Using DNA-Stable Isotope Probing to Identify MTBE- and TBA-Degrading Microorganisms in Contaminated Groundwater.

Although the anaerobic biodegradation of methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA) has been documented in the laboratory and the field, knowledge of the microorganisms and mechanisms involved is still lacking. In this study, DNA-stable isotope probing (SIP) was used to identify microorganisms involved in anaerobic fuel oxygenate biodegradation in a sulfate-reducing MTBE and TBA plume. Microorganisms were collected in the field using Bio-Sep® beads amended with 13C5-MTBE, 13C1-MTBE (only methoxy carbon labeled), or13C4-TBA. 13C-DNA and 12C-DNA extracted from the Bio-Sep beads were cloned and 16S rRNA gene sequences were used to identify the indigenous microorganisms involved in degrading the methoxy group of MTBE and the tert-butyl group of MTBE and TBA. Results indicated that microorganisms were actively degrading 13C-labeled MTBE and TBA in situ and the 13C was incorporated into their DNA. Several sequences related to known MTBE- and TBA-degraders in the Burkholderiales and the Sphingomonadales orders were detected in all three13C clone libraries and were likely to be primary degraders at the site. Sequences related to sulfate-reducing bacteria and iron-reducers, such as Geobacter and Geothrix, were only detected in the clone libraries where MTBE and TBA were fully labeled with 13C, suggesting that they were involved in processing carbon from the tert-butyl group. Sequences similar to the Pseudomonas genus predominated in the clone library where only the methoxy carbon of MTBE was labeled with 13C. It is likely that members of this genus were secondary degraders cross-feeding on 13C-labeled metabolites such as acetate.

An ex situ evaluation of TBA- and MTBE-baited bio-traps.

Aquifer microbial communities can be investigated using Bio-traps(®) ("bio-traps"), passive samplers containing Bio-Sep(®) beads ("bio-beads") that are deployed in monitoring wells to be colonized by bacteria delivered via groundwater flow through the well. When bio-beads are "baited" with organic contaminants enriched in (13)C, stable isotope probing allows assessment of the composition and activity of the microbial community. This study used an ex situ system fed by groundwater continuously extracted from an adjacent monitoring well within an experimentally-created aerobic zone treating a tert-butyl alcohol (TBA) plume. The goal was to evaluate aspects of bio-trap performance that cannot be studied quantitatively in situ. The measured groundwater flow through a bio-trap housing suggests that such traps might typically "sample" about 1.8 L per month. The desorption of TBA or methyl tert-butyl ether (MTBE) bait from bio-traps during a typical deployment duration of 6 weeks was approximately 90% and 45%, respectively, of the total initial bait load, with initially high rate of mass loss that decreased markedly after a few days. The concentration of TBA in groundwater flowing by the TBA-baited bio-beads was estimated to be as high as 3400 mg/L during the first few days, which would be expected to inhibit growth of TBA-degrading microbes. Initial inhibition was also implied for the MTBE-baited bio-trap, but at lower concentrations and for a shorter time. After a few days, concentrations in groundwater flowing through the bio-traps dropped below inhibitory concentrations but remained 4-5 orders of magnitude higher than TBA or MTBE concentrations within the aquifer at the experimental site. Desorption from the bio-beads during ex situ deployment occurred at first as predicted by prior sorption analyses of bio-beads but with apparent hysteresis thereafter, possibly due to mass transfer limitations caused by colonizing microbes. These results suggest that TBA- or MTBE-baited bio-traps could be baited at lower initial total mass loading with no detriment to trapping ability. The bio-traps were able to collect detectable amounts of microbial DNA and thus allow some insight into the sparse microbial community present in the aquifer during remediation of the low concentration plume.

Prevalence of lysogeny among soil bacteria and presence of 16S rRNA and trzN genes in viral-community DNA.

Bacteriophages are very abundant in the biosphere, and viral infection is believed to affect the activity and genetic diversity of bacterial communities in aquatic environments. Lysogenic conversion, for example, can improve host fitness and lead to phage-mediated horizontal gene transfer. However, little is known about lysogeny and transduction in the soil environment. In this study we employed atrazine-impregnated Bio-Sep beads (a cell immobilization matrix) to sample active microbiota from soils with prior pesticide exposure history. Once recovered from soil, the bead communities were induced with mitomycin C (MC), and viral and bacterial abundances were determined to evaluate the incidence of inducible prophage in soil bacteria. The inducible fraction calculated within bead communities was high (ca. 85%) relative to other studies in aquatic and sedimentary environments. Moreover, the bacterial genes encoding 16S rRNA and trzN, a chlorohydrolase gene responsible for dehalogenation of atrazine, were detected by PCR in the viral DNA fraction purified from MC-induced bead communities. A diverse collection of actinobacterial 16S rRNA gene sequences occurred within the viral DNA fraction of induced, water-equilibrated beads. Similar results were observed in induced atrazine-equilibrated beads, where 77% of the cloned sequences were derived from actinobacterial lineages. Heterogeneous 16S rRNA gene sequences consisting of fragments from two different taxa were detected in the clone libraries. The results suggest that lysogeny is a prevalent reproductive strategy among soil bacteriophages and that the potential for horizontal gene transfer via transduction is significant in soil microbial communities.