The genome sequence of Desulfatibacillum alkenivorans AK-01: a blueprint for anaerobic alkane oxidation.

Desulfatibacillum alkenivorans AK-01 serves as a model organism for anaerobic alkane biodegradation because of its distinctive biochemistry and metabolic versatility. The D. alkenivorans genome provides a blueprint for understanding the genetic systems involved in alkane metabolism including substrate activation, CoA ligation, carbon-skeleton rearrangement and decarboxylation. Genomic analysis suggested a route to regenerate the fumarate needed for alkane activation via methylmalonyl-CoA and predicted the capability for syntrophic alkane metabolism, which was experimentally verified. Pathways involved in the oxidation of alkanes, alcohols, organic acids and n-saturated fatty acids coupled to sulfate reduction and the ability to grow chemolithoautotrophically were predicted. A complement of genes for motility and oxygen detoxification suggests that D. alkenivorans may be physiologically adapted to a wide range of environmental conditions. The D. alkenivorans genome serves as a platform for further study of anaerobic, hydrocarbon-oxidizing microorganisms and their roles in bioremediation, energy recovery and global carbon cycling.

Bacterial community structure and bamA gene diversity in anaerobic degradation of toluene and benzoate under denitrifying conditions.

AIM: To characterize the microbial community structure and bamA gene diversity involved in anaerobic degradation of toluene and benzoate under denitrifying conditions. METHODS AND RESULTS: Nitrate-reducing enrichment cultures were established on either toluene, benzoate or without additional substrate. Bacterial community structures were characterized by 16S rRNA gene-based PCR-DGGE analysis. bamA gene diversity was analysed using DGGE and cloning methods. The results showed that bamA gene related to bamA of Thauera chlorobenzoica was dominant in toluene and benzoate cultures. However, a greater diversity of sequences was obtained in benzoate cultures. Low diversity of bamA gene was observed, and some similarities of bamA were also found between active cultures and backgrounds, suggesting that potential natural attenuation of aromatic compounds might occur. CONCLUSIONS: The combined analysis of 16S rRNA and bamA genes suggests that the species related to genera Thauera dominated toluene- and benzoate-degrading cultures. The combination of multiple methods (DGGE and cloning) provides a more complete picture of bamA gene diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report of bamA gene in denitrifying enrichments using DGGE and cloning analysis.

Dual biomarkers of anaerobic hydrocarbon degradation in historically contaminated groundwater.

This study reports that ongoing in situ anaerobic hydrocarbon biodegradation at a manufactured gas plant impacted site is occurring, 9 years after the initial investigation. Groundwater samples from the site monitoring wells (MW) were analyzed for biomarkers by GC-MS, end-point PCR, and quantitative PCR (qPCR). Metabolic biomarkers included specific intermediates of anaerobic naphthalene and/or 2-methylnaphthalene degradation: 2-naphthoic acid (2-NA); 5,6,7,8-tetrahydro-2-NA (TH-2-NA); hexahydro-2-NA (HH-2-NA); and carboxylated-2-methylnaphthalene (MNA). The analogues of gene bssA, encoding alpha subunit of enzyme benzylsuccinate synthase, were used as a genetic biomarker. Results indicate 1-2 orders of magnitude higher abundance of total bacteria in the impacted wells than in the unimpacted wells. End-point PCR analysis of bssA gene, with degenerate primers, indicated the presence of hydrocarbon degrading bacteria within the plume. In qPCR analysis, using primers based on toluene-degrading denitrifying or sulfate-reducing/methanogenic bacteria, bssA genes were detected only in MW-24, located downstream from the source. Metabolic biomarkers were detected in multiple wells. The highest abundance of 2-NA (6.7 µg/L), TH-2-NA (2.6 µg/L), HH-2-NA, and MNA was also detected in MW-24. The distribution of two independent biomarkers indicates that the site is enriched for anaerobic hydrocarbon biodegradation and provides strong evidence in support of natural attenuation.

Anaerobic biodegradation of n-hexadecane by a nitrate-reducing consortium.

Nitrate-reducing enrichments, amended with n-hexadecane, were established with petroleum-contaminated sediment from Onondaga Lake. Cultures were serially diluted to yield a sediment-free consortium. Clone libraries and denaturing gradient gel electrophoresis analysis of 16S rRNA gene community PCR products indicated the presence of uncultured alpha- and betaproteobacteria similar to those detected in contaminated, denitrifying environments. Cultures were incubated with H(34)-hexadecane, fully deuterated hexadecane (d(34)-hexadecane), or H(34)-hexadecane and NaH(13)CO(3). Gas chromatography-mass spectrometry analysis of silylated metabolites resulted in the identification of [H(29)]pentadecanoic acid, [H(25)]tridecanoic acid, [1-(13)C]pentadecanoic acid, [3-(13)C]heptadecanoic acid, [3-(13)C]10-methylheptadecanoic acid, and d(27)-pentadecanoic, d(25)-, and d(2)(4)-tridecanoic acids. The identification of these metabolites suggests a carbon addition at the C-3 position of hexadecane, with subsequent beta-oxidation and transformation reactions (chain elongation and C-10 methylation) that predominantly produce fatty acids with odd numbers of carbons. Mineralization of [1-(14)C]hexadecane was demonstrated based on the recovery of (14)CO(2) in active cultures.

Identification of critical members in a sulfidogenic benzene-degrading consortium by DNA stable isotope probing.

Stable isotope probing (SIP) was used to identify the active members in a benzene-degrading sulfidogenic consortium. SIP-terminal restriction fragment length polymorphism analysis indicated that a 270-bp peak incorporated the majority of the (13)C label and is a sequence closely related to that of clone SB-21 (GenBank accession no. AF029045). This target may be an important biomarker for anaerobic benzene degradation in the field.

Transformation of galena to pyromorphite produces bioavailable sulfur for neutrophilic chemoautotrophy.

The aqueous concentration of lead [Pb(II)] in geochemical environments is controlled by the solubility of Pb-bearing minerals and their weathering products. In contaminated soils, a common method for in situ stabilization of Pb(II) is the addition of phosphate to convert more redox sensitive sulfide minerals into sparingly soluble pyromorphite [Pb5 (PO4 )3 X]. In this study, we conducted experimental studies to investigate the fate of reduced sulfur during the conversion of galena [PbS] to chloropyromorphite [Pb5 (PO4 )3 Cl]. Powder X-ray diffraction analysis indicated that the reaction of phosphate with galena under oxic conditions resulted in the oxidation of sulfide and formation of elemental sulfur [S8 ]. Under oxic abiotic conditions, the S8 was retained in the solid phase, and negligible concentrations of sulfur as sulfide and thiosulfate were detected in the aqueous phase and only a small amount of sulfate. When PbS reacted in the presence of the chemoautotrophic organism Bosea sp. WAO, the S8 in the secondary mineral was oxidized to sulfate. Strain WAO produced significantly more sulfate from the secondary S8 than from the primary galena. Microscopic analysis of mineral-microbe aggregates on mineral-embedded slide cultures showed that the organism was colocalized and increased in biomass over time on the secondary mineral surface supporting a microbial role. The results of this study indicate that stimulation of sulfur-oxidizing activity may be a direct consequence of phosphate amendments to Pb(II)-contaminated soils.

A STE12 homolog is required for mating but dispensable for filamentation in candida lusitaniae.

Candida lusitaniae is a dimorphic yeast that is emerging as an opportunistic fungal pathogen. In contrast to Candida albicans, which is diploid and asexual, C. lusitaniae has been reported to have a sexual cycle. We have employed genetic approaches to demonstrate that C. lusitaniae is haploid and has a sexual cycle involving mating between MATa and MATalpha cells under nutrient deprivation conditions. By degenerate PCR, we identified a C. lusitaniae homolog (Cls12) of the Ste12 transcription factor that regulates mating, filamentation, and virulence in Saccharomyces cerevisiae, C. albicans, and Cryptococcus neoformans. Comparison of the CLS12 DNA and protein sequences to other STE12 homologs and transformation experiments with selectable markers from S. cerevisiae (URA3, KanMX, HphMX) and C. albicans (CaURA3) provide evidence that the CUG codon encodes serine instead of leucine in C. lusitaniae, as is also the case in C. albicans. The C. lusitaniae CLS12 gene was disrupted by biolistic transformation and homologous recombination. C. lusitaniae cls12 mutant strains were sterile but had no defect in filamentous growth. Our findings reveal both conserved and divergent roles for the C. lusitaniae STE12 homolog in regulating differentiation of this emerging fungal pathogen.

Biodegradation of toxic and environmental pollutants.

Organic chemicals that are toxic to humans and to the environment can be transformed and metabolized by a variety of microorganisms. Such chemicals include trichloroethylene, chloroform, carbon tetrachloride, toluene, phenols, chlorinated phenols, polychlorinated biphenyls and polyaromatic hydrocarbons. This review focuses on some of the most important recent developments in the biodegradation of these toxic chemicals. Depending on the compound and the organism, the extent of our understanding ranges from the molecular level to the conceptual.

Biogeochemical environments of streambed-sediment pore waters with and without arsenic enrichment in a sedimentary rock terrain, New Jersey Piedmont, USA.

Release of arsenic (As) from sedimentary rocks has resulted in contamination of groundwater in aquifers of the New Jersey Piedmont Physiographic Province, USA; the contamination also may affect the quality of the region's streamwater to which groundwater discharges. Biogeochemical mechanisms involved in the release process were investigated in the streambeds of Six Mile Run and Pike Run, tributaries to the Millstone River in the Piedmont. At Six Mile Run, streambed pore water and shallow groundwater were low or depleted in oxygen, and contained As at concentrations greater than 20 µg/L. At Pike Run, oxidizing conditions were present in the streambed, and the As concentration in pore water was 2.1 µg/L. The 16S rRNA gene and the As(V) respiratory reductase gene, arrA, were amplified from DNA extracted from streambed pore water at both sites and analyzed, revealing that distinct bacterial communities that corresponded to the redox conditions were present at each site. Anaerobic enrichment cultures were inoculated with pore water from gaining reaches of the streams with acetate and As(V). As(V) was reduced by microbes to As(III) in enrichments with Six Mile Run pore water and groundwater, whereas no reduction occurred in enrichments with Pike Run pore water. Cloning and sequencing of the arrA gene indicated 8 unique operational taxonomic units (OTUs) at Six Mile Run and 11 unique OTUs at Pike Run, which may be representative of the arsenite oxidase gene arxA. Low-oxygen conditions at Six Mile Run have favored microbial As reduction and release, whereas release was inhibited by oxidizing conditions at Pike Run.

Anaerobic degradation of benzene in BTX mixtures dependent on sulfate reduction.

Enrichment cultures from marine sediments mineralized benzene while using sulfate as the terminal electron acceptor. Parallel cultures using river marsh sediment displayed no activity. Mineralization was confirmed by release of 14CO2 from radiolabeled benzene. The dependence on sulfate reduction was demonstrated by stoichiometric balances and the use of specific inhibitors. This work supports recent observations that anaerobic benzene degradation takes place coupled to sulfate reduction.

Anaerobic degradation of halogenated benzoic acids coupled to denitrification observed in a variety of sediment and soil samples.

Denitrifying enrichment cultures utilizing monochlorinated benzoic acids as a carbon source were established using sediments and soils from a variety of sources as inocula. Enrichment cultures from most of the sites readily degraded 3- and 4-chlorobenzoate within 2-4 weeks. Upon refeeding, 3- and 4-chlorobenzoate were rapidly depleted, and stable denitrifying cultures were obtained by repeated dilution and refeeding of the substrates. 2-Chlorobenzoate, however, was only slowly metabolized and this activity was only observed in a few sites. Denitrifying consortia were maintained on either 3- or 4-chlorobenzoate as the sole source of carbon and energy and chlorobenzoate utilization was dependent on denitrification. These cultures were also capable of utilizing the corresponding brominated and iodinated benzoic acids, but the activity was specific to the position of the halogen substituent. Removal of halogen was stoichiometric, indicating that dehalogenation occurred at some step in metabolism.

Microbial reductive dechlorination of pre-existing PCBs and spiked 2,3,4,5,6-pentachlorobiphenyl in anaerobic slurries of a contaminated sediment of Venice Lagoon (Italy).

Abstract Reductive dechlorination of polychlorinated biphenyls (PCBs) pre-existing (at approximately 1 mg kg(-1)) in a marine sediment of Porto Marghera (Venice Lagoon, Italy) was investigated in anaerobic slurries developed in water of the same contaminated site. Some microcosms were pasteurized whereas others were amended with 2-bromoethanesulfonic acid, molybdate or eubacteria-inhibiting antibiotics (without and in the presence of exogenous carbon sources) to preliminarily characterize the microbial populations involved in the process. Bioconversion of highly chlorinated PCBs into tri- and di-chlorinated, ortho-substituted biphenyls was detected from the 11th week of incubation both in the non-amended and in the pasteurized microcosms, where a significant consumption of sulfate and no methane production were observed. Conversely, no significant PCB transformation was detected in the microcosms with molybdate, where no sulfate consumption and a significant methane evolution occurred. Neither was PCB transformation observed in the microcosms supplemented with antibiotics and exogenous carbon sources, where a strong methane evolution and no sulfate consumption were recorded until the 11th week. The addition of exogenous 2,3,4,5,6-pentachlorobiphenyl showed preferential dechlorination at the meta and para positions, and did not significantly influence the onset of pre-existing PCB dechlorination. These results indicate that endogenous PCBs pre-existing in the marine sediment underwent reductive dechlorination. They also suggest that the process was not 'primed' upon 2,3,4,5,6-pentachlorobiphenyl addition, and was likely to be mediated by sulfate-reducing, spore-forming bacteria.

Anaerobic biodegradation of alkanes by enriched consortia under four different reducing conditions.

Enrichments of alkane-degrading microorganisms were established under sulfate-reducing, denitrifying, iron-reducing, and methanogenic conditions using an estuarine sediment. The sulfate-reducing and denitrifying enrichments mineralized [1-14C]hexadecane to 14CO2. Degradation was coupled to sulfate reduction and denitrification, as indicated by the ratios of hexadecane degraded to the electron acceptors reduced. Dependence of hexadecane degradation by the sulfate-reducing enrichments on sulfate reduction was also shown. The results indicate the existence of diverse microbial communities capable of alkane degradation in the sediment studied.

Effect of sediment toxicity on anaerobic microbial metabolism.

Mineralization of a readily biodegradable aromatic compound (benzoate) by intrinsic microorganisms in the anoxic sediment was used to quantify the inhibitory effect of heavily contaminated sediment from the Arthur Kill estuary (NY/NJ Harbor system, USA) on the anaerobic metabolism by naturally present bacterial populations. In anoxic microcosms, the effect of varying ratios of contaminated sediment:site water and contaminated sediment:noncontaminated sediment (Flax Pond, Stony Brook, NY, USA) were investigated. In all cases, increasing the ratio of Arthur Kill sediment in the microcosms showed an inhibitory effect on the rate of 14C-benzoate mineralization as measured by the evolution of 14CO2. This inhibitory effect could be alleviated through dilution of the sediment with noncontaminated sediment, resulting in some cases in mineralization rates that were greater by an order of magnitude. The toxicity of the sediment was confirmed by whole-sediment Microtox bioassay. Analysis of the sediment revealed high (>200 mg/kg) levels of Pb, Cu, Zn, and Cr, suggesting that heavy metals may contribute to overall sediment toxicity.

The needs of families of infants discharged home with continuous oxygen therapy.

Forty-four parents of 48 infants who had been discharged home with continuous oxygen therapy described their experiences, needs, and resources in a semistructured interview. In addition, 20 professionals in contact with these infants were interviewed to determine their perceptions of discharge preparations, teaching, relief care, coordination of medical care, and expertise of community professionals regarding high-risk infants on oxygen. Both the professionals and parents reported a need for improved discharge teaching and community support services. Researchers concluded that individual needs must be considered in arranging supportive interventions, as needs vary across families, time, and geographic locations.

Microbial transformations of arsenic: mobilization from glauconitic sediments to water.

In the Inner Coastal Plain of New Jersey, arsenic (As) is released from glauconitic sediment to carbon- and nutrient-rich shallow groundwater. This As-rich groundwater discharges to a major area stream. We hypothesize that microbes play an active role in the mobilization of As from glauconitic subsurface sediments into groundwater in the Inner Coastal Plain of New Jersey. We have examined the potential impact of microbial activity on the mobilization of arsenic from subsurface sediments into the groundwater at a site on Crosswicks Creek in southern New Jersey. The As contents of sediments 33-90 cm below the streambed were found to range from 15 to 26.4 mg/kg, with siderite forming at depth. Groundwater beneath the streambed contains As at concentrations up to 89 µg/L. Microcosms developed from site sediments released 23 µg/L of As, and active microbial reduction of As(V) was observed in microcosms developed from site groundwater. DNA extracted from site sediments was amplified with primers for the 16S rRNA gene and the arsenate respiratory reductase gene, arrA, and indicated the presence of a diverse anaerobic microbial community, as well as the presence of potential arsenic-reducing bacteria. In addition, high iron (Fe) concentrations in groundwater and the presence of iron-reducing microbial genera suggests that Fe reduction in minerals may provide an additional mechanism for release of associated As, while arsenic-reducing microorganisms may serve to enhance the mobility of As in groundwater at this site.

Arsenic transformation and mobilization from minerals by the arsenite oxidizing strain WAO.

Analysis of arsenic concentrations in New Jersey well water from the Newark Basin showed up to 15% of the wells exceed 10 microg L(-1), with a maximum of 215 microg L(-1). In some geologic settings in the basin, this mobile arsenic could be from the weathering of pyrite (FeS2) found in black shale that contains up to 4% arsenic by weight. We hypothesized that under oxic conditions at circumneutral pH, the microbially mediated oxidation of sulfide in the pyrite lattice would lead to the release of pyrite-bound arsenic. Moreover, the oxidation of aqueous As(III) to As(V) by aerobic microorganisms could further enhance arsenic mobilization from the solid phase. Enrichment cultures under aerobic, As(III)-oxidizing conditions were established under circumneutral pH with weathered black shale from the Newark Basin as the inoculum source. Strain WAO, an autotrophic inorganic-sulfur and As(III)-oxidizer, was isolated and phylogenetically and physiologically characterized. Arsenic mobilization studies from arsenopyrite (FeAsS) mineral, conducted with strain WAO at circumneutral pH, showed microbially enhanced mobilization of arsenic and complete oxidation of released arsenic and sulfur to stoichiometric amounts of arsenate and sulfate. In addition, WAO preferentially colonized pyrite on the surface of arsenic-bearing, black shale thick sections. These findings support the hypothesis that microorganisms can directly mobilize and transform arsenic bound in mineral form at circumneutral pH and suggest that the microbial mobilization of arsenic into groundwater may be important in other arsenic-impacted aquifers.

The arsenite oxidase genes (aroAB) in novel chemoautotrophic arsenite oxidizers.

Six novel bacterial strains were isolated from the environment which can oxidize arsenite [As(III)] to the less mobile form arsenate [As(V)] coupled to CO(2) fixation under either aerobic or denitrifying conditions. PCR primers were designed to the conserved molybdopterin domain of the large subunit of arsenite oxidase in order to identify the arsenite oxidase genes from these isolates. The amino acid sequences for the arsenite oxidases reported here were 72-74% identical to that of strain NT-26, the only previously reported autotrophic arsenite oxidizer. Indeed the autotrophic arsenite oxidase genes form a distinct phylogenetic group, separated from previously described heterotrophic arsenite oxidase genes, with the exception of the heterotroph Agrobacterium tumefaciens. The arsenite oxidase primers described here represent a powerful culture-independent tool to assess the diversity of arsenite oxidase genes in environmental bacteria.

Anaerobic arsenite oxidation by novel denitrifying isolates.

Autotrophic microorganisms have been isolated that are able to derive energy from the oxidation of arsenite [As(III)] to arsenate [As(V)] under aerobic conditions. Based on chemical energetics, microbial oxidation of As(III) can occur in the absence of oxygen, and may be relevant in some environments. Enrichment cultures were established from an arsenic contaminated industrial soil amended with As(III) as the electron donor, inorganic C as the carbon source and nitrate as the electron acceptor. In the active enrichment cultures, oxidation of As(III) was stoichiometrically coupled to the reduction of NO(3) (-). Two autotrophic As(III)-oxidizing strains were isolated that completely oxidized 5 mM As(III) within 7 days under denitrifying conditions. Based on 16S rRNA gene sequencing results, strain DAO1 was 99% related to Azoarcus and strain DAO10 was most closely related to a Sinorhizobium. The nitrous oxide reductase (nosZ) and the RuBisCO Type II (cbbM) genes were successfully amplified from both isolates underscoring their ability to denitrify and fix CO(2) while coupled to As(III) oxidation. Although limited work has been done to examine the diversity of anaerobic autotrophic oxidizers of As(III), this process may be an important component in the biological cycling of arsenic within the environment.

Microbial reductive dechlorination of weathered and exogenous co-planar polychlorinated biphenyls (PCBs) in an anaerobic sediment of Venice Lagoon.

The occurrence of reductive dechlorination processes towards pre-existing PCBs and five exogenous coplanar PCBs were investigated in a contaminated sediment of Porto Marghera (Venice Lagoon, Italy) suspended, under strictly anaerobic conditions, in water collected from the same site. PCB dechlorination started after five months of incubation, when sulfate initially occurring in the microcosms was completely depleted and methanogenesis was in progress. It was ascribed to sulfate-reducing bacteria. Several pre-existing hexa-, penta- and tetra-chlorinated biphenyls were slowly bioconverted into tri- and di-, ortho-substituted PCBs from the 5th to the 16th month of experiment. Spiked coplanar PCBs, i.e., 3,3',4,4'-tetrachlorobiphenyl, 3,3',4,4',5- and 2,3',4,4',5-pentachlorobiphenyls, 3,3',4,4',5,5'- and 2,3,3',4,4',5-hexachlorobiphenyls, were extensively transformed (by about 90%) into lower chlorinated congeners, such as 3,3',5,5'-/2,3',4,4'-tetrachlorobiphenyl, 3,3',5-, 2,4,4'-, 2,3',4- and 2,3',5-trichlorobiphenyl, 3,4-/3,4'- and 3,3'-dichlorobiphenyl and 2-chlorobiphenyl. The reductive dechlorination of spiked PCBs did not influence significantly the biotransformation rate and extent of pre-existing PCBs.