A pilot field study was conducted to assess the impact of bioaugmentation with two plasmid pJP4-bearing microorganisms: the natural host, Ralstonia eutropha JMP134, and a laboratory-generated strain amenable to donor counterselection, Escherichia coli D11. The R. eutropha strain contained chromosomal genes necessary for mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D), while the E. coli strain did not. The soil system was contaminated with 2,4-D alone or was cocontaminated with 2,4-D and Cd. Plasmid transfer to indigenous populations, plasmid persistence in soil, and degradation of 2,4-D were monitored over a 63-day period in the bioreactors. To assess the impact of contaminant reexposure, aliquots of bioreactor soil were reamended with additional 2,4-D. Both introduced donors remained culturable and transferred plasmid pJP4 to indigenous recipients, although to different extents. Isolated transconjugants were members of the Burkholderia and Ralstonia genera, suggesting multiple, if not successive, plasmid transfers. Upon a second exposure to 2,4-D, enhanced degradation was observed for all treatments, suggesting microbial adaptation to 2,4-D. Upon reexposure, degradation was most rapid for the E. coli D11-inoculated treatments. Cd did not significantly impact 2,4-D degradation or transconjugant formation. This study demonstrated that the choice of donor microorganism might be a key factor to consider for bioaugmentation efforts. In addition, the establishment of an array of stable indigenous plasmid hosts at sites with potential for reexposure or long-term contamination may be particularly useful.

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The abundance, distribution, and phylogenetic diversity of members of the Fe(III)-reducing family Geobacteraceae were studied along a gradient of metal contaminants in Lake Coeur d'Alene, Idaho. Partial 16S rRNA gene fragments were amplified by PCR using primers directed toward conserved regions of the gene within the family Geobacteraceae. Analysis of amplicons separated by denaturing gradient gel electrophoresis (DGGE) suggested within-site variation was as great as between-site variation. Amplicons were cloned and grouped by RFLP type and DGGE migration distance and representatives were sequenced. Grouping clones with 3% or less sequence dissimilarity, 15 distinct phylotypes were identified compared to 16 distinct DGGE bands. Only 1 phylotype was recovered from all sites. This clone, B14, is most closely related to Geobacter metallireducens and constituted a greater portion of the pristine community than of the contaminated communities. A second phylotype, Q2, predominated in the contaminated communities and was notably absent from the pristine libraries. Clone Q2 presents a high degree of sequence similarity to two Geobacter spp. previously isolated from this region of Lake Coeur d'Alene. Six phylotypes were unique to the contaminated sediments, whereas two were found only in the pristine sediments. Indices of diversity (Shannon and Simpson) were consistently higher when calculated with DGGE data than when clone library data were used. Most-probable-number PCR and real-time PCR suggested that the Geobacteraceae phylotypes were spread relatively evenly across all three sites along the gradient. Our data indicate that the Geobacteraceae are diverse and abundant in Lake Coeur d'Alene sediments, regardless of metals content. These results provide insight into the ability of dissimilatory Fe(III)-reducing bacteria to colonize habitats with elevated metal concentrations, and they have important implications for the management and remediation of metal-contaminated sites.

Methane hydrate found in marine sediments is thought to contain gigaton quantities of methane and is considered an important potential fuel source and climate-forcing agent. Much of the methane in hydrates is biogenic, so models that predict the presence and distribution of hydrates require accurate rates of in situ methanogenesis. We estimated the in situ methanogenesis rates in Hydrate Ridge (HR) sediments by coupling experimentally derived minimal rates of methanogenesis to methanogen biomass determinations for discrete locations in the sediment column. When starved in a biomass recycle reactor, Methanoculleus submarinus produced ca. 0.017 fmol methane/cell/day. Quantitative PCR (QPCR) directed at the methyl coenzyme M reductase subunit A gene (mcrA) indicated that 75% of the HR sediments analyzed contained <1,000 methanogens/g. The highest numbers of methanogens were found mostly from sediments <10 m below seafloor. By considering methanogenesis rates for starved methanogens (adjusted to account for in situ temperatures) and the numbers of methanogens at selected depths, we derived an upper estimate of <4.25 fmol methane produced/g sediment/day for the samples with fewer methanogens than the QPCR method could detect. The actual rates could vary depending on the real number of methanogens and various seafloor parameters that influence microbial activity. However, our calculated rate is lower than rates previously reported for such sediments and close to the rate derived using geochemical modeling of the sediments. These data will help to improve models that predict microbial gas generation in marine sediments and determine the potential influence of this source of methane on the global carbon cycle.Subseafloor sediments near continental margins are often rich in dissolved methane as well as methane hydrates where pressures and temperatures are sufficient to maintain gases in this solid form (35,53). Because this methane, whether dissolved in sediment pore waters or present as a hydrate, occupies a large volume globally and is inherently unstable, these formations are of considerable interest as a potential source of energy, a mechanism for climate change, and a factor in seafloor stability (21,34).Much of the methane in subseafloor hydrate formations and the surrounding sediments is biogenic (33), and therefore, the conceptual and computational models describing hydrate occurrence, distribution, and abundance benefit from the knowledge of accurate in situ methane formation rates. With information about the primary biological methane supply, models may be able to provide estimates of the timescale for hydrate accumulation (64) and the transport of methane into the hydrate stability zone, to confine where in the sediments methane production can occur (23), and to simulate the formation of hydrate deposits (17).Determining accurate rates for microbial activities in subsurface environments, such as those that contain hydrates, is difficult. Subsurface microbial activities are believed to occur at exceedingly low ...

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