Isolation and characterization of a halophilic Modicisalibacter sp. strain Wilcox from produced water.

We report the isolation a halophilic bacterium that degrades both aromatic and aliphatic hydrocarbons as the sole sources of carbon at high salinity from produced water. Phylogenetic analysis of 16S rRNA-gene sequences shows the isolate is a close relative of Modicisalibacter tunisiensis isolated from an oil-field water in Tunisia. We designate our isolate as Modicisalibacter sp. strain Wilcox. Genome analysis of strain Wilcox revealed the presence of a repertoire of genes involved in the metabolism of aliphatic and aromatic hydrocarbons. Laboratory culture studies corroborated the predicted hydrocarbon degradation potential. The strain degraded benzene, toluene, ethylbenzene, and xylenes at salinities ranging from 0.016 to 4.0 M NaCl, with optimal degradation at 1 M NaCl. Also, the strain degraded phenol, benzoate, biphenyl and phenylacetate as the sole sources of carbon at 2.5 M NaCl. Among aliphatic compounds, the strain degraded n-decane and n-hexadecane as the sole sources of carbon at 2.5 M NaCl. Genome analysis also predicted the presence of many heavy metal resistance genes including genes for metal efflux pumps, transport proteins, and enzymatic detoxification. Overall, due to its ability to degrade many hydrocarbons and withstand high salt and heavy metals, strain Wilcox may prove useful for remediation of produced waters.

Genome Sequence of Lignin-Degrading Arthrobacter sp. Strain RT-1, Isolated from Termite Gut and Rumen Fluid.

Here, we report the genome sequence of Arthrobacter sp. strain RT-1, isolated from a cocktail of termite gut and rumen fluid. Strain RT-1 degrades a variety of lignin monomers and dimers as the growth substrates. The genome annotation predicted the genes necessary for the catabolism of lignin-derived aromatic compounds.

Microbial community structure analysis of a benzoate-degrading halophilic archaeal enrichment.

A benzoate-degrading archaeal enrichment was developed using sediment samples from Rozel Point at Great Salt Lake, UT. The enrichment degraded benzoate as the sole carbon source at salinity ranging from 2.0 to 5.0 M NaCl with highest rate of degradation observed at 4.0 M. The enrichment was also tested for its ability to grow on other aromatic compounds such as 4-hydroxybenzoic acid (4-HBA), gentisic acid, protocatechuic acid (PCA), catechol, benzene and toluene as the sole sources of carbon and energy. Of these, the culture only utilized 4-HBA as the carbon source. To determine the initial steps in benzoate degradation pathway, a survey of ring-oxidizing and ring-cleaving genes was performed using degenerate PCR primers. Results showed the presence of 4-hydroxybenzoate 3-monooxygenase (4-HBMO) and protocatechuate 3, 4-dioxygenase (3,4-PCA) genes suggesting that the archaeal enrichment might degrade benzoate to 4-HBA that is further converted to PCA by 4-HBMO and, thus, formed PCA would undergo ring-cleavage by 3,4-PCA to form intermediates that enter the Krebs cycle. Small subunit rRNA gene-based diversity survey revealed that the enrichment consisted entirely of class Halobacteria members belonging to the genera Halopenitus, Halosarcina, Natronomonas, Halosimplex, Halorubrum, Salinarchaeum and Haloterrigena. Of these, Halopenitus was the dominant group accounting for almost 91 % of the total sequences suggesting their potential role in degrading oxygenated aromatic compounds at extreme salinity.

Genome Sequences of the Lignin-Degrading Pseudomonas sp. Strain YS-1p and Rhizobium sp. Strain YS-1r Isolated from Decaying Wood.

Pseudomonas sp. strain YS-1p and Rhizobium sp. strain YS-1r were isolated from a lignin-degrading enrichment culture. The isolates degraded lignin-derived monomers, dimers, alkali lignin, and, to a smaller extent (3% to 5%), lignin in switch grass and alfalfa. Genome analysis revealed the presence of a variety of lignin-degrading genes.

Arhodomonas sp. strain Seminole and its genetic potential to degrade aromatic compounds under high-salinity conditions.

Arhodomonas sp. strain Seminole was isolated from a crude oil-impacted brine soil and shown to degrade benzene, toluene, phenol, 4-hydroxybenzoic acid (4-HBA), protocatechuic acid (PCA), and phenylacetic acid (PAA) as the sole sources of carbon at high salinity. Seminole is a member of the genus Arhodomonas in the class Gammaproteobacteria, sharing 96% 16S rRNA gene sequence similarity with Arhodomonas aquaeolei HA-1. Analysis of the genome predicted a number of catabolic genes for the metabolism of benzene, toluene, 4-HBA, and PAA. The predicted pathways were corroborated by identification of enzymes present in the cytosolic proteomes of cells grown on aromatic compounds using liquid chromatography-mass spectrometry. Genome analysis predicted a cluster of 19 genes necessary for the breakdown of benzene or toluene to acetyl coenzyme A (acetyl-CoA) and pyruvate. Of these, 12 enzymes were identified in the proteome of toluene-grown cells compared to lactate-grown cells. Genomic analysis predicted 11 genes required for 4-HBA degradation to form the tricarboxylic acid (TCA) cycle intermediates. Of these, proteomic analysis of 4-HBA-grown cells identified 6 key enzymes involved in the 4-HBA degradation pathway. Similarly, 15 genes needed for the degradation of PAA to the TCA cycle intermediates were predicted. Of these, 9 enzymes of the PAA degradation pathway were identified only in PAA-grown cells and not in lactate-grown cells. Overall, we were able to reconstruct catabolic steps for the breakdown of a variety of aromatic compounds in an extreme halophile, strain Seminole. Such knowledge is important for understanding the role of Arhodomonas spp. in the natural attenuation of hydrocarbon-impacted hypersaline environments.

Recent studies in microbial degradation of petroleum hydrocarbons in hypersaline environments.

Many hypersaline environments are often contaminated with petroleum compounds. Among these, oil and natural gas production sites all over the world and hundreds of kilometers of coastlines in the more arid regions of Gulf countries are of major concern due to the extent and magnitude of contamination. Because conventional microbiological processes do not function well at elevated salinities, bioremediation of hypersaline environments can only be accomplished using high salt-tolerant microorganisms capable of degrading petroleum compounds. In the last two decades, there have been many reports on the biodegradation of hydrocarbons in moderate to high salinity environments. Numerous microorganisms belonging to the domain Bacteria and Archaea have been isolated and their phylogeny and metabolic capacity to degrade a variety of aliphatic and aromatic hydrocarbons in varying salinities have been demonstrated. This article focuses on our growing understanding of bacteria and archaea responsible for the degradation of hydrocarbons under aerobic conditions in moderate to high salinity conditions. Even though organisms belonging to various genera have been shown to degrade hydrocarbons, members of the genera Halomonas Alcanivorax, Marinobacter, Haloferax, Haloarcula, and Halobacterium dominate the published literature. Despite rapid advances in understanding microbial taxa that degrade hydrocarbons under aerobic conditions, not much is known about organisms that carry out similar processes in anaerobic conditions. Also, information on molecular mechanisms and pathways of hydrocarbon degradation in high salinity is scarce and only recently there have been a few reports describing genes, enzymes and breakdown steps for some hydrocarbons. These limited studies have clearly revealed that degradation of oxygenated and non-oxygenated hydrocarbons by halophilic and halotolerant microorganisms occur by pathways similar to those found in non-halophiles.

Proteogenomic elucidation of the initial steps in the benzene degradation pathway of a novel halophile, Arhodomonas sp. strain Rozel, isolated from a hypersaline environment.

Lately, there has been a special interest in understanding the role of halophilic and halotolerant organisms for their ability to degrade hydrocarbons. The focus of this study was to investigate the genes and enzymes involved in the initial steps of the benzene degradation pathway in halophiles. The extremely halophilic bacteria Arhodomonas sp. strain Seminole and Arhodomonas sp. strain Rozel, which degrade benzene and toluene as the sole carbon source at high salinity (0.5 to 4 M NaCl), were isolated from enrichments developed from contaminated hypersaline environments. To obtain insights into the physiology of this novel group of organisms, a draft genome sequence of the Seminole strain was obtained. A cluster of 13 genes predicted to be functional in the hydrocarbon degradation pathway was identified from the sequence. Two-dimensional (2D) gel electrophoresis and liquid chromatography-mass spectrometry were used to corroborate the role of the predicted open reading frames (ORFs). ORFs 1080 and 1082 were identified as components of a multicomponent phenol hydroxylase complex, and ORF 1086 was identified as catechol 2,3-dioxygenase (2,3-CAT). Based on this analysis, it was hypothesized that benzene is converted to phenol and then to catechol by phenol hydroxylase components. The resulting catechol undergoes ring cleavage via the meta pathway by 2,3-CAT to form 2-hydroxymuconic semialdehyde, which enters the tricarboxylic acid cycle. To substantiate these findings, the Rozel strain was grown on deuterated benzene, and gas chromatography-mass spectrometry detected deuterated phenol as the initial intermediate of benzene degradation. These studies establish the initial steps of the benzene degradation pathway in halophiles.

Phototrophic phylotypes dominate mesothermal microbial mats associated with hot springs in Yellowstone National Park.

The mesothermal outflow zones (50-65°C) of geothermal springs often support an extensive zone of green and orange laminated microbial mats. In order to identify and compare the microbial inhabitants of morphologically similar green-orange mats from chemically and geographically distinct springs, we generated and analyzed small-subunit ribosomal RNA (rRNA) gene amplicons from six mesothermal mats (four previously unexamined) in Yellowstone National Park. Between three and six bacterial phyla dominated each mat. While many sequences bear the highest identity to previously isolated phototrophic genera belonging to the Cyanobacteria, Chloroflexi, and Chlorobi phyla, there is also frequent representation of uncultured, unclassified members of these groups. Some genus-level representatives of these dominant phyla were found in all mats, while others were unique to a single mat. Other groups detected at high frequencies include candidate divisions (such as the OP candidate clades) with no cultured representatives or complete genomes available. In addition, rRNA genes related to the recently isolated and characterized photosynthetic acidobacterium "Candidatus Chloracidobacterium thermophilum" were detected in most mats. In contrast to microbial mats from well-studied hypersaline environments, the mesothermal mats in this study accrue less biomass and are substantially less diverse, but have a higher proportion of known phototrophic organisms. This study provides sequences appropriate for accurate phylogenetic classification and expands the molecular phylogenetic survey of Yellowstone microbial mats.

Biodegradation of vinyl chloride and cis-dichloroethene by a Ralstonia sp. strain TRW-1.

An aerobic bacterium, Ralstonia sp. strain TRW-1, that assimilates vinyl chloride (VC) or ethene (ETH) as the sole carbon source was isolated from a chloroethene-degrading enrichment culture. Phylogenetic analysis of 16S rDNA sequence of the isolate revealed almost 99% sequence similarity to Ralstonia pickettii. To our knowledge, this is the first report describing the isolation of a member of Ralstonia that can degrade VC as the growth substrate. The measured growth yield values for VC and ETH were 11.27 and 18.90 g protein/mole, respectively. The estimated half-velocity constant K (m) values for VC and ETH were 9.09+/-2.97 and 5.73+/-2.96 muM, respectively. These values are almost three- to tenfold higher than for other VC-assimilating Mycobacterium sp. The strain also degrades cis-dichloroethene (cis-DCE) in mineral salts medium containing yeast-extract, beef-extract, casamino acids, or peptone. This ability of the strain TRW-1 to degrade cis-DCE in the presence of a nontoxic, water-soluble substrate is relevant to in-situ remediation of cis-DCE-contaminated aquifers.

Bioaugmentation potential of a vinyl chloride-assimilating Mycobacterium sp., isolated from a chloroethene-contaminated aquifer.

An aerobic bacterium, Mycobacterium sp. strain TRW-2 that assimilated vinyl chloride (VC) or ethene (ETH) as the sole carbon source was isolated from a chloroethene-degrading enrichment culture. The strain TRW-2 also degraded cis-dichloroethene (cis-DCE) in mineral salts medium, but only when VC was present as the primary carbon source. However, no degradation of trans-dichloroethene or trichloroethene occurred in either the presence or absence of added VC. The measured growth yield values were 6.53 and 14.1g protein/mol of VC and ETH utilized, respectively. Inoculation by strain TRW-2 in microcosms prepared with aquifer samples resulted in rapid degradation of VC, whereas native bacteria degraded negligible amounts of VC within the same time period, thus suggesting bioaugmentation potential of the isolate. Phylogenetic analysis of the 16S rDNA sequence of the isolate revealed 98% sequence similarity to the members of the genus Mycobacterium. In summary, the isolate's ability to degrade VC, cis-DCE, and ETH and also its ability to survive and degrade VC in the presence of other microorganisms is relevant to the remediation of VC-impacted aquifers.

Aerobic biodegradation of benzene and toluene under hypersaline conditions at the Great Salt Plains, Oklahoma.

The Great Salt Plains is a 65-km(2) hypersaline habitat of geological origin located in north-central Oklahoma. Contamination of such ecosystems by petroleum compounds is expected from non-point sources and due to increased human activities. Little information exists about the ability of halophilic and halotolerant bacteria present in such ancient and uncontaminated environments to degrade aromatic hydrocarbons. An enrichment culture was established from soil samples obtained from the salt flats using benzene as the sole carbon and energy source. The enrichment degraded benzene at varied salt concentrations ranging from 0 to 4M. Studies showed that roughly 33% of the (14)C-benzene was converted to (14)CO(2), indicating the mineralization capacity of native bacteria. Bacterial community structure analysis using denaturing gradient gel electrophoresis showed that different phylotypes were dominant at different salt concentrations.

Aerobic biodegradation of vinyl chloride by a highly enriched mixed culture.

Lower chlorinated compounds such as cis-dichloroethene (cis-DCE) and vinyl chloride (VC) often accumulate in chloroethene-contaminated aquifers due to incomplete reductive dechlorination of higher chlorinated compounds. A highly enriched aerobic culture that degrades VC as a growth substrate was obtained from a chloroethene-contaminated aquifer material. The culture rapidly degraded 50-250 microM aqueous VC to below GC detection limit with a first-order rate constant of 0.2 day(-1). Besides VC, the culture also degraded ethene as the sole carbon source. In addition, the culture degraded cis-DCE, but only in the presence of VC. However, no degradation of trans-DCE or TCE occurred either in the presence or absence of VC. The ability of the TRW culture to degrade cis-DCE is significant for natural attenuation since both VC and cis-DCE are often found in chloroethene-contaminated groundwater. Experiments examining the effect of oxygen threshold on VC degradation showed that the culture was able to metabolize VC efficiently at extremely low concentrations of dissolved oxygen (DO). Complete removal of 150 micromoles of VC occurred in the presence of only 0.2 mmol of oxygen (1.8 mg/L DO). This is important since most groundwater environments contain low DO (1-2 mg/L). Studies showed that the culture was able to withstand long periods of VC starvation. For example, the culture was able to assimilate VC with minimal lag time even after 5 months of starvation. This is impressive from the point of its sustenance under field conditions. Overall the culture is robust and degrades VC to below the detection limit rendering this culture suitable for field application.

Biodegradation of benzene by halophilic and halotolerant bacteria under aerobic conditions.

A highly enriched halophilic culture was established with benzene as the sole carbon source by using a brine soil obtained from an oil production facility in Oklahoma. The enrichment completely degraded benzene, toluene, ethylbenzene, and xylenes within 1 to 2 weeks. Also, [14C]benzene was converted to 14CO2, suggesting the culture's ability to mineralize benzene. Community structure analysis revealed that Marinobacter spp. were the dominant members of the enrichment.

Acetate versus hydrogen as direct electron donors to stimulate the microbial reductive dechlorination process at chloroethene-contaminated sites.

A study to evaluate the dechlorination end points and the most promising electron donors to stimulate the reductive dechlorination process at the chloroethene-contaminated Bachman Road site in Oscoda, MI, was conducted. Aquifer materials were collected from inside the plume and used to establish microcosms under a variety of electron donor conditions using chlorinated ethenes as electron acceptors. All microcosms that received an electron donor showed dechlorination activity, but the end points depended on the sampling location, indicating a heterogeneous distribution of the dechlorinating populations in the aquifer. Interestingly, several microcosms that received acetate as the only electron donor completely dechlorinated PCE to ethene. All acetate-amended microcosms rapidly converted PCE to cis-DCE, whereas PCE dechlorination in H2-fed microcosms only occurred after a pronounced lag time and after acetate had accumulated by H2/CO2 acetogenic activity. The microcosm experiments were corroborated by defined co-culture experiments, which demonstrated that H2 sustained PCE to cis-DCE dechlorination by acetotrophic populations in the presence of H2/CO2 acetogens. In sediment-free nonmethanogenic enrichment cultures derived from ethene-producing microcosms, acetate alone supported complete reductive dechlorination of chloroethenes to ethene, although the addition of H2 resulted in higher cis-DCE and VC dechlorination rates. Measurements of H2 production and consumption suggested that syntrophic acetate-oxidizing population(s) were active in the enrichment cultures. These findings demonstrated that either acetate or H2 alone can be sufficient to promote complete