A Single-Culture Bioprocess of Methanothermobacter thermautotrophicus to Upgrade Digester Biogas by CO2-to-CH4 Conversion with H2

We optimized and tested a postbioprocessing step with a single-culture archaeon to upgrade biogas (i.e., increase methane content) from anaerobic digesters via conversion of CO2 into CH4 by feeding H2 gas. We optimized a culture of the thermophilic methanogen Methanothermobacter thermautotrophicus using: (1) a synthetic H2/CO2 mixture; (2) the same mixture with pressurization; (3) a synthetic biogas with different CH4 contents and H2; and (4) an industrial, untreated biogas and H2. A laboratory culture with a robust growth (dry weight of 6.4–7.4？g/L; OD600 of 13.6–15.4), a volumetric methane production rate of 21？L/L culture-day, and a H2 conversion efficiency of 89% was moved to an industrial anaerobic digester facility, where it was restarted and fed untreated biogas with a methane content of ~70% at a rate such that CO2 was in excess of the stoichiometric requirements in relation to H2. Over an 8-day operating period, the dry weight of the culture initially decreased slightly before stabilizing at an elevated level of ~8？g/L to achieve a volumetric methane production rate of 21？L/L culture-day and a H2 conversion efficiency of 62%. While some microbial contamination of the culture was observed via microscopy, it did not affect the methane production rate of the culture. 1. Introduction Organic waste streams contain energy that is stored in biomass, which had originally been harnessed from the sun by photosynthesis. To prevent environmental problems during the release of these waste streams, biological treatment is necessary. At the same time, there is a growing interest in recovering this stored energy in more useful forms by converting the complex biomass into bioenergy sources that are direct replacements of fossil fuels [1]. The traditional route for this conversion with relatively energy-dense wastes is via methane fermentation in anaerobic digesters [2, 3]. Anaerobic digesters consist of an open culture of microbial consortia (referred to here as a reactor microbiome) with a dynamic food web that includes bacterial hydrolysis, acidogenesis, and acetogenesis, as well as archaeal methanogenesis [1]. Anaerobic digestion is an ideal process for two reasons: (1) the product methane bubbles freely out of solution without costly separation; and (2) the anaerobic reactor microbiome harvests the maximum amount of free energy without oxygen by maximizing the production of methane (resulting in high conversion efficiencies) [4]. However, during digestion, both methane and carbon dioxide must be produced to balance the high oxidation number (i.e., number of