Methane, the second most important greenhouse gas, is regulated primarily by microbial processes [1]. A renewed interest in methane as a gas substrate for the production of biofuels is spearheaded by its abundance in shale gas [2–5]. At the same time, concerns related with methane’s role as a potent greenhouse gas drives the need to mitigate its adverse environmental impact [6]. Advances in the characterization of microbial consortia in anoxic sediments have revealed the potential of transforming methane into various products through biological routes [7–10].
The global methane cycle is predominantly controlled by anaerobic methanotrophic archaea (ANME) in anoxic environments [11, 12] and aerobic methanotrophic bacteria at the anoxic–oxic interface of habitats [13, 14]. Aerobic methanotrophy [15], proceeds via the oxidation of methane to methanol by a methane monooxygenase and then to formaldehyde by methanol dehydrogenase, which is subsequently integrated into central carbon metabolism through the ribulose monophosphate or the serine pathway [16]. This scheme, however, requires an initial activation cost in the form of NAD(P)H, which is replenished at the expense of carbon efficiency. Shaped by the paucity of available energy, the anaerobic methanotrophy has been shown to exhibit better carbon and energy efficiency [17, 18]. However, in contrast to the aerobic route, anaerobic methanotrophy is relatively poorly characterized as a result of the difficulties in culturing ANMEs in the lab [11] arising from syntrophy requirements. In such environments, the anaerobic methanotroph oxidizes methane and the microbial partner reduces an electron acceptor, often an inorganic ion such as NO3? [19] or SO42? [20]. Despite these difficulties, recent metagenomics analysis of ANMEs has partially revealed the methanotrophic pathways, observed in most methanogenic archaea, demonstrating the phylogenetic relationship between ANMEs and methanogens [21, 22]. Of particular interest is the methanogenic archaeon Methanosarcina acetivorans for which trace methane oxidation has been observed [23, 24] implying that it possesses the necessary pathways and electron flow systems to accomplish methanotrophy. However, a complete reversal of methanogenesis pathway is thermodynamically infeasible unless coupled with an electron-accepting pathway [25, 26].
M. acetivorans, a strictly anaerobic marine methanogen possessing one of the largest known archaeal genomes [27], has emerged as a model archaeon owing to the availability of genetic tools [28] and versatility in substrate utilization [29, 30]. While the pathways describing the metabolism on native substrates have been extensively studied [31–34], relevant pathways and electron flows for methane oxidation by M. acetivorans remain largely uncharacterized. Two genome-scale metabolic (GSM) models, iVS941 [35] and iMB745 [36], for this organism have been proposed. Both models, however, are not up to date with the current literature on the stoichiometry of ion transport across the membrane and ATP synthesis [37–42]. Recent findings on the electron flow mechanisms of M. acetivorans cell extracts grown with methane (unpublished observations, Zhen Yan and James G. Ferry) motivates an update in the existing genome-scale models to incorporate recent findings and to allow for the analysis of methane utilization in silico.
In this paper we make use of a revamped GSM for M. acetivorans to postulate pathways for reversing methanogenesis while maintaining overall thermodynamic feasibility. We first generated an up-to-date GSM model for M. acetivorans by combining information from two earlier models (i.e., iVS941 and iMB745) along with the most recent data from literature and databases. M. acetivorans has transcriptome and proteome profiles that differ depending on growth substrate [31, 34]. We augmented the updated gene-protein-reaction (GPR) associations with regulatory (i.e., ?R) switches to incorporate proteomics data to the updated metabolic reconstruction by switching on/off reactions for different substrates. Using the model as a starting point a thermodynamically feasible pathway is proposed for the co-utilization of methane and bicarbonate in the presence of Fe3+, NO3?, SO42?, and MnO2 as external electron acceptors. Overall ?G ? 0 is imposed as a constraint to ensure thermodynamic feasibility of methanogenesis reversal in the presence of an external electron acceptor. The interplay between externally supplied electron acceptors and various by-products is analyzed. The feasibility of methanotrophy by resting cells is assessed when all carbons coming from methane and bicarbonate are converted into acetate, formate, CO2, and methyl sulfide, the known byproducts of M. acetivorans’ metabolism [30, 43] some of which were also observed recently by Wood et al. [44] as end products of methanotrophy by the archaeon.