Complete Genome Sequence of an Anaerobic Benzene-Degrading Bacterium, Azoarcus sp. Strain DN11.

Here, we present the complete genome sequence of Azoarcus sp. strain DN11, a denitrifying bacterium capable of anaerobic benzene degradation. The DN11 genome is 4,956,835 bp long with a G+C content of 66.3%. Genome analysis suggested the possibility that DN11 utilizes three proposed pathways for anaerobic benzene degradation, namely, methylation, hydroxylation, and carboxylation pathways.

Ecological impact assessment of a bioaugmentation site on remediation of chlorinated ethylenes by multi-omics analysis.

Bioremediation may affect the ecological system around bioremediation sites. However, little is known about how microbial community structures change over time after the initial injection of degraders. In this study, we have assessed the ecological impact of bioaugmentation using metagenomic and metatranscriptomic approaches to remove trichlorinated ethylene/cis-dichloroethylene (TCE/cDCE) by Rhodococcus jostii strain RHA1 as an aerobic chemical compound degrader. Metagenomic analysis showed that the number of organisms belonging to the genus Rhodococcus, including strain RHA1, increased from 0.1% to 76.6% of the total microbial community on day 0 at the injection site. Subsequently, the populations of strain RHA1 and other TCE/cDCE-degrading bacteria gradually decreased over time, whereas the populations of the anaerobic dechlorinators Geobacter and Dehalococcoides increased at later stages. Metatranscriptomic analysis revealed a high expression of aromatic compound-degrading genes (bphA1-A4) in strain RHA1 after RHA1 injection. From these results, we concluded that the key dechlorinators of TCE/cDCE were mainly aerobic bacteria, such as RHA1, until day 1, after which the key dechlorinators changed to anaerobic bacteria, such as Geobacter and Dehalococcocides, after day 6 at the injection well. Based on the α-diversity, the richness levels of the microbial community were increased after injection of strain RHA1, and the microbial community composition had not been restored to that of the original composition during the 19 days after treatment. These results provide insights into the assessment of the ecological impact and bioaugmentation process of RHA1 at bioremediation sites.

Complete genome sequencing of Dehalococcoides sp. strain UCH007 using a differential reads picking method.

A novel Dehalococcoides sp. strain UCH007 was isolated from the groundwater polluted with chlorinated ethenes in Japan. This strain is capable of dechlorinating trichloroethene, cis-1,2-dichloroethene and vinyl chloride to ethene. Dehalococcoides bacteria are hardly cultivable, so genome sequencing has presented a challenge. In this study, we developed a differential reads picking method for mixed genomic DNA obtained from a co-culture, and applied it to the sequencing of strain UCH007. The genome of strain UCH007 consists of a 1,473,548-bp chromosome that encodes 1509 coding sequences including 29 putative reductive dehalogenase genes. Strain UCH007 is the first strain in the Victoria subgroup found to possess the pceA, tceA and vcrA genes.

The Impact of Injections of Different Nutrients on the Bacterial Community and Its Dechlorination Activity in Chloroethene-Contaminated Groundwater.

Dehalococcoides spp. are currently the only organisms known to completely reduce cis-1,2-dichloroethene (cis-DCE) and vinyl chloride (VC) to non-toxic ethene. However, the activation of fermenting bacteria that generate acetate, hydrogen, and CO2 is considered necessary to enhance the dechlorination activity of Dehalococcoides and enable the complete dechlorination of chloroethenes. In the present study, we stimulated chloroethene-contaminated groundwater by injecting different nutrients prepared from yeast extract or polylactate ester using a semicontinuous culture system. We then evaluated changes in the bacterial community structure and their relationship with dechlorination activity during the biostimulation. The populations of Dehalococcoides and the phyla Bacteroidetes, Firmicutes, and Spirochaetes increased in the yeast extract-amended cultures and chloroethenes were completely dechlorinated. However, the phylum Proteobacteria was dominant in polylactate ester-amended cultures, in which almost no cis-DCE and VC were dechlorinated. These results provide fundamental information regarding possible interactions among bacterial community members involved in the dechlorination process and support the design of successful biostimulation strategies.

Complete Genome Sequences of Sulfurospirillum Strains UCH001 and UCH003 Isolated from Groundwater in Japan.

Sulfurospirillum strains UCH001 and UCH003 were isolated from anaerobic cis-1,2-dichloroethene-dechlorinating microbial consortia derived from groundwater in Japan. Here, we report the complete genome sequences of strains UCH001 and UCH003.

Degradative capacities and bioaugmentation potential of an anaerobic benzene-degrading bacterium strain DN11.

Azoarcus sp. strain DN11 is a denitrifying bacterium capable of benzene degradation under anaerobic conditions. The present study evaluated strain DN11 for its application to bioaugmentation of benzene-contaminated underground aquifers. Strain DN11 could grow on benzene, toluene, m-xylene, and benzoate as the sole carbon and energy sources under nitrate-reducing conditions, although o- and p-xylenes were transformed in the presence of toluene. Phenol was not utilized under anaerobic conditions. Kinetic analysis of anaerobic benzene degradation estimated its apparent affinity and inhibition constants to be 0.82 and 11 microM, respectively. Benzene-contaminated groundwater taken from a former coal-distillation plant site was anaerobically incubated in laboratory bottles and supplemented with either inorganic nutrients (nitrogen, phosphorus, and nitrate) alone, or the nutrients plus strain DN11, showing that benzene was significantly degraded only when DN11 was introduced. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments, and quantitative PCR revealed that DN11 decreased after benzene was degraded. Following the decrease in DN11 16S rRNA gene fragments corresponding to bacteria related to Owenweeksia hongkongensis and Pelotomaculum isophthalicum, appeared as strong bands, suggesting possible metabolic interactions in anaerobic benzene degradation. Results suggest that DN11 is potentially useful for degrading benzene that contaminates underground aquifers at relatively low concentrations.

Rapid intrinsic biodegradation of benzene, toluene, and xylenes at the boundary of a gasoline-contaminated plume under natural attenuation.

A groundwater plume contaminated with gasoline constituents [mainly benzene, toluene, and xylenes (BTX)] had been treated by pumping and aeration for approximately 10 years, and the treatment strategy was recently changed to monitored natural attenuation (MNA). To gain information on the feasibility of using MNA to control the spread of BTX, chemical and microbiological parameters in groundwater samples obtained inside and outside the contaminated plume were measured over the course of 73 weeks. The depletion of electron acceptors (i.e., dissolved oxygen, nitrate, and sulfate) and increase of soluble iron were observed in the contaminated zone. Laboratory incubation tests revealed that groundwater obtained immediately outside the contaminated zone (the boundary zone) exhibited much higher potential for BTX degradation than those in the contaminated zone and in uncontaminated background zones. The boundary zone was a former contaminated area where BTX were no longer detected. Denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction (PCR)-amplified bacterial 16S rRNA gene fragments revealed that DGGE profiles for groundwater samples obtained from the contaminated zone were clustered together and distinct from those from uncontaminated zones. In addition, unique bacterial rRNA types were observed in the boundary zone. These results indicate that the boundary zone in the contaminant plumes served as a natural barrier for preventing the BTX contamination from spreading out.

RNA-based stable isotope probing and isolation of anaerobic benzene-degrading bacteria from gasoline-contaminated groundwater.

Stable isotope probing (SIP) of benzene-degrading bacteria in gasoline-contaminated groundwater was coupled to denaturing gradient gel electrophoresis (DGGE) of DNA fragments amplified by reverse transcription-PCR from community 16S rRNA molecules. Supplementation of the groundwater with [(13)C(6)]benzene together with an electron acceptor (nitrate, sulfate, or oxygen) showed that a phylotype affiliated with the genus Azoarcus specifically appeared in the (13)C-RNA fraction only when nitrate was supplemented. This phylotype was also observed as the major band in DGGE analysis of bacterial 16S rRNA gene fragments amplified by PCR from the gasoline-contaminated groundwater. In order to isolate the Azoarcus strains, the groundwater sample was streaked on agar plates containing nonselective diluted CGY medium, and the DGGE analysis was used to screen colonies formed on the plates. This procedure identified five bacterial isolates (from 60 colonies) that corresponded to the SIP-identified Azoarcus phylotype, among which two strains (designated DN11 and AN9) degraded benzene under denitrifying conditions. Incubation of these strains with [(14)C]benzene showed that the labeled carbon was mostly incorporated into (14)CO(2) within 14 days. These results indicate that the Azoarcus population was involved in benzene degradation in the gasoline-contaminated groundwater under denitrifying conditions. We suggest that RNA-based SIP identification coupled to phylogenetic screening of nonselective isolates facilitates the isolation of enrichment/isolation-resistant microorganisms with a specific function.

Physiological and molecular characterization of a microbial community established in unsaturated, petroleum-contaminated soil.

The microbial communities established in soil samples from an unsaturated, petroleum-contaminated zone and from an adjacent uncontaminated site were characterized by physiological and molecular approaches. Possible electron acceptors such as sulfate and nitrate had been completely depleted in these soil samples. Slurries of these soil samples were incubated in bottles in the presence of hydrocarbon indicators (benzene, toluene, xylene and decane), and the degradation of these compounds was examined. Supplementation with electron acceptors stimulated hydrocarbon degradation, although the stimulatory effect was small in the contaminated soil. The initial degradation rates in the contaminated soil under fermentative/methanogenic conditions were comparable to those under aerobic conditions. The microbial populations in the original soil samples were analysed by cloning and sequencing of polymerase chain reaction (PCR)-amplified bacterial and archaeal 16S rRNA gene fragments, showing that the sequences retrieved from these soils were substantially different. For instance, Epsilonproteobacteria, Gammaproteobacteria, Crenarchaeota and Methanosarcinales could only be detected at significant levels in the contaminated soil. Denaturing gradient gel electrophoresis (DGGE) analyses of 16S rRNA gene fragments amplified by PCR from the incubated soil-slurry samples showed that supplementation of the electron acceptors resulted in a shift in the major populations, while the DGGE profiles after incubating the contaminated soil under the fermentative/methanogenic conditions were not substantially changed. These results suggest that petroleum contamination of the unsaturated zone resulted in the establishment of a fermentative/methanogenic community with substantial hydrocarbon-degrading potential.

Development of a PCR method for the detection and quantification of benzoyl-CoA reductase genes and its application to monitored natural attenuation.

Benzoyl coenzyme A reductase (BCR) catalyzes dearomatization of benzoyl coenzyme A (benzoyl-CoA), which is the central step in the anaerobic degradative pathways for a variety of aromatic compounds. This study developed a PCR method for the detection and quantification of BCR genes in bacterial strains and environmental samples. PCR primers were designed by aligning known BCR genes in Thauera, Azoarcus and Rhodopseudomonas species, and their utility was assessed by amplifying BCR fragments from aromatic-hydrocarbon degrading anaerobes and other bacteria. BCR fragments with the expected sizes were obtained from denitrifying and phototrophic aromatics degraders. The positive signals were also obtained from Geobacter metallireducens and xylene-degrading sulfate-reducing bacterium (strain mXyS1) but not from other aromatics-degrading sulfate-reducing bacteria and aerobic bacteria. When the PCR was used for analyzing a natural attenuation (NA) site, the positive signal was obtained only from gasoline-contaminated groundwater; sequence analysis of these amplicons revealed that most of them exhibited substantial similarities to the known BCRs. Quantitative competitive PCR analysis estimated BCR-gene copies to account for 10-40% of bacterial 16S rRNA gene copies in the contaminated groundwater, indicating that bacteria possessing BCR genes were highly enriched in the contaminated groundwater. In microcosm bioremediation tests using the contaminated groundwater, the copy number of BCR gene was approximately 10-fold increased in the course of aromatics degradation under denitrifying conditions but not under sulfidogenic conditions. These results suggest the utility of the PCR method for assessing the potential of denitrifying bacteria for aromatic-compound degradation in groundwater.