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The authors descibe the use of real-time PCR in detecting and quantifying DNA targets. The real-time PCR detection of Brucella abortus has been carried out via evaluation of 5'-exonuclease, hybridization probe analysis and SYBR Green I approaches.

Microbial enrichments from Cr(VI) contaminated and uncontaminated US Department of Energy Hanford Site sediments produced Cr(VI) reducing consortia when grown in the presence of Cr(VI) with acetate, D-xylose or glycerol as a carbon and energy source. Eight of the nine isolates from the consortia were Gram positive and four of these were identified by 16S rRNA sequence homology and membrane fatty acid composition as belonging to the genus Cellulomonas. Two strains, ES6 and WS01, were further examined for their ability to reduce Cr(VI) under growth and non-growth conditions. During fermentative growth on D-xylose, ES6 and WS01 decreased aqueous Cr(VI) concentrations from 0.04 mM Cr(VI) to below the detection limit (0.002 mM Cr(VI)) in less than three days and retained their ability to reduce Cr(VI) even after four months of incubation. Washed ES6 and WS01 cells also reduced Cr(VI) under non-growth conditions for over four months, both with and without the presence of an exogenous electron donor. K-edge XANES spectroscopy confirmed the reduction of Cr(VI) to Cr(III). The ability to reduce Cr(VI) after growth had stopped and in the absence of an external electron donor, suggests that stimulation of these types of organisms may lead to effective long-term, in situ passive reactive barriers for Cr(VI) removal. Our results indicate that Cr(VI) reduction by indigenous Cellulomonas spp. may be a potential method of in situ bioremediation of Cr(VI) contaminated sediment and groundwater.

Methanotrophic bacteria play an important role in global cycling of carbon and co-metabolism of contaminants. Methanotrophs from pristine regions of the Snake River Plain Aquifer (SRPA; Idaho, USA) were studied in order to gain insight into the native groundwater communities' genetic potential to carry out TCE co-metabolism. Wells were selected that were proximal to a TCE plume believed to be undergoing natural attenuation. Methane concentrations ranged from 1 to >1000 nM. Carbon isotope ratios and diversity data together suggest that the SRPA contains active communities of methanotrophs that oxidize microbially produced methane. Microorganisms removed from groundwater by filtration were used as inocula for enrichments or frozen immediately and DNA was subsequently extracted for molecular characterization. Primers that specifically target methanotroph 16S rRNA genes or genes that code for subunits of soluble or particulate methane monooxygenase, mmoX and pmoA, respectively, were used to characterize the indigenous methanotrophs via PCR, cloning, RFLP analysis, and sequencing. Type I methanotroph clones aligned with Methylomonas, Methylocaldum, and Methylobacter sequences and a distinct 16S rRNA phylogenetic lineage grouped near Methylobacter. The majority of clone sequences in type II methanotroph 16S rRNA, pmoA, and mmoX gene libraries grouped closely with sequences in the Methylocystis genus. A subset of the type II methanotroph clones from the aquifer had sequences that aligned most closely to Methylosinus trichosporium OB3b and Methylocystis spp., known TCE-co-metabolizing methanotrophs.

Little is known regarding plasmid fate within contaminated soils. Column studies were used to evaluate dissemination of plasmid pJP4 under unsaturated or saturated flow conditions in a 2,4-dichlorophenoxyacetic acid (2,4-D) contaminated soil. Columns were destructively sampled following 1 week of percolation to assess the vertical distribution of donors, transconjugants, and 2,4-D concentrations within the soil. In unsaturated soil, pJP4 was detected in both culturable donor and transconjugant cells within soil 10.5 cm from the inoculated end of the column. In saturated soil, no transconjugants were detected; however, donors were found throughout the entire length of the column (30.5 cm). These results suggest that donor transport, particularly in conjunction with plasmid transfer to indigenous recipients, allows for significant dispersal of introduced genes through contaminated soil.

Changes in microbial populations were evaluated following inoculation of contaminated soil with a 3-chlorobenzoate degrader. Madera sandy loam was amended with 0, 500, or 1,000 microg 3-chlorobenzoate g(-1) dry soil. Selected microcosms were inoculated with the degrader Comamonas testosteroni BR60. Culturable bacterial degraders were enumerated on minimal salts media containing 3-chlorobenzoate. Culturable heterotrophic bacteria were enumerated on R2A. Isolated degraders were grouped by enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction fingerprints and identified based on 16S ribosomal-DNA sequences. Bioaugmentation increased the rate of degradation at both levels of 3-chlorobenzoate. In both the 500 and 1,000 microg 3-chlorobenzoate g(-1) dry soil inoculated microcosms, degraders increased from the initial inoculum and decreased following degradation of 3-CB. Inoculation delayed the development of indigenous 3-chlorobenzoate degrading populations. It is unclear if inoculation altered the composition of indigenous degrader populations. In the uninoculated soil, degraders increased from undetectable levels to 6.6 x 10(7) colony-forming-units g(-1) dry soil in the 500 microg 3-chlorobenzoate g(-1) dry soil microcosms, but none were detected in the 1,000 microg 3-chlorobenzoate g(-1) dry soil microcosms. Degraders isolated from uninoculated soil were identified as one of two distinct Burkholderia species. In the uninoculated soil, numbers of culturable heterotrophic bacteria initially decreased following addition of 1,000 microg 3-chlorobenzoate g(-1) dry soil. Inoculation with C. testosteroni reduced this negative impact on culturable bacterial numbers. The results indicate that bioaugmentation may not only increase the rate of 3-chlorobenzoate degradation but also reduce the deleterious effects of 3-chlorbenzoate on indigenous soil microbial populations.