Inhibition of Microbial Methane Oxidation by 2-Chloro-6-Methylpyridine

Several pyridine derivatives including the pesticide nitrapyrin [2-chloro-6-(trichloromethyl) pyridine] are strong inhibitors of methane monooxygenase, a key enzyme of aerobic methane (CH4) oxidation. In this study we examined the effects of 2-chloro-6-methylpyridine (2C6MP) concentration on aerobic CH4 oxidation and the development of populations of putative methanotrophs in sediment from Old Woman Creek, a freshwater estuary in Huron Co., Ohio. Experimental systems were prepared in serum bottles containing minimal medium with a headspace containing 20% O2 and 10% CH4. The microcosms were spiked with 2C6MP to achieve concentrations of 0, 0.1, 1, or 10 mM and inoculated with sediment. When headspace CH4 concentrations decreased from 10% to < 2%, subsamples were taken for DNA extraction and sequencing of 16S rRNA gene amplicons. There was minimal effect of 2C6MP on CH4 oxidation at concentrations of 0.1, and 1 mM, but complete inhibition for > 20 months was observed at 10 mM. ANOSIM of weighted UniFrac distances between groups of triplicate samples supported a primary distinction of the inoculum relative to the enrichments (R=0.999) and a secondary distinction between bottles containing 2C6MP versus those without (R=0.464 [0.1 mM]; R=0.894 [1 mM]). The inoculum was dominated by members of the Proteobacteria (49.9{+/-}1.5%), and to a lesser extent by Bacteroidetes (8.8{+/-}0.2%), Acidobacteria (8.9{+/-}0.4%), and Verrucomicrobia (4.4{+/-}0.3%). In enrichments with or without 2C6MP, Proteobacteria expanded to comprise 65-70% of the total. In the absence of inhibitor, members of the Methylococcaceae and Methylophilaceae increased in relative abundance from < 0.1% of the inoculum to 8.5{+/-}1.0% and 13.4{+/-}2.3%, of the total community respectively. At both 0.1 and 1 mM concentrations of the inhibitor, the Methylococcaceae were much less abundant, representing 3.3{+/-}0.5% and 2.8{+/-}3.3% respectively. No inhibition of the Methylophilaceae was seen at the lower concentration of 2C6MP, but at the higher concentration this taxon was only 7.8{+/-}1.1% of the total. In contrast, members of the Crenotrichaceae, another group of methane oxidizers, increased in relative abundance with greater amounts of inhibitor, representing 8.6{+/-}3.6% of the total at 0.1 mM and 12.0{+/-}4.5% at 1 mM, compared to only 4.1{+/-}0.4% when no inhibitor was present. These results clearly show changes in the populations of putative aerobic methanotrophs relative to the amount of 2C6MP present.