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.     

Genomic Analysis and Comparative Hexavalent Chromium Reduction Potential of Predominant Bacillus species Isolated from Chromite Mine Soil

Ten predominant bacteria (CSB 1-10), isolated from chromite mine soil of Sukinda, Odisha, were characterized by means of biochemical and 16S rRNA gene sequencing. All of the bacterial isolates were Gram-positive, spore-forming, and motile rods with diameter ranging from 1.57–2.79 μm and identified as Bacillus species. Based on 16S rRNA gene sequencing and phylogenetic tree construction, 10 Bacillus species were grouped into two clusters: Bacillus subtilis cluster with four species (two of B. amyloliquefaciens, and one each of B. tequilensis and B. mojavensis); and Bacillus cereus cluster containing six species of B. cereus. Secondary rRNA structure predicted for all 10 bacteria using 16S rRNA sequences revealed some degree of genetic variations among the species. Among the isolated bacteria, CSB-9 was found to be the most efficient chromate reducing strain (0.8 × 10^?4 mg mg^?1 h^?1) in comparison to the others. Chromate reduction of the bacteria was associated with the contribution of extracellular enzyme production, and highest enzyme activity (0.9 ± 0.09 U mL^?1) was observed in CSB-9 (B. amyloliquefaciens). The present study revealed the dominance of Bacillus species in the chromite mine soil and their potential for bioremediation of hexavalent chromium from the polluted environments.     

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     

Aerobic Reduction of Hexavalent Chromium in Soil by Indigenous Microorganisms

Surface soil containing 25,100 mg/kg total Cr [12,400 mg/kg Cr(VI)] obtained from a Superfund site was used in laboratory microcosm studies to evaluate the potential for aerobic reduction of Cr(VI) by the indigenous soil microbial community. Hexavalent chromium in soil was reduced by as much as 33% (from 1840 to 1240 mg/L) within 21 days under enrichment conditions. Reduction of Cr(VI) in this system was biologically mediated and depended on the availability of usable energy sources. Mass balance studies suggested that the microbial populations removed Cr(VI) from the soil solutions by reduction. Indigenous microbial soil communities even with no history of Cr(VI) contamination were capable of mediating this process. However, Cr(VI) removal was not observed when microbial populations from a sewage sludge sample were added to the soil microcosms. The results suggest that Cr(VI)-reducing microbial populations are widespread in soil, and thus the potential exists for in situ remediation of environmentally significant levels of Cr( VI) contamination.     

Degradation of Urea Herbicides by Cell-Free Extracts of Bacillus sphaericus

N′-methoxy phenylurea herbicides are degraded by induced cells of Bacillus sphaericus ATCC 12123 by liberating carbon dioxide from the ureido portion of the molecule and leaving the corresponding aniline moieties. Cell-free extracts of B. sphaericus inactivate these herbicides in the same way as reported for whole cells. A 6.6-fold purification of the crude extract was achieved by a combination of salt fractionation with ammonium sulfate and column chromatography on diethyl-aminoethyl cellulose.     

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.     

The Synthesis In Vitro of Flagellin by a Cell-Free Extract from Bacillus Pumilus

Abstract

Bacterial flagella are constructed mainly, or perhaps exclusively, of protein subunits, the flagellins. Demonstration is given in this report for the in vitro incorporation of radioactive amino acids into flagellin, it also appears that part of such incorporation reflects de novo synthesis of flagellin moleculus. Cell-free extracts were prepared from flagellated cells of Bacillus pumilus, by digestion of the cell wall with lysozyme, lysis in the Standard buffer of NIRENBERG and MATTHAEI (1961), treatment with deoxyribonuclease and centrifugation at 15.000×g. The reaction mixtures contained the cell-free extract, one or more [¹⁴C]-amino acids and the usual components required for cell-free protein synthesis. After incubation at 37° carrier flagellin was added and the pH of the reaction mixture adjusted to 2. Flagellin, which is soluble at this pH, was purified by disc electrophoresis or by reconstitution of flagellar filament at pH 5.4 followed by electrophoresis on a column of ethanolized cellulose. When an amino acid absent from B. pumilus flagellin (such as tyrosine) was used, the amount of radioactivity incorporated into the flagellin fraction was negligible as compared to that incorporated when radioactive leucine, arginine and lysine were used. The identity of the purified radioactive protein was established more conclusively by tryptic digestion and chromatographic separation of the resulting peptides. The ninhydrin positive peaks were shown to be coincident with the radioactive peaks. The radioactive peaks disappeared when a cell-free extract from non-flagellated mutant cells was used. The incorporation of radioactive methionine in the N-terminal position of the molecule indicated that at least some of the molecules had been synthesized de novo.     

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.     

Oxidation and Reduction of D-Xylose by Cell-Free Extract of Pichia quercuum

The fermentation mechanism of the simultaneous production of D-xylonic acid and xylitol from D-xylose by Pichia quercuum was studied by using a cell-free enzyme preparation. Nicotinamide adenine dinucleotide phosphate (NADP)-dependent D-xylose dehydrogenase activity and NADP-dependent D-xylose reductase activity were detected, and the oxido-reduction reaction of D-xylose was able to couple through regeneration of NADP and NADPH to produce D-xylonic acid and xylitol.     

Kinetics of Manganese Oxidation by Cell-Free Extracts of Bacteria Isolated from Manganese Concretions from Soil

A study of the kinetics of Mn²⁺ oxidation catalyzed by cell extracts of two bacterial isolates (E₁, Pseudomonas III [new isolate] and E₄, Citrobacter freundii) isolated from the core of manganese concretions from Greek soils is presented. The reaction velocity of Mn²⁺ oxidation was determined from the rate of consumption of Mn²⁺. The oxidation of Mn²⁺ was followed by measuring changes in Mn²⁺ concentration by activation analysis and by atomic absorption spectrophotometry. The reaction velocity was directly proportional to cell extract concentration when the reaction time was 1 h. At longer reaction times, the relationship deviated from linearity because substrate concentration became limiting. The rate of Mn²⁺ oxidation increased with the Mn²⁺ concentration. Analysis of the results by application of the integrated Michaelis equation for determining Michaelis constants and maximal velocities either in the presence (K_m = 3.33 μmol/ml and V_max = 1.25 μmol/ml·h) or in the absence of maleate buffer (K_m = 2.52 μmol/ml and V_max = 2.04 μmol/ml·h) indicated a strong affinity between the oxidizing system and manganese. All results in this study are consistent with an enzymatic manganese-oxidizing system and give an indication of the mechanism of biological Mn²⁺ oxidation in soil which differs from that in the marine environment.     

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 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.     

Reduction of Hexavalent Chromium by Immobilized Viable Cells of Arthrobacter sp. SUK 1201

Arthrobacter sp. SUK 1201, a potent isolate reported from chromite mine overburden of Orissa, India, has been evaluated for Cr(VI) reduction with immobilized whole cells. For whole-cell immobilization, Ba-alginate was found to be most effective, and the Cr(VI) reduction potential was maximum in minimal salts (MS) medium with cells immobilized in 2% alginate. Fourier transform infrared spectra of depolymerized cells has failed to detect any sign of complexation of Cr(VI) or its reduced products with the cell mass. Reduction efficiency of the beads increased with increase in cell load, but decreased with increase in Cr(VI) concentration in the medium. Glycerol was the most potent electron donor for chromate reduction, followed by glucose and peptone. Optimum pH for Cr(VI) reduction was 7.0, and the process was inhibited by metal ions such as Ni(II), Co(II), Cd(II), Zn(II), and Mn(II) but not by Cu(II) and Fe(III). Similarly, CCCP (carbonyl cyanide-m-chlorophenylhydrazone), DCC (N,N,-dicyclohexylcarbodiimide), sodium azide, and sodium fluoride were inhibitory in nature, whereas chromate reduction was unaffected in the presence of DNP (2,4-dinitrophenol). Moreover, immobilized cells of SUK 1201 remained biologically active for four consecutive cycles, accompanied with an initial increase in cell number in the beads, although a decline in chromate reduction was recorded from the second cycle onward. Immobilized cells of Arthrobacter sp. SUK 1201, therefore, could be a potential tool for long-term uses in chromium detoxification.     

Reductive Dechlorination of Tetrachloroethene Following Abiotic Versus Biotic Reduction of Hexavalent Chromium

A feasibility evaluation identified chemical reduction and biostimulation as a potential remedy for a plume containing hexavalent chromium (Cr(VI)) and tetrachloroethene (PCE) at an industrial site in southern California. The objectives of this laboratory study were to determine the stoichiometry of calcium polysulfide (CaS_x) reaction with Cr(VI) in the presence of sediment, the effect of CaS_x on the potential for in situ biological reductive dechlorination of PCE, and the potential to reduce Cr(VI) and PCE by addition of only an electron donor. Approximately 1 L of CaS_x solution (containing 50 g S^2-/L) was required per 1000 L of groundwater containing 45 mg/L of Cr(VI) (i.e., 1.8 mol S^2- per mol Cr(VI)). The sediment also exerted a sulfide demand (≥0.38 g S^{2 -} per kg sediment), but at a slower rate than the Cr(VI). In microcosms prepared with lactate, corn syrup, soybean oil, or methanol, but no CaS_x, the Cr(VI) was biologically reduced in the treatments with lactate and corn syrup, but much more slowly than with CaS_x. Even after 20 months of incubation, no significant reductive dechlorination of PCE occurred in any of the microcosms, including those in which the Cr(VI) was removed with CaS_x. Bioaugmentation was tested with the microcosms that received lactate and corn syrup (following 20 months of incubation), using an enrichment culture that actively dechlorinates trichloroethene. PCE dechlorination began within 1 month in the lactate-only treatment; in the corn syrup-amended treatment, PCE dechlorination occurred in only one of the three bottles. However, no PCE dechlorination occurred following bioaugmentation of the lactate and corn syrup microcosms that were initially treated with CaS_x, indicating that CaS_x (and/or its reaction products) exerted a negative impact on the chlororespiring microbes. This outcome highlights the need to evaluate sites on a case-by-case basis when in situ chemical treatment is applied prior to microbial reductive dechlorination.     

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.     

Production of a Novel Extracellular Polysaccharide by a Bacillus Strain Isolated from Soil

A bacterium that was isolated from soil and identified as Bacillus circulans was found to produce a highly viscous extracellular polysaccharide when it was grown aerobically in a medium containing glucose as a sole source of carbon. The product was characterized by TLC and GC analyses as a novel heteropolysaccharide consisted of rhamnose, mannose, galactose, and mannuronic acid as sugar components. A maximal yield of polysaccharide reached about 2 g/liter by jar-fermentor culture at 30°C for 48 hr with a medium containing 1% glucose, 0.05% asparagine, 0.005% yeast extract, and small amounts of inorganic salts. Some culture conditions for the production of polysaccharide were investigated with flask culture; an optimal production was attained with a medium containing 0.1–1 % glucose and 0.01–0.05% asparagine, pH 7–8, at 30°C under aerobic conditions.     

UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-L-lysine synthetase from Bacillus sphaericus: activation by potassium phosphate

During the course of purification of UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-L-lysine synthetase, we observed a marked stimulation of the enzymatic activity in the presence of phosphate ions. This activation effect was studied with enzyme purified 979-fold from Bacillus sphaericus. Each salt tested stimulated the activity of the synthetase. The order of activation by different anions was HPO4(2-) greater than Cl- greater than SO4(2-). In every case, the potassium salt gave higher activity than the corresponding sodium salt. The activation in the presence of phosphate was quite pronounced (almost sevenfold with K2HPO4) and occurred at a relatively low concentration. The Ka for K2HPO4 was found to be 3.4 mM and the Hill coefficient was calculated to be 1.0. This would suggest that there is one phosphate-binding site per active centre. The presence of phosphate did not affect either the pH optimum of this enzyme or the optimum concentration of Mg2+ required. The presence of phosphate has little or no effect on the Km of any of the substrates. Thus, it appears that the presence of phosphate changes the enzyme conformation to a catalytically more active form. The activation of this enzyme in the presence of phosphate anion is all the more interesting because phosphate is a product of the reaction catalyzed by this enzyme.     

Selective Isolation of Bacillus sphaericus from Soil by Use of Acetate as the Only Major Source of Carbon

A simple chemically defined medium containing sodium acetate (37 or 74 mM) as the only major source of carbon was inoculated with soil from various locations. The bacteria which grew most rapidly at 30°C were almost entirely strains of Bacillus sphaericus and Arthrobacter species. Pasteurization of the soils made the medium selective for B. sphaericus. Several new strains were isolated, and the peptidoglycans of their cell walls were examined.     

Structure of the mosquitocidal toxin from Bacillus sphaericus

The catalytic domain of a mosquitocidal toxin prolonged by a C-terminal 44 residue linker connecting to four ricin B-like domains was crystallized. Three crystal structures were established at resolutions between 2.5A and 3.0A using multi-wavelength and single-wavelength anomalous X-ray diffraction as well as molecular replacement phasing techniques. The chainfold of the toxin fragment corresponds to those of ADP-ribosylating enzymes. At pH 4.3 the fragment is associated in a C(7)-symmetric heptamer in agreement with an aggregate of similar size observed by size-exclusion chromatography. In two distinct crystal forms, the heptamers formed nearly spherical, D(7)-symmetric tetradecamers. Another crystal form obtained at pH 6.3 contained a recurring C(2)-symmetric tetramer, which, however, was not stable in solution. On the basis of the common chainfold and NAD(+)-binding site of all ADP-ribosyl transferases, the NAD(+)-binding site of the toxin was assigned at a high confidence level. In all three crystal forms the NAD(+) site was occupied by part of the 44 residue linker, explaining the known inhibitory effect of this polypeptide region. The structure showed that the cleavage site for toxin activation is in a highly mobile loop that is exposed in the monomer. Since it contains the inhibitory linker as a crucial part of the association contact, the observed heptamer is inactive. Moreover, the heptamer cannot be activated by proteolysis because the activation loop is at the ring center and not accessible for proteases. Therefore the heptamer, or possibly the tetradecamer, seems to represent an inactive storage form of the toxin.