Biodegradation is an important mechanism for contaminant destruction at monitored natural attenuation sites. However, obtaining evidence of in situ biodegradation can be difficult for some contaminants if their metabolites are transient in the environment and/or little is known about the microorganisms and degradation pathways involved. Although many pathways for aerobic benzene biodegradation are well-documented, comparatively less is known about anaerobic benzene biodegradation. Early literature indicated that benzene was recalcitrant in anaerobic environments, but benzene biodegradation has since been demonstrated under iron- reducing, nitrate-reducing, sulfate-reducing, perchlorate-reducing, and methanogenic conditions. Hydroxylation, methylation, and carboxylation have been proposed as potential anaerobic pathways, but many of the genes involved have yet to be identified. Stable isotope probing (SIP) is a versatile molecular biological tool that can be used to provide conclusive proof of in situ biodegradation without requiring prior knowledge of the microorganisms or pathways involved.

The current study includes statistical analysis of SIP results from 300 field samples collected from benzene sites around the world, including the United States, Australia, Canada, China, Saudi Arabia, and the United Kingdom. The samples included in the current analysis were Bio-Traps amended with a specially synthesized form of benzene containing carbon-13 (13C). Since 13C is rare, carbon originating from labeled contaminant can be readily
distinguished from carbon from other sources (predominantly carbon-12). Following in-well deployment, the Bio-Traps were analyzed for 13C enrichment
in dissolved inorganic carbon (DIC) and microbial phospholipid fatty acids (PLFA). 13C incorporation into DIC conclusively demonstrates benzene
mineralization during the deployment period. PLFA are a main component of cell membranes, and 13C-enriched PLFA indicate that benzene was
metabolized and incorporated into microbial biomass under current field conditions. Furthermore, 13C incorporation into specific fatty acids associated
with anaerobic microbial groups indicates that anaerobes were actively involved in the degradation of the 13C-labeled benzene or one of its metabolites.

Primary Author / Conference Presenter:
Kerry Sublette, PhD
University of Tulsa
Tulsa, Oklahoma, USA

Dora Taggart, Microbial Insights, Inc., Knoxville, TX
Kate Clark, Microbial Insights, Inc., Knoxville, TN