Inhibition of nitrate reduction by chromium (VI) in anaerobic soil microcosms.
ABSTRACT: Chromium is often found as a cocontaminant at sites polluted with organic compounds. For nitrate-respiring microbes, Cr(VI) may be not only directly toxic but may also specifically interfere with N reduction. In soil microcosms amended with organic electron donors, Cr(VI), and nitrate, bacteria oxidized added carbon, but relatively low doses of Cr(VI) caused a lag and then lower rates of CO(2) accumulation. Cr(VI) strongly inhibited nitrate reduction; it occurred only after soluble Cr(VI) could not be detected. However, Cr(VI) additions did not eliminate Cr-sensitive populations; after a second dose of Cr(VI), bacterial activity was strongly inhibited. Differences in microbial community composition (assayed by PCR-denaturing gradient gel electrophoresis) driven by different organic substrates (glucose and protein) were smaller than when other electron acceptors had been used. However, the selection of bacterial phylotypes was modified by Cr(VI). Nine isolated clades of facultatively anaerobic Cr(VI)-resistant bacteria were closely related to cultivated members of the phylum Actinobacteria or Firmicutes. In Bacillus cereus GNCR-4, the nature of the electron donor (fermentable or nonfermentable) affected Cr(VI) resistance level and anaerobic nitrate metabolism. Our results indicate that carbon utilization and nitrate reduction in these soils were contingent upon the reduction of added Cr(VI). The amount of Cr(VI) required to inhibit nitrate reduction was 10-fold less than for aerobic catabolism of the same organic substrate. We speculate that the resistance level of a microbial process is directly related to the diversity of microbes capable of conducting it.
Project description:Chromium (VI) is toxic to microorganisms and can inhibit the biodegradation of organic pollutants in contaminated soils. We used microcosms amended with either glucose or protein (to drive bacterial community change) and Fe(III) (to stimulate iron-reducing bacteria) to study the effect of various concentrations of Cr(VI) on anaerobic bacterial communities. Microcosms were destructively sampled based on microbial activity (measured as evolution of CO2) and analyzed for the following: (i) dominant bacterial community by PCR-denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA gene; (ii) culturable Cr-resistant bacteria; and (iii) enrichment of iron-reducing bacteria of the Geobacteraceae family by real-time PCR. The addition of organic C stimulated the activities of anaerobic communities. Cr(VI) amendment resulted in lower rates of CO2 production in glucose microcosms and a slow mineralization phase in protein-amended microcosms. Glucose and protein amendments selected for different bacterial communities. This selection was modified by the addition of Cr(VI), since some DGGE bands were intensified and new bands appeared in Cr(VI)-amended microcosms. A second dose of Cr(VI), added after the onset of activity, had a strong inhibitory effect when higher levels of Cr were added, indicating that the developing Cr-resistant communities had a relatively low tolerance threshold. Most of the isolated Cr-resistant bacteria were closely related to previously studied Cr-resistant anaerobes, such as Pantoea, Pseudomonas, and Enterobacter species. Geobacteraceae were not enriched during the incubation. The studied Cr(VI)-contaminated soil contained a viable anaerobic bacterial community; however, Cr(VI) altered its composition, which could affect the soil biodegradation potential.
Project description:Electroplating wastewater contains both Cr (VI) and sulfate. So Cr (VI) removal under sulfate-rich condition is quite complicated. This study mainly investigates the pathways for Cr (VI) removal under biological sulfate-reducing condition in the up-flow anaerobic sludge bed (UASB) reactor. Two potential pathways are found for the removal of Cr (VI). The first one is the sulfidogenesis-induced Cr (VI) reduction pathway (for 90% Cr (VI) removal), in which Cr (VI) is reduced by sulfide generated from biological reduction of sulfate. The second one leads to direct reduction of Cr (VI) which is utilized by bacteria as the electron acceptor (for 10% Cr (VI) removal). Batch test results confirmed that sulfide was oxidized to elemental sulfur instead of sulfate during Cr (VI) reduction. The produced extracellular polymeric substances (EPS) provided protection to the microbes, resulting in effective removal of Cr (VI). Sulfate-reducing bacteria (SRB) genera accounted for 11.1% of the total bacterial community; thus they could be the major organisms mediating the sulfidogenesis-induced reduction of Cr (VI). In addition, chromate-utilizing genera (e.g. Microbacterium) were also detected, which were possibly responsible for the direct reduction of Cr (VI) using organics as the electron donor and Cr (VI) as the electron acceptor.
Project description:Bioremediation of Cr(VI) and nitrate is considered as a promising and cost-effective alternative to chemical and physical methods. However, organo-Cr(III) complexes in effluent generally causes environmental concerns due to second-pollution. Here, Cr(VI) reduction and immobilization efficiencies of anaerobic activated sludge were investigated. Anaerobic activated sludge showed strong reduction ability of Cr(VI) and possessed a great potential of Cr(III) immobilization. Almost 100.0?mg?l-1 Cr(VI) could be completely reduced and immobilized by anaerobic activated sludge in a sequencing batch reactor in 24?h. And most generated Cr(III) was accumulated outside of sludge cells. Extracellular polymeric substances (EPS) could bind to Cr(VI) and form EPS-Cr(VI) interaction to reduce the toxic effect of Cr(VI) and promote the Cr(VI) reduction. Protein-like and humic-like substances were responsible for binding with Cr(VI), meanwhile the process was a thermodynamically favorable binding reaction. Then Cr(VI) was reduced to Cr(III) by membrane-associated chromate reductase of sludge. Eventually, the generated Cr(III) might exist as poly-nuclear Cr(III) complexes adhered to sludge surfaces.
Project description:Whole-genome DNA microarrays were used to examine the gene expression profile of Shewanella oneidensis MR-1 during U(VI) and Cr(VI) reduction. The same control, cells pregrown with nitrate and incubated with no electron acceptor, was used for the two time points considered and for both metals. U(VI)-reducing conditions resulted in the upregulation (> or = 3-fold) of 121 genes, while 83 genes were upregulated under Cr(VI)-reducing conditions. A large fraction of the genes upregulated [34% for U(VI) and 29% for Cr(VI)] encode hypothetical proteins of unknown function. Genes encoding proteins known to reduce alternative electron acceptors [fumarate, dimethyl sulfoxide, Mn(IV), or soluble Fe(III)] were upregulated under both U(VI)- and Cr(VI)-reducing conditions. The involvement of these upregulated genes in the reduction of U(VI) and Cr(VI) was tested using mutants lacking one or several of the gene products. Mutant testing confirmed the involvement of several genes in the reduction of both metals: mtrA, mtrB, mtrC, and menC, all of which are involved in Fe(III) citrate reduction by MR-1. Genes encoding efflux pumps were upregulated under Cr(VI)- but not under U(VI)-reducing conditions. Genes encoding proteins associated with general (e.g., groL and dnaJ) and membrane (e.g., pspBC) stress were also upregulated, particularly under U(VI)-reducing conditions, pointing to membrane damage by the solid-phase reduced U(IV) and Cr(III) and/or the direct effect of the oxidized forms of the metals. This study sheds light on the multifaceted response of MR-1 to U(VI) and Cr(VI) under anaerobic conditions and suggests that the same electron transport pathway can be used for more than one electron acceptor.
Project description:Chromium and uranium are highly toxic metals that contaminate many natural environments. We investigated their mechanisms of toxicity under anaerobic conditions using nitrate-reducing Pseudomonas stutzeri RCH2, which was originally isolated from a chromium-contaminated aquifer. A random barcode transposon site sequencing library of RCH2 was grown in the presence of the chromate oxyanion (Cr[VI][Formula: see text]) or uranyl oxycation (U[VI][Formula: see text]). Strains lacking genes required for a functional nitrate reductase had decreased fitness as both metals interacted with heme-containing enzymes required for the later steps in the denitrification pathway after nitrate is reduced to nitrite. Cr[VI]-resistance also required genes in the homologous recombination and nucleotide excision DNA repair pathways, showing that DNA is a target of Cr[VI] even under anaerobic conditions. The reduced thiol pool was also identified as a target of Cr[VI] toxicity and psest_2088, a gene of previously unknown function, was shown to have a role in the reduction of sulfite to sulfide. U[VI] resistance mechanisms involved exopolysaccharide synthesis and the universal stress protein UspA. As the first genome-wide fitness analysis of Cr[VI] and U[VI] toxicity under anaerobic conditions, this study provides new insight into the impact of Cr[VI] and U[VI] on an environmental isolate from a chromium contaminated site, as well as into the role of a ubiquitous protein, Psest_2088.
Project description:Pelosinus spp. are fermentative firmicutes that were recently reported to be prominent members of microbial communities at contaminated subsurface sites in multiple locations. Here we report metabolic characteristics and their putative genetic basis in Pelosinus sp. strain HCF1, an isolate that predominated anaerobic, Cr(VI)-reducing columns constructed with aquifer sediment. Strain HCF1 ferments lactate to propionate and acetate (the methylmalonyl-coenzyme A [CoA] pathway was identified in the genome), and its genome encodes two [NiFe]- and four [FeFe]-hydrogenases for H(2) cycling. The reduction of Cr(VI) and Fe(III) may be catalyzed by a flavoprotein with 42 to 51% sequence identity to both ChrR and FerB. This bacterium has unexpected capabilities and gene content associated with reduction of nitrogen oxides, including dissimilatory reduction of nitrate to ammonium (two copies of NrfH and NrfA were identified along with NarGHI) and a nitric oxide reductase (NorCB). In this strain, either H(2) or lactate can act as a sole electron donor for nitrate, Cr(VI), and Fe(III) reduction. Transcriptional studies demonstrated differential expression of hydrogenases and nitrate and nitrite reductases. Overall, the unexpected metabolic capabilities and gene content reported here broaden our perspective on what biogeochemical and ecological roles this species might play as a prominent member of microbial communities in subsurface environments.
Project description:Hexavalent Chromium [Cr(VI)] is a widespread contaminant found in soil, sediment, and ground water in several DOE sites, including Hanford 100 H area. In order to stimulate microbially mediated reduction of Cr(VI) at this site, a poly-lactate hydrogen release compound was injected into the chromium contaminated aquifer. Targeted enrichment of dominant nitrate-reducing bacteria post injection resulted in the isolation of Pseudomonas stutzeri strain RCH2. P. stutzeri strain RCH2 was isolated using acetate as the electron donor and is a complete denitrifier. Experiments with anaerobic washed cell suspension of strain RCH2 revealed it could reduce Cr(VI) and Fe(III). The genome of strain RCH2 was sequenced using a combination of Illumina and 454 sequencing technologies and contained a circular chromosome of 4.6 Mb and three plasmids. Global genome comparisons of strain RCH2 with six other fully sequenced P. stutzeri strains revealed most genomic regions are conserved, however strain RCH2 has an additional 244 genes, some of which are involved in chemotaxis, Flp pilus biogenesis and pyruvate/2-oxogluturate complex formation.
Project description:Desulfovibrio spp. are capable of heavy metal reduction and are well-studied systems for understanding metal fate and transport in anaerobic environments. Desulfovibrio vulgaris Hildenborough was grown under environmentally relevant conditions (i.e., temperature, nutrient limitation) to elucidate the impacts on Cr(VI) reduction on cellular physiology. Growth at 20 °C was slower than 30 °C and the presence of 50 ?M Cr(VI) caused extended lag times for all conditions, but once growth resumed the growth rate was similar to that without Cr(VI). Cr(VI) reduction rates were greatly diminished at 20 °C for both 50 and 100 ?M Cr(VI), particularly for the electron acceptor limited (EAL) condition in which Cr(VI) reduction was much slower, the growth lag much longer (200 h), and viability decreased compared to balanced (BAL) and electron donor limited (EDL) conditions. When sulfate levels were increased in the presence of Cr(VI), cellular responses improved via a shorter lag time to growth. Similar results were observed between the different resource (donor/acceptor) ratio conditions when the sulfate levels were normalized (10 mM), and these results indicated that resource ratio (donor/acceptor) impacted D. vulgaris response to Cr(VI) and not merely sulfate limitation. The results suggest that temperature and resource ratios greatly impacted the extent of Cr(VI) toxicity, Cr(VI) reduction, and the subsequent cellular health via Cr(VI) influx and overall metabolic rate. The results also emphasized the need to perform experiments at lower temperatures with nutrient limitation to make accurate predictions of heavy metal reduction rates as well as physiological states in the environment.
Project description:Columns containing Hanford 100H aquifer sediment continuously infused with 5 mM lactate, 5 uM Cr(VI), and either 7.5 mM sulfate or 12 mM nitrate as an electron acceptor. A two-chip study using total RNA extracted from unfiltered effluent from columns (nitrate or sulfate infused).
Project description:Microbial reduction of toxic hexavalent chromium (Cr(VI)) in-situ is a plausible bioremediation strategy in electron-acceptor limited environments. However, higher [Cr(VI)] may impose stress on syntrophic communities and impact community structure and function. The study objectives were to understand the impacts of Cr(VI) concentrations on community structure and on the Cr(VI)-reduction potential of groundwater communities at Hanford, WA. Steady state continuous flow bioreactors were used to grow native communities enriched with lactate (30 mM) and continuously amended with Cr(VI) at 0.0 (No-Cr), 0.1 (Low-Cr) and 3.0 (High-Cr) mg/L. Microbial growth, metabolites, Cr(VI), 16S rRNA gene sequences and GeoChip based functional gene composition were monitored for 15 weeks. Temporal trends and differences in growth, metabolite profiles, and community composition were observed, largely between Low-Cr and High-Cr bioreactors. In both High-Cr and Low-Cr bioreactors, Cr(VI) levels were below detection from week 1 until week 15. With lactate enrichment, native bacterial diversity substantially decreased as Pelosinus spp., and Sporotalea spp., became the dominant groups, but did not significantly differ between Cr concentrations. The Archaea diversity also substantially decreased after lactate enrichment from Methanosaeta (35%), Methanosarcina (17%) and others, to mostly Methanosarcina spp. (95%). Methane production was lower in High-Cr reactors suggesting some inhibition of methanogens. Several key functional genes were distinct in Low-Cr bioreactors compared to High-Cr. Among the Cr resistant microbes, Burkholderia vietnamiensis, Comamonas testosterone and Ralstonia pickettii proliferated in Cr amended bioreactors. In-situ fermentative conditions facilitated Cr(VI) reduction, and as a result 3.0 mg/L Cr(VI) did not impact the overall bacterial community structure.