Mechanisms of Microbial Methane Production from Sub-Bituminous Australian Coal

Abstract

Biogenic methane production from coal necessarily involves a community of microorganisms acting in concert. The large, lignin-like molecules in coal cannot be metabolised by methanogens and other microorganisms are required to catalyse the breakdown of coal to acetate, hydrogen and carbon dioxide. This thesis investigated the microbial processes involved in the nutrient and oxygen-stimulated methane production at the Lithgow State Coal Mine (LSCM) in the Eastern NSW coalfields. Sub-bituminous coal from the LSCM was found to contain long chain aliphatic compounds ranging from C11 to C27 and aromatic compounds, such as methylated naphthalenes, fluorene and phenanthrene. When treated with oxidative chemicals and enzymes mimicking microbial coal degradation, compounds such as acetate, propionate and formate were produced. Methane production and microbial community changes were studied during a field trial with wells drilled into the LSCM coal seam. Microbial analysis using the 16S rRNA showed the presence of a diverse range of methanogens, including Methanosarcina, Methanoregula and Methanosaeta, associated with LSCM coal. An increase in Methanosarcina abundance was observed to coincide with the increase in methane production in the nutrient-only treated well, while the calcium peroxide (CaO2) + nutrient well saw a shift in methanogen composition from Methanosarcina-dominated to Methanoregula-dominated. This may potentially represent a change in substrate utilisation from the methanogenic community in this well where Methanoregula, using a different substrate, was able to out-compete Methanosarcina. High abundances of Desulfovibrio were also observed as well as a number of bacteria potentially capable of hydrocarbon degradation, such as Dechloromonas, Georgfuchsia and Bradyrhizobium under anaerobic conditions. A PCR approach for detection of anaerobic hydrocarbon degradation genes showed the presence of a number of genes in the benzoyl-CoA reduction pathway, which is central to many anaerobic aromatic hydrocarbon degradation processes. Metagenomic analysis of microbial communities in the field trial revealed a number of relevant pathways for the biogasification of coal, including anaerobic hydrocarbon degradation pathways, dissimilatory sulphate and nitrate reduction and all three known pathways of methanogenesis. Six microbial genomes (for two species of Methanosarcina, two Alteromonas, unclassified Bacteroidetes and unclassified Firmicutes) were binned from the metagenomic data obtained from the field trial and this is the first time genomes have been isolated from a coal associated community. Methanosarcina genomes showed the presence of all three major methanogenic metabolisms as well as the ability to fix nitrogen, an important survival mechanism in conditions of low nutrients. The complete pathway for dissimilatory nitrate reduction and denitrification were also observed in the binned genome for Alteromonas. This work has shown the production of methane from sub-bituminous coal can be stimulated by the addition of nutrients, which activates a set of microorganisms involved in anaerobic hydrocarbon degradation, methanogenesis, sulphate and nitrate reduction and fermentation. This work has provided important insights into the microbial community dynamics and the metabolic processes occurring in-situ, during the biogasification of coal.