Acetogens and acetoclastic methanosarcinales govern methane formation in abandoned coal mines

Abstract

In abandoned coal mines, methanogenic archaea are responsible for the production of substantial amounts of methane. The present study aimed to directly unravel the active methanogens mediating methane release as well as active bacteria potentially involved in the trophic network. Therefore, the stable-isotope-labeled precursors of methane, [(13)C]acetate and H(2)-(13)CO(2), were fed to liquid cultures from hard coal and mine timber from a coal mine in Germany. Guided by methane production rates, samples for DNA stable-isotope probing (SIP) with subsequent quantitative PCR and denaturing gradient gel electrophoretic (DGGE) analyses were taken over 6 months. Surprisingly, the formation of [(13)C]methane was linked to acetoclastic methanogenesis in both the [(13)C]acetate- and the H(2)-(13)CO(2)-amended cultures of coal and timber. H(2)-(13)CO(2) was used mainly by acetogens related to Pelobacter acetylenicus and Clostridium species. Active methanogens, closely affiliated with Methanosarcina barkeri, utilized the readily available acetate rather than the thermodynamically more favorable hydrogen. Thus, the methanogenic microbial community appears to be highly adapted to the low-H(2) conditions found in coal mines.

Fig. 1.

Long-term incubation of weathered hard coal and mine timber amended with ¹³C-labeled acetate or H₂-CO₂. Shown are microbial methane formation (A) and acetate (B) and hydrogen (C) depletion/formation. Measurements were carried out in 5 replicates.

Fig. 2.

Quantitative PCR distribution and DGGE community profiles of density-resolved bacterial and archaeal DNA in SIP centrifugation gradients after 3 and 6 months of incubation of hard coal with [¹³C]acetate (A) or H₂-¹³CO₂ (B). Each letter on the right of a DGGE profile indicates the same band throughout the gradient. Band letters correspond to those in Table 1. The same band letter indicates the same organism.

Fig. 3.

Quantitative PCR distribution and DGGE community profiles of density-resolved bacterial and archaeal DNA in SIP centrifugation gradients after 3 and 6 months of incubation of mine timber with [¹³C]acetate (A) or H₂-¹³CO₂ (B). Each letter on the right of a DGGE profile indicates the same band throughout the gradient. Band letters correspond to those in Table 2. The same band letter indicates the same organism.