Molecular Analyses of the Methane-Oxidizing Microbial Community in Rice Field Soil by Targeting the Genes of the 16S rRNA, Particulate Methane Monooxygenase, and Methanol Dehydrogenase

Abstract

ABSTRACT Rice field soil with a nonsaturated water content induced CH 4 consumption activity when it was supplemented with 5% CH 4 . After a lag phase of 3 days, CH 4 was consumed rapidly until the concentration was less than 1.8 parts per million by volume (ppmv). However, the soil was not able to maintain the oxidation activity at near-atmospheric CH 4 mixing ratios (i.e., 5 ppmv). The soil microbial community was monitored by performing denaturing gradient gel electrophoresis (DGGE) during the oxidation process with different PCR primer sets based on the 16S rRNA gene and on functional genes. A universal small-subunit (SSU) ribosomal DNA (rDNA) primer set and 16S rDNA primer sets specifically targeting type I methylotrophs (members of the γ subdivision of the class Proteobacteria [γ- Proteobacteria ]) and type II methylotrophs (members of the α- Proteobacteria ) were used. Functional PCR primers targeted the genes for particulate methane monooxygenase ( pmoA ) and methanol dehydrogenase ( mxaF ), which code for key enzymes in the catabolism of all methanotrophs. The yield of PCR products amplified from DNA in soil that oxidized CH 4 was the same as the yield of PCR products amplified from control soil when the universal SSU rDNA primer set was used but was significantly greater when primer sets specific for methanotrophs were used. The DGGE patterns and the sequences of major DGGE bands obtained with the universal SSU rDNA primer set showed that the community structure was dominated by nonmethanotrophic populations related to the genera Flavobacterium and Bacillus and was not influenced by CH 4 . The structure of the methylotroph community as determined with the specific primer sets was less complex; this community consisted of both type I and type II methanotrophs related to the genera Methylobacter , Methylococcus , and Methylocystis . DGGE profiles of PCR products amplified with functional gene primer sets that targeted the mxaF and pmoA genes revealed that there were pronounced community shifts when CH 4 oxidation began. High CH 4 concentrations stimulated both type I and II methanotrophs in rice field soil with a nonsaturated water content, as determined with both ribosomal and functional gene markers.