Carbonate Mineral Sustained Methanogenesis Under Alkaline Conditions

Abstract:

A majority of the carbon on Earth (ca. 80%) is sequestered in the form of sedimentary carbonates. Under alkaline conditions, carbonate minerals are considered metastable and therefore inaccessible for microbial metabolism. Here we demonstrate the use of calcium carbonate as a CO₂ source for methanogenesis at alkaline pH. An anaerobic enrichment culture was initiated from alkaline saline wetland soil in anoxic minimal medium (pH 8.3) and continuously maintained with hydrogen as an electron donor and calcium carbonate as the sole source of inorganic carbon. In subsequent experiments, the enriched microbial consortium was cultivated in minimal medium with 10 mmol L^-1 of calcium carbonate and 7.4 mmol L^-1 of headspace hydrogen and incubated at 30ºC for 45 days. Parallel reactors, either inoculated with autoclaved cultures or uninoculated, served as negative controls. Over the course of the incubation headspace methane increased with a simultaneous decrease in hydrogen. An increase in dissolved calcium was measured in reactors inoculated with live cultures relative to negative controls. No significant change in pH was observed over the experimental period indicating microbially-catalyzed calcite dissolution. Shotgun metagenomic sequencing and genome assembly of the enrichment culture confirmed the presence of a methanogen (Methanobacterium sp.), alongside several other community members including an Acetoanaerobium sp., Pseudomonas sp., Desulfovibrio sp., and Tessaracoccus sp. Genomic data also confirm the Methanobacterium sp. has a complete pathway for hydrogenotrophic methanogenesis. Together, these results indicate that microbially-catalyzed carbonate mineral dissolution can support methane generation at alkaline pH.