CrCl₃ or K₂CrO₄ ml⁻¹). Severe reduction (20%) in seed germination was observed in Cr(VI) stress. Plant height decreased (36%) with Cr(VI) when compared with chromium-free control, while O. intermedium inoculation resulted a 20% increment in this parameter as compared to non-inoculated chromium-free control. CrT-3 inoculation resulted a 69% increment in auxin content as compared to non-inoculated control. O. intermedium caused 30% decrease in chromium uptake in sunflower plant roots under Cr(VI) stress as compared to chromium-free control plants.     

Long-term exposure to chromium(VI) oxide leads to defects in sulfate transport system in chinese hamster ovary cells

Chromium(VI) resistant Chinese hamster ovary (CHO) cell lines were established in this study by exposing parental CHO-K1 cells to sequential increases in CrO₃ concentration. The final concentration of CrO₃ used for selection was 7 μM for Cr7 and 16 μM for Cr16 cells. Cr16-1 was a subclone derived from Cr16 cells. Next, these resistant cells were cultured in media without CrO₃ for more than 6 months. The resistance of these cells to CrO₃ was determined by colony-forming ability following a 24-h treatment. The LD₅₀ of CrO₃ for chromium(VI) resistant cells was at least 25-fold higher than that of the parental cells. The cellular growth rate, chromosome number, and the hprt mutation frequency of these chromium(VI) resistant cells were quite similar to their parental cells. The glutathione level, glutathione S-transferase, catalase activity, and metallothionine mRNA level in Cr7 and Cr16-1 cells were not significantly different from their parental cells. Furthermore, Cr16-1 cells were as sensitive as CHO-K1 cells to free-radical generating agents, including hydrogen peroxide, nickel chloride, and methanesulfonate methyl ester, and emetine, i.e., a protein synthesis inhibitor. The uptake of chromium(VI) and the remaining amount of this metal in these resistant and the parental cell lines were assayed by atomic absorption spectrophotometry. Experimental results indicated that a vastly smaller amount of CrO₃ entered the resistant cell lines than their parental cells did. A comparison was made of the sulfate uptake abilities of CHO-K1 and chromium(VI) resistant cell lines. These results revealed that the uptake of sulfate anion was substantially reduced in Cr7 and Cr16-1 cells. Extracellular chloride reduced sulfate uptake in CHO-K1 but not in Cr16-1 cells. Therefore, the major causative for chromium(VI) resistance in these resistant cells could possibly be due to the defects in SO₄^2-/C1^? transport system for uptake chromium(VI).     

Chromium-resistant bacteria and cyanobacteria: impact on Cr(VI) reduction potential and plant growth

Two chromium-resistant bacterial strains, Bacillus cereus S-6 and Ochrobactrum intermedium CrT-1, and two cyanobacterial strains, Oscillatoria sp. and Synechocystis sp., were used in this study. At initial chromate concentrations of 300 and 600 μg K₂CrO₄ mL⁻¹, and an inoculum size of 9.6×10⁷ cells mL⁻¹, B. cereus S-6 completely reduced Cr(VI), while O. intermedium CrT-1 reduced Cr(VI) by 98% and 70%, respectively after 96 h. At 100 μg K₂CrO₄ mL⁻¹, Synechocystis sp. MK(S) and Oscillatoria sp. BJ2 reduced 62.1% and 39.9% of Cr(VI), respectively, at 30°C and pH 8. Application of hexavalent chromate salts adversely affected wheat seedling growth and anatomical characters. However, bacterial inoculation alleviated the toxic effects, as reflected by significant improvements in growth as well as anatomical parameters. Cyanobacterial strains also led to some enhancement of various growth parameters in wheat seedlings.

     

The lipidome of the photosynthetic bacterium Rhodobacter sphaeroides R26 is affected by cobalt and chromate ions stress

A detailed characterization of membrane lipids of the photosynthetic bacterium Rhodobacter (R.) sphaeroides was accomplished by thin-layer chromatography coupled with matrix-assisted laser desorption ionization mass spectrometry. Such an approach allowed the identification of the main membrane lipids belonging to different classes, namely cardiolipins (CLs), phosphatidylethanolamines, phosphatidylglycerols (PGs), phosphatidylcholines, and sulfoquinovosyldiacylglycerols (SQDGs). Thus, the lipidomic profile of R. sphaeroides R26 grown in abiotic stressed conditions by exposure to bivalent cobalt cation and chromate oxyanion, was investigated. Compared to bacteria grown under control conditions, significant lipid alterations take place under both stress conditions; cobalt exposure stress results in the relative content increase of CLs and SQDGs, most likely compensating the decrease in PGs content, whereas chromate stress conditions result in the relative content decrease of both PGs and SQDGs, leaving CLs unaltered. For the first time, the response of R. sphaeroides to heavy metals as Co²⁺ and CrO₄ ^2? is reported and changes in membrane lipid profiles were rationalised.     

Study on the interaction of chromate with bovine serum albumin by spectroscopic method

The interaction between two chromates [sodium chromate (Na₂CrO₄) and potassium chromate K₂CrO₄)] and bovine serum albumin (BSA) in physiological buffer (pH 7.4) was investigated by the fluorescence quenching technique. The results of fluorescence titration revealed that two chromates could strongly quench the intrinsic fluorescence of BSA through a static quenching procedure. The apparent binding constants K and number of binding sites n of chromate with BSA were obtained by the fluorescence quenching method. The thermodynamic parameters enthalpy change (ΔH), entropy change (ΔS) were negative, indicating that the interaction of two chromates with BSA was driven mainly by van der Waals forces and hydrogen bonds. The process of binding was a spontaneous process in which Gibbs free energy change was negative. The distance r between donor (BSA) and acceptor (chromate) was calculated based on Forster’s non-radiative energy transfer theory. The results of UV–Vis absorption, synchronous fluorescence, three-dimensional fluorescence and circular dichroism (CD) spectra showed that two chromates induced conformational changes of BSA.     

Toxicity of chromium to the marine planktonic copepodAcartia clausi,Giesbrecht

The toxicity of chromium to the marine planktonic copepodAcartia clausi, Giesbrecht was studied. The LC50 48 h values (concentration of chromium lethal to 50% of the test animals) vary with the experimental temperature, the form of chromium compound tested and the annual generation to which theAcartia specimens belong. The elevation of temperature resulted in a considerable increase ofAcartia's sensitivity to chromium. Cr⁶⁺ in the form of Na₂CrO₄ was more toxic toA. clausi than in the form of CrO₃ · Cr³⁺ in the form of Cr(NO₃)₃ 9 H₂O precipitated to the bottom and was not toxic toAcartia.Acartia specimens belonging to the summer generation were more sensitive to chromium than those belonging to the winter or autumn generation. The exposure ofAcartia to sublethal concentrations of chromium resulted in a reduction of its longevity proportional to the chromium concentrations used.Furthermore, whenA. clausi was exposed to sublethal chromium concentrations it showed a decrease of feeding capacity and increase of respiratory rates, which became more pronounced with increasing chromium concentrations.     

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).     

Comparison of the uptake and distribution of chromate in rats and mice

The purpose of this study was to evaluate species differences in tissue accumulation of chromium. Rats and mice were orally exposed to Cr(VI) (potassium chromate) via drinking water (8 mg/d/kg body wt for 4 or 8 wk) or by ip injection (0.3 and 0.8 mg/d/kg, for 4 or 14 d). Chromium concentrations were measured by atomic absorption spectrophotometry, and tissues were compared for exposure route and species differences. After oral exposure, irrespective of treatment duration, liver concentrations of chromium were three to four times higher in mice than rats, whereas kidney concentrations were about 50% lower. However, after ip injection, kidney and blood concentrations in rats were two- and four-fold, higher, respectively. Both rats and mice showed high values of Cr concentration in the bone. After single ip injection of Na₂ ⁵¹CrO₄; Cr concentrations were higher in the blood of rats than mice both after 24 and 72 h. Red blood cell concentrations of Cr were also greater in rats than mice by approximately threefold, whereas white blood cell Cr concentrations were higher in mice than rats. There was also a twofold greater binding of Cr/μmol of hemoglobin in rats compared to mice. These data indicate that species differences exist for Cr metabolism and that they differ with respect to the route of exposure. These results may be owing to species differences in the reduction of Cr and different binding of Cr to hemoglobin.     

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.     

Assessment of Pollution-Induced Microbial Community Tolerance to Heavy Metals in Soil Using Ammonia-Oxidizing Bacteria and Biolog Assay

This study attempted to investigate if the tolerance of soil bacterial communities in general, and autotrophic ammonia-oxidizing bacteria (AOB) in particular, evolved as a result of prolonged exposure to metals, and could be used as an indigenous bioindicator for soil metal pollution. A soil contaminated with copper, chromium, and arsenic (CCA) was mixed with an uncontaminated garden soil (GS3) to make five test soils with different metal concentrations. A modified potential ammonium oxidation assay was used to determine the metal tolerance of the AOB community. Tolerance to Cr, Cu, and As was tested at the beginning and after up to 13 months of incubation. Compared with the reference GS3 soil, the five CCA soils showed significantly higher tolerance to Cr no matter which form of Cr (Cr³⁺, CrO₄ ^2?, or Cr₂O₇ ^2?) was tested, and the Cr tolerance correlated with the total soil Cr concentration. However, the tolerance to Cu²⁺, As³⁺, and As⁵⁺ did not differ significantly between the GS3 soil and the five CCA soils. Community level physiological profiles using Biolog microtiter plates were also used to examine the chromate tolerance of the bacterial communities extracted after six months of exposure. Our results showed that the bacterial community tolerance was altered and increased as the soil Cr concentration was increased, indicating that the culturable microbial community and the AOB community responded in a similar manner.     

Pharmacological blockade of gap junctions induces repetitive surging of extracellular potassium within the locust CNS

The maintenance of cellular ion homeostasis is crucial for optimal neural function and thus it is of great importance to understand its regulation. Glial cells are extensively coupled by gap junctions forming a network that is suggested to serve as a spatial buffer for potassium (K⁺) ions. We have investigated the role of glial spatial buffering in the regulation of extracellular K⁺ concentration ([K⁺]_o) within the locust metathoracic ganglion by pharmacologically inhibiting gap junctions. Using K⁺-sensitive microelectrodes, we measured [K⁺]_o near the ventilatory neuropile while simultaneously recording the ventilatory rhythm as a model of neural circuit function. We found that blockade of gap junctions with either carbenoxolone (CBX), 18β-glycyrrhetinic acid (18β-GA) or meclofenamic acid (MFA) reliably induced repetitive [K⁺]_o surges and caused a progressive impairment in the ability to maintain baseline [K⁺]_o levels throughout the treatment period. We also show that a low dose of CBX that did not induce surging activity increased the vulnerability of locust neural tissue to spreading depression (SD) induced by Na⁺/K⁺-ATPase inhibition with ouabain. CBX pre-treatment increased the number of SD events induced by ouabain and hindered the recovery of [K⁺]_o back to baseline levels between events. Our results suggest that glial spatial buffering through gap junctions plays an essential role in the regulation of [K⁺]_o under normal conditions and also contributes to a component of [K⁺]_o clearance following physiologically elevated levels of [K⁺]_o.