Integrated Ecogenomics Study for Bioremediation of Cr(VI) at Hanford 100H Area

Hexavalent chromium is a widespread contaminant found in groundwater. In order to stimulate microbially mediated Cr(VI)-reduction, a poly-lactate compound was injected into Cr(VI)-contaminated aquifers at site 100H at Hanford. Investigation of bacterial community composition using high-density DNA microarray analysis of 16S rRNA gene products revealed a stimulation of Pseudomonas, Desulfovibrio and Geobacter species amongst others. Enrichment of these organisms coincided with continued Cr(VI) depletion. Functional gene-array analysis of DNA from monitoring well indicated high abundance of genes involved in nitrate-reduction, sulfate-reduction, iron-reduction, methanogenesis, chromium tolerance/reduction. Clone-library data revealed Psedomonas was the dominant genus in these samples. Based on above results, we conducted lab investigations to study the dominant anaerobic culturable microbial populations present at this site and their role in Cr(VI)-reduction. Enrichments using defined anaerobic media resulted in isolation of an iron-reducing, a sulfate-reducing and a nitrate-reducing isolate among several others. Preliminary 16S rDNA sequence analysis identified the isolates as Geobacter metallireducens, Pseudomonas stutzeri and Desulfovibrio vulgaris species respectively. The Pseudomonas isolate utilized acetate, lactate, glycerol and pyruvate as alternative carbon sources, and reduced Cr(VI). Anaerobic washed cell suspension of strain HLN reduced almost 95?M Cr(VI) within 4 hr. Further, with 100?M Cr(VI) as sole electron-acceptor, cells grew to 4.05 x 107 /ml over 24 h after an initial lag, demonstrating direct enzymatic Cr(VI) reduction coupled to growth. These results demonstrate that Cr(VI)-immobilization at Hanford 100H site could be mediated by direct microbial metabolism in addition to indirect chemical reduction of Cr(VI) by end-products of microbial activity

Enhanced crude oil depletion by constructed bacterial consortium comprising bioemulsifier producer and petroleum hydrocarbon degraders.

The aim of this work was to study the production of bioemulsifier by Rhodococcus erythropolis OSDS1, and the improvement of crude oil depletion efficiency using a consortium of petroleum hydrocarbon degraders and OSDS1. The results showed that R. erythropolis OSDS1 produced highly stable bioemulsifier under various salinity (0-35 g/L NaCl) and pH (5.0-9.0) conditions; more than 90% of the initial emulsification activity was retained after 168 h. Emulsification capacity of the bioemulsifier on different petroleum hydrocarbons was diesel > mineral oil/crude oil > gasoline. A mixed bacterial consortium combining OSDS1 and four other petroleum hydrocarbon degraders was constructed. GC-MS results revealed that the constructed consortium achieved 85.26% depletion efficiency of crude oil in 15 days, which was significantly higher than that of individual strains. During the process, alkane hydroxylase gene (alkB) was successfully amplified from the consortium, confirming presence of crude oil degrading enzymes

Draft Genome Sequence for Desulfovibrio africanus Strain PCS.

Desulfovibrio africanus strain PCS is an anaerobic sulfate-reducing bacterium (SRB) isolated from sediment from Paleta Creek, San Diego, CA. Strain PCS is capable of reducing metals such as Fe(III) and Cr(VI), has a cell cycle, and is predicted to produce methylmercury. We present the D. africanus PCS genome sequence

Microbial Response to the MC-252 Oil and Corexit 9500 in the Gulf of Mexico

The Deepwater Horizon spill released over 4.1 million barrels of crude oil into the Gulf of Mexico. In an effort to mitigate large oil slicks, the dispersant Corexit 9500 was sprayed onto surface slicks and injected directly at the wellhead at water depth of 1,500 m. Several research groups were involved in investigating the fate of the MC-252 oil using newly advanced molecular tools to elucidate microbial interactions with oil, gases, and dispersant. Microbial community analysis by different research groups revealed that hydrocarbon degrading bacteria belonging to Oceanospirillales, Colwellia, Cycloclasticus, Rhodobacterales, Pseudoalteromonas, and methylotrophs were found enriched in the contaminated water column. Presented here is a comprehensive overview of the ecogenomics of microbial degradation of MC-252 oil and gases in the water column and shorelines. We also present some insight into the fate of the dispersant Corexit 9500 that was added to aid in oil dispersion process. Our results show the dispersant was not toxic to the indigenous microbes at concentrations added, and different bacterial species isolated in the aftermath of the spill were able to degrade the various components of Corexit 9500 that included hydrocarbons, glycols, and dioctyl sulfosuccinate

Adaptation of the Biolog Phenotype MicroArrayTM Technology to Profile the Obligate Anaerobe Geobacter metallireducens

The Biolog OmniLog? Phenotype MicroArray (PM) plate technology was successfully adapted to generate a select phenotypic profile of the strict anaerobe Geobacter metallireducens (G.m.). The profile generated for G.m. provides insight into the chemical sensitivity of the organism as well as some of its metabolic capabilities when grown with a basal medium containing acetate and Fe(III). The PM technology was developed for aerobic organisms. The reduction of a tetrazolium dye by the test organism represents metabolic activity on the array which is detected and measured by the OmniLog(R) system. We have previously adapted the technology for the anaerobic sulfate reducing bacterium Desulfovibrio vulgaris. In this work, we have taken the technology a step further by adapting it for the iron reducing obligate anaerobe Geobacter metallireducens. In an osmotic stress microarray it was determined that the organism has higher sensitivity to impermeable solutes 3-6percent KCl and 2-5percent NaNO3 that result in osmotic stress by osmosis to the cell than to permeable non-ionic solutes represented by 5-20percent ethylene glycol and 2-3percent urea. The osmotic stress microarray also includes an array of osmoprotectants and precursor molecules that were screened to identify substrates that would provide osmotic protection to NaCl stress. None of the substrates tested conferred resistance to elevated concentrations of salt. Verification studies in which G.m. was grown in defined medium amended with 100mM NaCl (MIC) and the common osmoprotectants betaine, glycine and proline supported the PM findings. Further verification was done by analysis of transcriptomic profiles of G.m. grown under 100mM NaCl stress that revealed up-regulation of genes related to degradation rather than accumulation of the above-mentioned osmoprotectants. The phenotypic profile, supported by additional analysis indicates that the accumulation of these osmoprotectants as a response to salt stress does not occur in G.m. and response to stress must occur by other mechanisms. The Phenotype MicroArray technology can be reliably used as a rapid screening tool for characterization in anaerobic microbial ecology

Development of Extraction Techniques for the Detection of Signature Lipids from Oil

Pure cultures, including Desulfovibrio vulgaris and Methanococcus maripaludus, were combined with model oil samples and oil/diesel mixtures to optimize extraction techniques of signature lipids from oil in support of investigation of microbial communities in oil deposit samples targets for microbial enhanced hydrocarbon recovery. Several techniques were evaluated, including standard phospholipid extraction, ether linked lipid for Archaeal bacterial detection, and high pressure extractiontechniques. Recovery of lipids ranged from 50-80percent as compared to extraction of the pure culture. Extraction efficiency was evaluated by the use of internal standards. Field samples will also be tested for recovery of signature lipids with optimized extraction techniques

Resource limitation modulates the fate of dissimilated nitrogen in a dual-pathway Actinobacterium

Respiratory ammonification and denitrification are two evolutionarily unrelated dissimilatory nitrogen (N) processes central to the global N cycle, the activity of which is thought to be controlled by carbon (C) to nitrate (NO₃⁻) ratio. Here we find that Intrasporangium calvum C5, a novel menaquinone-based dual-pathway denitrifier/respiratory ammonifier, disproportionately utilizes ammonification rather than denitrification when grown under carbon or nitrate limitation, not C:NO3- ratio. Instead, C:NO₃⁻ ratio is a confounding variable for resource limitation. We find that the protein atomic composition for denitrification modules (NirK) are significantly cost minimized for C and N compared to ammonification modules (NrfA), indicating that resource limitation is a major selective pressure imprinted in the architecture of these proteins. The evolutionary precedent for these findings suggests ecological and biogeochemical importance as evidenced by higher growth rates when I. calvum grows predominantly using its ammonification pathway and by assimilating its end-product (ammonium) for growth under ammonium-deplete conditions. Genomic analysis of I. calvum further reveals a versatile ecophysiology to cope with nutrient stress and redox conditions. Metabolite and transcriptional profiles during growth indicate that transcript abundances encoding for its nitrite reducing enzyme modules, NrfAH and NirK, significantly increase in response to nitrite production. Mechanistically, our results suggest that pathway selection is driven by intracellular redox potential (redox poise), which may be lowered during resource limitation, thereby decreasing catalytic activity of upstream electron transport steps needed for denitrification enzymes. Our work advances our understanding of the biogeochemical flexibility of N-cycling organisms, pathway evolution, and ecological food-webs

High-Quality Draft Genome Sequence of Desulfovibrio carbinoliphilus FW-101-2B, an Organic Acid-Oxidizing Sulfate-Reducing Bacterium Isolated from Uranium(VI)-Contaminated Groundwater.

Desulfovibrio carbinoliphilus subsp. oakridgensis FW-101-2B is an anaerobic, organic acid/alcohol-oxidizing, sulfate-reducing δ-proteobacterium. FW-101-2B was isolated from contaminated groundwater at The Field Research Center at Oak Ridge National Lab after in situ stimulation for heavy metal-reducing conditions. The genome will help elucidate the metabolic potential of sulfate-reducing bacteria during uranium reduction

Conservation of Modules but not Phenotype in Bacterial Response to Environmental Stress

Microbes live in changing environments and change their phenotype via gene regulation in response. Although this transcriptional response is important for fitness, very little is known about how it evolves in microbes. We started by asking a number of high-level questions about the evolution of transcriptional phenotype: (1) To what extent is transcriptional response conserved, i.e. do conserved genes respond similarly to the same condition; (2) To what extent are transcriptional modules conserved; and (3) Does there exist a general stress response to a variety of stressors? To illuminate these questions, we analyzed more than 500 microarray experiments across the bacterial domain. We looked for conservation of transcriptional regulation both in close sister species and vastly divergent clades. In addition, we produced and analyzed an extensive in-house compendium of environmental stress data in three metal-reducing bacteria