Hydrogenotrophic Methanogenesis and Autotrophic Growth of Methanosarcina thermophila

Abstract

Although Methanosarcinales are versatile concerning their methanogenic substrates, the ability of Methanosarcina thermophila to use carbon dioxide (CO₂) for catabolic and anabolic metabolism was not proven until now. Here, we show that M. thermophila used CO₂ to perform hydrogenotrophic methanogenesis in the presence as well as in the absence of methanol. During incubation with hydrogen, the methanogen utilized the substrates methanol and CO₂ consecutively, resulting in a biphasic methane production. Growth exclusively from CO₂ occurred slowly but reproducibly with concomitant production of biomass, verified by DNA quantification. Besides verification through multiple transfers into fresh medium, the identity of the culture was confirmed by 16s RNA sequencing, and the incorporation of carbon atoms from ¹³CO₂ into ¹³CH₄ molecules was measured to validate the obtained data. New insights into the physiology of M. thermophila can serve as reference for genomic analyses to link genes with metabolic features in uncultured organisms.

Figure 1

Decreasing methanol (MeOH) concentration (a) in the medium, cumulative CH₄ (a), H₂ (b), and CO₂ (b) in the headspace of a Methanosarcina thermophila culture with an initially either N₂/CO₂- or H₂/CO₂-containing headspace within 23 days of incubation (means; whiskers: standard deviation).

Figure 2

Acetate and DNA content in the culture fluid and cumulative CH₄ production by Methanosarcina thermophila in an organic carbon-free medium with a H₂/CO₂ headspace (means; whiskers: standard deviation).

Figure 3

Cumulative CH₄ production, cumulative H₂ consumption, and cumulative CO₂ consumption by Methanosarcina thermophila growing in a medium containing only CO₂ as methanogenic substrate (means; whiskers: standard deviation).

Figure 4

Linear correlation between CH₄ production and CO₂/H₂ consumption by Methanosarcina thermophila during 26 days of incubation (Pearson correlation: CO₂: p ≤ 0.01, R² = 0.88; H₂: p ≤ 0.01, R² = 0.96).