Doctoral Dissertations

Date of Award

5-1994

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Ecology and Evolutionary Biology

Major Professor

Gary S. Sayler

Committee Members

Gary Stacey, Barbara T. Walton, David C. White

Abstract

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 Km 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.

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