Proteomic and targeted qPCR analyses of subsurface microbial communities for presence of methane monooxygenase
The Test Area North (TAN) site at the Idaho National Laboratory near Idaho Falls, ID, USA, sits over a trichloroethylene (TCE) contaminant plume in the Snake River Plain fractured basalt aquifer. Past observations have provided evidence that TCE at TAN is being transformed by biological natural attenuation that may be primarily due to co-metabolism in aerobic portions of the plume by methanotrophs. TCE co-metabolism by methanotrophs is the result of the broad substrate specificity of microbial methane monooxygenase which permits non-specific oxidation of TCE in addition to the primary substrate, methane. Arrays of experimental approaches have been utilized to understand the biogeochemical processes driving intrinsic TCE co-metabolism at TAN. In this study, aerobic methanotrophs were enumerated by qPCR using primers targeting conserved regions of the genes pmoA and mmoX encoding subunits of the particulate MMO (pMMO) and soluble MMO (sMMO) enzymes, respectively, as well as the gene mxa encoding the downstream enzyme methanol dehydrogenase. Identification of proteins in planktonic and biofilm samples from TAN was determined using reverse phase ultra-performance liquid chromatography (UPLC) coupled with a quadrupole-time-of-flight (QToF) mass spectrometer to separate and sequence peptides from trypsin digests of the protein extracts. Detection of MMO in unenriched water samples from TAN provides direct evidence of intrinsic methane oxidation and TCE co-metabolic potential of the indigenous microbial population. Mass spectrometry is also well suited for distinguishing which form of MMO is expressed in situ either soluble or particulate. Using this method, pMMO proteins were found to be abundant in samples collected from wells within and adjacent to the TCE plume at TAN.
KeywordsProteomics Methanotrophs Co-metabolism Methane monooxygenase Trichloroethylene
This research was funded by the Office of Science, Office of Biological and Environmental Research, Environmental Remediation Sciences Division, of the U.S. Department of Energy under Contract Numbers DE-FG02-06ER64198 (to the University of Idaho), DE-AC02-05CH11231 (to Lawrence Berkeley National Laboratory), and DEAC07-05ID14517 (to the Idaho National Laboratory).
- Atlas RM (1993) Handbook of microbiological media. CRC Press, Boca RatonGoogle Scholar
- Lee SW, Im J, DiSpirito AA, Bodrossy L, Barcelona MJ, Semrau JD (2009) Effect of nutrient and selective inhibitor amendments on methane oxidation, nitrous oxide production, and key gene presence and expression in landfill cover soils: characterization of the role of methanotrophs, nitrifiers, and denitrifiers. Appl Microbiol Biotechnol 85:389–403PubMedCrossRefGoogle Scholar
- Richard D, Parke R, John HP, Katharine GF, Jorge WSD (2006) Development and applications of microbial ecogenomic indicators for monitoring water quality: report of a workshop assessing the state of the science, research needs and future directions. Environ Monit Assess 116:459–479CrossRefGoogle Scholar
- Sorenson K (2008) Coupling of realistic rate estimates with genomics for assessing contaminant attenuation and long-term plume containment. doi: 10.2172/836439. URL:http://www.osti.gov/energycitations/purl.cover.jsp?purl=/836439-DI67Yx/native/