Since fossil sources for fuel and platform chemicals will become limited in the near future, it is important to develop new concepts for energy supply and production of basic reagents for chemical industry. One alternative to crude oil and fossil natural gas could be the biological conversion of CO2 or small organic molecules to methane via methanogenic archaea. This process has been known from biogas plants, but recently, new insights into the methanogenic metabolism, technical optimizations and new technology combinations were gained, which would allow moving beyond the mere conversion of biomass. In biogas plants, steps have been undertaken to increase yield and purity of the biogas, such as addition of hydrogen or metal granulate. Furthermore, the integration of electrodes led to the development of microbial electrosynthesis (MES). The idea behind this technique is to use CO2 and electrical power to generate methane via the microbial metabolism. This review summarizes the biochemical and metabolic background of methanogenesis as well as the latest technical applications of methanogens. As a result, it shall give a sufficient overview over the topic to both, biologists and engineers handling biological or bioelectrochemical methanogenesis.

A novel, strictly anaerobic, methanogenic archaeon, strain E03.2T, was isolated from a full-scale biogas plant in Germany. Cells were non-motile sarcina-like cocci, occurring in aggregates. Strain E03.2T grew autotrophically on H2 plus CO2, and additionally cells could utilize acetate, methanol, moni-, di- and trimethylamine as carbon and energy sources; however, growth or methanogenesis on formate was not observed. Yeast extract and vitamins stimulated growth but were not mandatory. The optimal growth temperature of strain E03.2T was approximately 45 °C; maximal growth rates were obtained at about pH 7.0 in the presence of approximately 6.8 mM NaCl. The DNA G+C content of strain E03.2T was 41.3 mol%. Phylogenetic analyses based on 16S rRNA gene and mcrA sequences placed strain E03.2T within the genus Methanosarcina. Based on 16S rRNA gene sequence similarity strain E03.2T was related to seven different species of the genus Methanosarcina, but most closely related to Methanosarcina thermophila TM-1T. Phenotypic, physiological and genomic characteristics indicated that strain E03.2T represents a novel species of the genus Methanosarcina, for which the name Methanosarcina flavescens sp. nov. is proposed. The type strain is E03.2T ( = DSM 100822T = JCM 30921T).

Significance: The reductive acetyl-coenzyme A (acetyl-CoA) pathway is the only carbon fixation pathway that can also be used for energy conservation like it is known for acetogenic bacteria. In methanogenic archaea, this pathway is extended with one route toward acetyl-CoA formation for anabolism and another route toward methane formation for catabolism. Which of these traits is ancestral in evolution has not been resolved. By diverging virtually all substrate carbon from methanogenesis to flow through acetyl-CoA, Methanosarcina acetivorans can be converted to an acetogenic organism. Being able to deconstruct methanogenic into the seemingly simpler acetogenic energy metabolism provides compelling evidence that methanogens are not nearly as metabolically limited as previously thought and suggests that methanogenesis might have evolved from the acetyl-CoA pathway.

Conversion of acetate to methane (aceticlastic methanogenesis) is an ecologically important process carried out exclusively by methanogenic archaea. An important enzyme for this process as well as for methanogenic growth on carbon monoxide is the five-subunit archaeal CO dehydrogenase/acetyl coenzyme A (CoA) synthase multienzyme complex (CODH/ACS) catalyzing both CO oxidation/CO ₂ reduction and cleavage/synthesis of acetyl-CoA. Methanosarcina acetivorans C2A contains two very similar copies of a six-gene operon ( cdh genes) encoding two isoforms of CODH/ACS (Cdh1 and Cdh2) and a single CdhA subunit, CdhA3. To address the role of the CODH/ACS system in M. acetivorans , mutational as well as promoter/reporter gene fusion analyses were conducted. Phenotypic characterization of cdh disruption mutants (three single and double mutants, as well as the triple mutant) revealed a strict requirement of either Cdh1 or Cdh2 for acetotrophic or carboxidotrophic growth, as well as for autotrophy, which demonstrated that both isoforms are bona fide CODH/ACS. While expression of the Cdh2-encoding genes was generally higher than that of genes encoding Cdh1, both appeared to be regulated differentially in response to growth phase and to changing substrate conditions. While dispensable for growth, CdhA3 clearly affected expression of cdh1 , suggesting that it functions in signal perception and transduction rather than in catabolism. The data obtained argue for a functional hierarchy and regulatory cross talk of the CODH/ACS isoforms.

The use of reporter gene fusions to assess cellular processes such as protein targeting and regulation of transcription or translation is established technology in archaeal, bacterial, and eukaryal genetics. Fluorescent proteins or enzymes resulting in chromogenic substrate turnover, likeβ-galactosidase, have been particularly useful for microscopic and screening purposes. However, application of such methodology is of limited use for strictly anaerobic organisms due to the requirement of molecular oxygen for chromophore formation or color development. We have developedβ-lactamase fromEscherichia coli(encoded bybla) in conjunction with the chromogenic substrate nitrocefin into a reporter system usable under anaerobic conditions for the methanogenic archaeonMethanosarcina acetivorans. By using a signal peptide of a putative flagellin fromM. acetivoransand different catabolic promoters, we could demonstrate growth substrate-dependent secretion ofβ-lactamase, facilitating its use in colony screening on agar plates. Furthermore, a series of fusions comprised of a constitutive promoter and sequences encoding variants of the synthetic tetracycline-responsive riboswitch (tc-RS) was created to characterize its influence on translation initiation inM. acetivorans. One tc-RS variant resulted in more than 11-fold tetracycline-dependent regulation ofblaexpression, which is in the range of regulation by naturally occurring riboswitches. Thus, tc-RS fusions represent the first solelycis-active, that is, factor-independent system for controlled gene expression in Archaea.