Trichloroethylene degradation by a type I methanotroph, Methylomonas Methanica 68-1, and molecular analysis of the population dynamics in the two methanotrophic species competition

The goal of this study was to determine whether type I and II methanotroph specific DNA probes could specifically detect and monitor the relevant populations in a methanotrophic mixed culture that degrades TOE. A model mixed culture was composed of a type I methanotroph, strain 68-1, isolated from a TCE-contaminated well and an extensively characterized type II methanotroph, Methylosinus trichosporium OB3b. The strain 68-1 was identified as Methylomonas methanica by pattern of intracytoplasmic membrane ultrastructure, 168 rRNA signature probe hybridizations, and PLFA and LPS-OHFA profiles. 68-1 could produce sMMO under copper-limiting condition, and oxidize naphthalene and degrade TOE more rapidly than M. trichosporium OB3b at room temperature despite its higher K_m values for these substrates. There was little genetic homology between the sMMO genes of 68-1 and OB3b, indicating the diversity of sMMO genes in methanotrophs. However, substrate specificity indicated a potential functional similarity between of the sMMOs of the type I and II methanotrophs. The putative sMMO gene fragment (4 kb) of 68-1 was cloned and partially identified by Southern and slot blots, and DNA sequencing. The DNA fragment cross-hybridized to the genomes of a few pink-pigment type I methanotrophs (not producing sMMO) and a type X methanotroph (producing sMMO), indicating the conservation of the putative gene at least within the RuMP pathway methanotrophs. The putative sMMO DNA probe was successfully used to monitor the 68-1 population in the mixed culture containing OB3b. The monitoring data were supported by plate count data and hybridization results using 16S rDNA-targeting oligodeoxynucleotide probes while AODC data generally complemented the sMMO gene probe results with certain levels of error. The 68-1 population was out-competed by OB3b population in both flask and continuous cultures under copper-limiting condition. The 68-1 population was also out-competed by OB3b under lower concentration of methane while higher methane concentration favored 68-1. Moreover, the OB3b population was dominant over 68-1 regardless of copper and nitrate concentrations. The putative sMMO gene probe was successfully used to detect the potential homologous methanotrophic genes and predict the population density in TCE-contaminated subsurface environments. Therefore, the putative sMMO gene from 68-1, if confirmed, could be used to monitor the populations of the potential type I sMMO producers growing or present in the various environments.