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

Abstract

AbstractSeveral 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 CH4oxidation 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% O2and 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 CH4concentrations 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 CH4oxidation 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 theProteobacteria(49.9±1.5%), and to a lesser extent byBacteroidetes(8.8±0.2%),Acidobacteria(8.9±0.4%), andVerrucomicrobia(4.4±0.3%). In enrichments with or without 2C6MP,Proteobacteriaexpanded to comprise 65–70% of the total. In the absence of inhibitor, members of theMethylococcaceaeandMethylophilaceaeincreased 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, theMethylococcaceaewere much less abundant, representing 3.3±0.5% and 2.8±3.3% respectively. No inhibition of theMethylophilaceaewas 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 theCrenotrichaceae, 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.