MOLECULAR CHARACTERIZATION OF METHANOTROPHS IN MIDDENDORF AQUIFER, SOUTH CAROLINA

Methanotrophs, a group of bacteria that can obtain energy by methane oxidation, play an important role in the global carbon cycle. They can also cometabolize halogenated hydrocarbons, and so have been used for bioremediation. Methanotrophs have been found in various environments, including pristine and contaminated groundwater. We report here a culture-independent study of methanotrophic diversity in the Middendorf aquifer, South Carolina.

We sampled groundwater from two municipal wells for chemical and microbiological analysis. Measured methane and dioxygen concentrations are in the ranges associated with methane oxidation. We characterized microbial diversity at each location by amplifying and sequencing Eubacteria- and Archaea-specific 16S rDNA genes. Most Eubacteria clones belong to a-, b-, d-, e-Proteobacteria, or Actinobacteria. A number of the a- and b-Proteobacteria clones closely matched known methanotrophs, such as Methylocystis.

To verify the presence of methanotrophs in our samples, we amplified and sequenced functional genes of particulate methane monooxygenase (pmoA) and methanol dehydrogenase (mxaF), key enzymes in methane oxidation. To compare the relative abundance of methanotrophs and other bacteria, we digested the Eubacteria 16S rDNA PCR products with restriction enzymes, and separated the resulting digestion fragments on polyacrylamide gels. Sequences of the dominant bands cut from the gels match methanotrophic clones from the samples, indicating the importance in the aquifer of methanotrophs. We also verified the dominance of methanotrophs by quantitative PCR, using sequence-specific primers.