Degradation of methyl bromide and methyl chloride in soil microcosms: Use of stable C isotope fractionation and stable isotope probing to identify reactions and the responsible microorganisms

Bacteria in soil microcosm experiments oxidized elevated levels of methyl chloride (MeCl) and methyl bromide (MeBr), the former compound more rapidly than the latter. MeBr was also removed by chemical reactions while MeCl was not. Chemical degradation dominated the early removal of MeBr and accounted for more than half of its total loss. Fractionation of stable carbon isotopes during chemical degradation of MeBr resulted in a kinetic isotope effect (KIE) of 59 ± 7‰. Soil bacterial oxidation dominated the later removal of MeBr and MeCl and was characterized by different KIEs for each compound. The KIE for MeBr oxidation was 69 ± 9‰ and the KIE for MeCl oxidation was 49 ± 3‰. Stable isotope probing revealed that different populations of soil bacteria assimilated added ¹³C-labeled MeBr and MeCl. The identity of the active MeBr and MeCl degrading bacteria in soil was determined by analysis of 16S rRNA gene sequences amplified from ¹³C-DNA fractions, which identified a number of sequences from organisms not previously thought to be involved in methyl halide degradation. These included Burkholderia, the major clone type in the ¹³C-MeBr fraction, and Rhodobacter, Lysobacter and Nocardioides the major clone types in the ¹³C-MeCl fraction. None of the 16S rRNA gene sequences for methyl halide oxidizing bacteria currently in culture (including Aminobacter strain IMB-1 isolated from fumigated soil) were identified. Functional gene clone types closely related to Aminobacter spp. were identified in libraries containing the sequences for the cmuA gene, which codes for the enzyme known to catalyze the initial step in the oxidation of MeBr and MeCl. The cmuA gene was limited to members of the alpha-Proteobacteria whereas the greater diversity demonstrated by the 16S rRNA gene may indicate that other enzymes catalyze methyl halide oxidation in different groups of bacteria.