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.

The effect of phenol concentration on phenol biodegradation at an industrial site in the south of Wales, United Kingdom, was investigated using standard Bio-Sep ® Bio-Traps ® and Bio-Traps ® coupled with stable isotope probing (SIP). Unlike many 13 C-amendments used in SIP studies (such as hydrocarbons) that physically and reversibly adsorb to the activated carbon component of the Bio-Sep ® beads, phenol is known to irreversibly chemisorb to activated carbon. Bio-Traps ® were deployed for 32 days in nine site groundwater monitoring wells representing a wide range of phenol concentrations. Bio-Traps ® amended with 13 C-phenol were deployed together with non-amended Bio-Traps ® in three wells. Quantitative polymerase chain reaction (qPCR) analysis of Bio-Traps ® post-deployment indicated an inhibitory effect of increasing phenol concentration on both total eubacteria and aerobic phenol-utilizing bacteria as represented by the concentration of phenol hydroxylase gene. Despite the chemisorption of phenol to the Bio-Sep ® beads, activated carbon stable isotope analysis showed incorporation of 13 C into biomass and dissolved inorganic carbon (DIC) in each SIP Bio-Trap ® indicating that chemisorbed amendments are bioavailable. However, there was a clear effect of phenol concentration on 13 C incorporation in both biomass and DIC confirming phenol inhibition. These results suggest that physical reductions of the phenol concentrations in some areas of the plume will be required before biodegradation of phenol can proceed at a reasonable rate. O

A microbial survey of hydrocarbon‐impacted groundwater and vadose zone at a Midwestern refinery employed molecular biological tools to elucidate the microbial processes involved in bioremediation occurring in the subsurface. qPCR analysis of bio‐traps incubated in groundwater indicated that a large and diverse microbial community was present throughout the site and suggested that mechanisms of benzene, toluene, ethylbenzene, and xylene (BTEX) biodegradation included aerobic oxidation, sulfate reduction, methanogenesis, and possibly Fe+3 reduction. To assess the role of vadose zone microorganisms in hydrocarbon attenuation, RNA was extracted from soil core samples, and reverse transcriptase‐qPCR (RT‐qPCR) analysis indicated that microbial activity in the vadose zone generally increased with depth, likely supported by hydrocarbons and methane volatilizing from the groundwater. Stable isotope probing (SIP) with 13C6‐benzene provided direct evidence of benzene biodegradation in six of the eight wells studied. The highest levels of 13C were detected in dissolved inorganic carbon (DIC) extracted from the two monitoring wells closest to the river. The influx of nutrients and oxygen coming from the river may help to maintain a robust population of hydrocarbon degraders in these wells. While qPCR analysis indicated that microorganisms with the genetic potential for hydrocarbon biodegradation were ubiquitous at the site, RT‐qPCR and SIP results were used to refine the site conceptual model by identifying areas where that genetic potential was actively being expressed and locations where biodegradation was lagging.

Two pilot tests of an aerobic in situ bioreactor (ISBR) have been conducted at field sites contaminated with petroleum hydrocarbons. The two sites differed with respect to hydrocarbon concentrations. At one site, concentrations were low but persistent, and at the other site concentrations were high enough to be inhibitory to biodegradation. The ISBR unit is designed to enhance biodegradation of hydrocarbons by stimulating indigenous microorganisms. This approach builds on existing Bio-Sep ® bead technology, which provides a matrix that can be rapidly colonized by the active members of the microbial community and serves to concentrate indigenous degraders.Oxygen and nutrients are delivered to the bioreactor to maintain conditions favorable for growth and reproduction, and contaminated groundwater is treated as it is circulated through the bed of Bio-Sep ® beads. Groundwater moving through the system also transports degraders released from Bio-Sep ® beads away from the bioreactor, potentially increasing biodegradation rates throughout the aquifer.Groundwater sampling, Bio-Traps, and molecular biological tools were used to assess ISBR performance during the two pilot tests. Groundwater monitoring indicated that contaminant concentrations decreased at both sites, and the microbial data suggested that these decreases were due to degradation by indigenous microorganisms rather than dilution or dispersion mechanisms.Taken together, these lines of evidence showed that the ISBR system effectively increased the number and activity of indigenous microbial degraders and enhanced bioremediation at the test sites. O