PNNL

Abstract

Gene probe hybridization was used to determine distribution and expression of co-metabolic genes at a contaminated site as it underwent in situ methanotrophic bioremediation of trichloroethylene (TCE). The bioremediation strategies tested consisted of a series of air, air:methane, and air:methane:nutrient pulses using a horizontal injection well. Sediment core samples (n=367) taken from 0 (surface)-43m depth were probed for genes coding for soluble methane monooxygenase (sMMO) and toluene dioxygenase (TOD), which are known to cometabolize TCE. The same samples were also probed for genes coding for methanol dehydrogenase (MDH) to access changes in methylotrophic bacterial populations. Hybridization results showed that the frequency of detection of sMMO genes were stimulated approximately 250% following 1% methane:air (v/v) injection. Subsequent 4% methane:air (v/v) injection resulted in an 85% decline probably due to nutrient limitations, since subsequent addition of nutrients (gaseous nitrogen and phosphorus) caused an increase in the frequency of detection of sMMO genes. Detection of TOD genes declined during the process becoming non-detectable by the final treatment. These patterns indicate methanotrophs displaced heterotrophs containing TOD genes. Active transcription of sMMO and TOD was evidenced by hybridization to mRNA. These analyses combined with studies showing the concomitant decline in TCE concentrations, increases in methanotroph viable counts, increased mineralization rates of TCE, and increases in chloride inventories provide multiple lines of evidence that TCE remediation was caused specifically by methanotrophs. This work suggests that sMMO genes are responsible for most, if not all, of the biodegradation of TCE observed. This study demonstrated that the use of nucleic acid analytical methods provided a gene specific assessment of the effects of in situ treatment technologies.