REMEDIATION Summer 2013 Concurrent and Complete Anaerobic Reduction and Microaerophilic Degradation of Mono-, Di-, and Trichlorobenzenes Matt Burns Kerry L. Sublette James Sobieraj Dora Ogles Stephen Koenigsberg Bio-Trap ® –based in situ microcosm studies were conducted to evaluate EHC-M ® stimulated degra- dation of mono-, di-, and trichlorobenzenes in anaerobic groundwater at a site in Michigan. The data show that the EHC-M ® amendment stimulated an overall increase in microbial activity and a shift in the microbial community structure, indicating more reduced conditions. Stable isotope probing with 13 C 6 -chlorobenzene demonstrated attenuation of chlorobenzene and subsequent separation and characterization of the 12 C- and 13 C-deoxyribonucleic acid (DNA) fractions were used to identify the attenuating microbes. These data clearly show the participation of an obligate aerobe in the chlorobenzene biodegradation process. Decreases in concentrations of trichlorobenzenes were also observed in comparison to a con- trol. Due to the thermodynamically favorable reducing conditions stimulated by EHC-M ® , the mechanism of degradation of the trichlorobenzenes is presumed to be reductive dehalogenation. However, on the strength of the DNA-based analysis of microbial community structure, concurrent microaerophilic degradation of chlorobenzene or its metabolites was defnitively demonstrated and cannot be ruled out for the other chlorobenzenes. O c 2013 Wiley Periodicals, Inc. INTRODUCTION Mono-, di-, and trichlorobenzenes are derivatives of benzene with one, two, or three chlorine atoms substituted for hydrogen, respectively, which have a wide range of industrial, agricultural, and commercial applications. Accordingly, sites with chlorinated benzene-afected soil and groundwater are diverse and numerous with releases from equipment containing oils, lubricants, and dielectric fuid being among the most frequent encountered. The US Environmental Protection Agency (US EPA) primary drinking water standards for chlorobenzenes vary by degree of chlorine substitution and confguration. Among the lower-molecular-weight chlorobenzenes, the drinking water maximum contaminant levels (MCLs) vary, with the 1,2,4-trichlorobenzene (70 micrograms/liter [μg/L]),1,4-dichlorobenzene (75 μg/L), and chlorobenzene (100 μg/L) having the lowest criteria for each level of chloro-substitution. In addition to human health concerns, chlorobenzenes have been shown to bio-accumulate (Malcolm et al., 2004). © 2013 Wiley Periodicals, Inc. Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/rem.21356 37