Joong-Jae Kim1, Roshan Khadka1, Patrick Hettiaratchi2, and Peter F. Dunfield1.
1Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
2Department of Civil Engineering, Center for Environmental Engineering Research and Education, University of Calgary, Calgary, Alberta, Canada.
A DNA-based detection and quantification protocol for methanotrophic bacteria was applied to develop a simple, efficient, and cost-effective strategy for monitoring the methane oxidation efficiency of a field methane biofilter. For the first step to develop a strategy for monitoring the methane oxidation efficiency of a field methane biofilter, molecular protocols for extraction and analyses of DNA from the biofilter system were optimized. The polymerase chain reaction (PCR) was used to amplify genes universal to all bacteria (16S rRNA) and a gene specific to methanotrophic bacteria (pmoA, encoding a subunit of the enzyme encoding methane monooxygenase). To estimate of the size of the methanotrophic population present in the sample as a possible index of biofilter activity, quantitative polymerase chain reaction (qPCR) was then used to quantify the number of target pmoA genes in different samples1. Results showed a trend of increasing methanotrophic populations during growth of methanotrophic bacteria in compost in model laboratory microcosms and mesocosms, and a correlation of pmoA counts to potential methane oxidation rates. A high throughput next-generation sequencing analysis was made of the microbial community in biofilter compost packing material based on 16S rRNA and pmoA genes. The methanotrophic community in the compost was composed of few species, e. g., Methylocystis sp., Methylosinus sp., Methylocaldum sp., and Methylobacter sp.. This indicated that inoculation of biofilters with other methanotrophic bacteria, such as psychrophilic methanotrophs, may be an effective method for improving biofilter performance in the field. Therefore, methanotrophic microbial consortia were enriched and identified for potential application to biofilter material to improve performance.
1. Kolb S, Knief C, Stubner S, Conrad R, (2003) Quantitative detection of methanotrophs in soil by novel pmoA-targeted real-time PCR assays, Appl Environ Microbiol. 69(5): 2423-9.