Experimental, Microbiome, and Geochemical Evidence for Methane Oxidation by Archaeal Ammonia Oxidizers in Hot Springs of Yellowstone National Park

Abstract:

Continental hot spring ecosystems provide high temperature extremes for aerobic metabolisms. We combined sequencing, microcosm, and geochemical/thermodynamic approaches to explore biological methane oxidation in sediments of hot springs in Yellowstone. Thermodynamic calculations from the aqueous compositions of hot spring fluids show that methane oxidation coupled to reduction of oxygen, nitrite, nitrate and sulfate are energy yielding. 16S rRNA gene amplicon sequencing of sediment samples from 66 hot springs yielded no sequences affiliated with known anaerobic methanotrophs. However, at 14 of the sites, sequences affiliated with known aerobic methanotrophs were detected. Microcosm activity assays were conducted at 17 of the 66 sequencing sites to quantify conversion of ¹⁴CH₄ to ¹⁴CO₂ and assimilation into biomass. These sites span pH (~2 to ~9) and temperature (~45 to ~90°C) and were conducted in springs where methanotrophs were and were not detected. Significant methane oxidation was observed at 14 of the 17 experimental sites. The highest rate of methane oxidation, 107 nmol C gdws^-1 day^-1, was measured in a spring (pH 4.0 and 53°C) where only Verrucomicrobia methanotroph sequences were detected.

Putative methanotroph sequences were detected at only 4 of the 14 sites where significant methane oxidation was measured, which suggests there may be alternate mechanisms for methane oxidation. One possible mechanism is through aerobic ammonia/ammonium oxidizers, which have been shown to be capable of carrying out methane oxidation in other environments. Energy calculations for ammonium oxidation show that it can yield up to ~4 orders of magnitude more energy than aerobic methane oxidation. Additionally, Yellowstone hot springs are enriched in ammonia/ammonium compared with methane. Putative archaeal ammonia oxidizer sequences were detected at 4 of the experimental sites where significant methane oxidation rates were measured. The highest methane oxidation rate associated with one of these four sites (pH 8.3 and 68°C) was 2.7 nmol C gdws^-1 day^-1. There are 8 experimental sites where significant methane oxidation was measured and the mechanisms for which remain to be explained. At these sites it is possible that there are uncharacterized microbial lineages capable of carrying out methane oxidation.