Correlation of Key Physiological Properties of Methanosarcina Isolates with Environment of Origin

Abstract

It is known that the physiology of Methanosarcina species can differ significantly, but the ecological impact of these differences is unclear. We recovered two strains of Methanosarcina from two different ecosystems with a similar enrichment and isolation method. Both strains had the same ability to metabolize organic substrates and participate in direct interspecies electron transfer but also had major physiological differences. Strain DH-1, which was isolated from an anaerobic digester, used H₂ as an electron donor. Genome analysis indicated that it lacks an Rnf complex and conserves energy from acetate metabolism via intracellular H₂ cycling. In contrast, strain DH-2, a subsurface isolate, lacks hydrogenases required for H₂ uptake and cycling and has an Rnf complex for energy conservation when growing on acetate. Further analysis of the genomes of previously described isolates, as well as phylogenetic and metagenomic data on uncultured Methanosarcina in anaerobic digesters and diverse soils and sediments, revealed a physiological dichotomy that corresponded with environment of origin. The physiology of type I Methanosarcina revolves around H₂ production and consumption. In contrast, type II Methanosarcina species eschew H₂ and have genes for an Rnf complex and the multiheme, membrane-bound c-type cytochrome MmcA, shown to be essential for extracellular electron transfer. The distribution of Methanosarcina species in diverse environments suggests that the type I H₂-based physiology is well suited for high-energy environments, like anaerobic digesters, whereas type II Rnf/cytochrome-based physiology is an adaptation to the slower, steady-state carbon and electron fluxes common in organic-poor anaerobic soils and sediments. IMPORTANCE Biogenic methane is a significant greenhouse gas, and the conversion of organic wastes to methane is an important bioenergy process. Methanosarcina species play an important role in methane production in many methanogenic soils and sediments as well as anaerobic waste digesters. The studies reported here emphasize that the genus Methanosarcina is composed of two physiologically distinct groups. This is important to recognize when interpreting the role of Methanosarcina in methanogenic environments, especially regarding H₂ metabolism. Furthermore, the finding that type I Methanosarcina species predominate in environments with high rates of carbon and electron flux and that type II Methanosarcina species predominate in lower-energy environments suggests that evaluating the relative abundance of type I and type II Methanosarcina may provide further insights into rates of carbon and electron flux in methanogenic environments.

Keywords: Methanosarcina; Rnf complex; anaerobic respiration; archaea; c-type cytochrome; direct interspecies electron transfer (DIET); extracellular electron transfer; methanogen.

FIG 1

Phylogenetic tree constructed from concatenated proteins from genomes of the 31 representative Methanosarcina species. Organisms highlighted in red represent type I Methanosarcina, while those highlighted in blue represent type II Methanosarcina species.

FIG 2

(A and B) Images of strain DH-1 (A) and strain DH-2 (B) obtained with scanning electron microscopy (lower panels), phase contrast (upper left), and fluorescence microscopy (upper right). Bar, 10 μm.

FIG 3

Ethanol consumption and methane and acetate production in defined cocultures established with G. metallireducens and either strain DH-1 (A) or DH-2 (B) on the fourth transfer of the cocultures. The mol CH₄/mol ethanol yields of 1.2 and 1.0 mol CH₄/mol for the cocultures with strains DH-1 and DH-2, respectively, are within the range of methane recoveries previously reported for G. metallireducens/Methanosarcina cocultures (18, 25, 26). Error bars represent triplicate samples.

FIG 4

Proportions of various Methanosarcina species based on mcrA, 16S rRNA, or both mcrA and 16S rRNA gene sequences from metagenomic libraries constructed from 20 different environments. Species were designated as type I or type II based on the physiology of pure culture isolates of the same species. Type I and type II species are designated with red and black symbols, respectively. All of the bioreactor environments had total organic carbon (TOC) concentrations of >5% (Table S3), while sediment TOC concentrations varied significantly. Bioreactor 1: low-salinity bioreactor (JGI GOLD IDs Ga0334882 to Ga0334890 mcrA); bioreactor 2: anaerobic solid waste digester (73, 74) (SRR8165483; mcrA); bioreactor 3: anaerobic digester in wastewater treatment plant (Gp0313021; mcrA); bioreactor 4: GAC-amended bioreactor treating municipal solid waste (MSW) (75) (SRR7687449 to SRR7687452; 16S rRNA and mcrA); bioreactor 5: bioreactor seeded with sewage sludge (76) (SRR5486931; mcrA); bioreactor 6: sewage sludge and household waste codigester (77) (ERR2586913 to ERR2586931; mcrA); bioreactor 7: cattle manure digester (78) (SRR3166092; mcrA); bioreactor 8: switchgrass digester (79) (JGI GOLD IDs Ga0134090 to Ga0134105; 16S rRNA and mcrA); bioreactor 9: bioreactor treating the dry organic fraction of MSW (80) (SRR5229592; 16S rRNA); bioreactor 10: anaerobic digester at WWTP (SRR3485656; 16S rRNA); sediment 1: mesotrophic meromictic freshwater lake, DOC ∼40 to 80 μM) (81) (IMG GOLD IDs Ga0247831 to Ga0247844); 16S rRNA and mcrA); sediment 2: paddy soil TOC 2.85% (82) (SRR11653212 to SRR11653222; 16S rRNA); sediment 3: estuary sediments, TOC >3.5% (31–33) (SRR1210425 to SRR1210426; mcrA); sediment 4: peat bog sediments, TOC 6 to 50% (36) (IMG GOLD ID Gp0348925; mcrA); sediment 5: mangrove sediment, TOC 0.7 to 11.4% (83, 84) (SRR3095812; mcrA); sediment 6: groundwater and sediments from uranium-contaminated aquifer, TOC <0.2% (38), mcrA (48); sediment 7: Amazon soil, TOC <2% (85) (SRR12110053 to SRR12110059; 16S rRNA); sediment 8: freshwater lake sediments, DOC ∼4 to 60 mg/liter) (86, 87) (IMG GOLD IDs Ga0031653 to Ga0031658; 16S rRNA); sediment 9: South Georgia marine sediments, TOC ∼0.65% (88) (SRR12815614 to SRR12815618; mcrA); sediment 10: Aarhus Bay marine sediments, TOC ∼2% (89) (SRR7119900 to SRR7119905; mcrA). Further details regarding organic carbon concentrations from various environments are available in Table S3 in the supplemental material.

FIG 5

Results from mixed principal-component analysis of 144 different Methanosarcina strains using the following observations: quantitative (number of multiheme c-type cytochromes) and qualitative (type I or type II, presence/absence of Rnf complex, presence/absence of Ech hydrogenase complex, presence/absence of MmcA homolog, organic content of environment from which organism was isolated/detected [high (>2% TOC) or low (<2% TOC)]). M1-M144 specifies the Methanosarcina strain described in Table S1; organisms with CheckM genome completeness scores of <90% were not included in the analysis.