In Situ Subsurface Cometabolic Transformation of Chlorinated Solvent Mixtures by Native and Bioaugmented Butane Utilizing Microorganisms

An aquifer test zone at Moffett Federal Airfield, CA, was used to assess the cometabolic transformation of chlorinated solvent mixtures by native and bioaugmented butane-utilizing microbes. Two parallel, but hydraulically separate, well legs were controlled by separate injection/ extraction systems and were fed pulses of butane and oxygen and were continually fed chlorinated aliphatic hydrocarbons \(CAHs\). Groundwater monitoring wells were located 1.0, 2.2, and 4.0 m from the respective injection wells and were semi-continuously analyzed for butane, CAHs, pH, anions, and dissolved oxygen. One well leg was bioaugmented with approximately 5 g dry weight of a butane-utilizing enrichment culture known to cometabolically transform 1,1-dichloroethene \(DCE\), 1,1-dichloroethane \(DCA\), and 1,1,1-trichloroethane \(TCA\). Periodically, samples were taken from the groundwater monitoring wells, or from two fully-penetrating wells placed 0.5 m and 1.5 m from the injection wells, for molecular microbial analyses including terminal restriction fragment length polymorphism \(T-RFLP\), real time quantitative PCR. Bioaugmentation stimulated butane uptake and CAH transformation with the greatest removal efficiencies observed within the first week, followed by declining CAH treatment efficiency over a period of days to months. DCE was at least partially transformed in both the indigenous and bioaugmented well legs. An injection concentration of 175 mg/L DCE was readily transformed in the bioaugmented well leg with a removal efficiency of greater than 90% while only 25% removal occurred in the indigenous well leg. Neither DCA nor TCA was transformed in the indigenous well leg, while both DCA and TCA transformation were obtained in the bioaugmented well leg. However, less than 80% TCA transformation efficiency was obtained and it was difficult to maintain TCA transformation over time. A clear and repeatable microbial community transition from dominance of organisms with a T-RFL of 277 bp, when restricted with MnlI, to an organisms with a T-RFL of 126 bp occurred upon bioaugmentation and stimulation of the aquifer test zone with butane and oxygen. Microbial community structure varied between the two parallel well legs while reasonably similar communities were observed along the flow path of the bioaugmented well leg. A Rhodococcus sp. present in the bioaugmentation culture was quantified in samples taken from the field using real time PCR. The organism was successfully transported at least 2 m through the subsurface, but was found to be present in insufficient quantity relative to the total microbial community to appear in T-RFLP analyses.