A unified and simple medium for growing model methanogens

Abstract

Apart from their archetypic use in anaerobic digestion (AD) methanogenic archaea are targeted for a wide range of applications. Using different methanogenic archaea for one specific application requires the optimization of culture media to enable the growth of different strains under identical environmental conditions, e.g., in microbial electrochemical technologies (MET) for (bio)electromethanation. Here we present a new culture medium (BFS01) adapted from the DSM-120 medium by omitting resazurin, yeast extract, casitone, and using a low salt concentration, that was optimized for Methanosarcina barkeri, Methanobacterium formicicum, and Methanothrix soehngenii. The aim was to provide a medium for follow-up co-culture studies using specific methanogens and Geobacter spp. dominated biofilm anodes. All three methanogens showed growth and activity in the BFS01 medium. This was demonstrated by estimating the specific growth rates ( $\mu$ ) and doubling times ( $t_d$ ) of each methanogen. The $\mu$ and $t_d$ based on methane accumulation in the headspace showed values consistent with literature values for M. barkeri and M. soehngenii. However, $\mu$ and $t_d$ based on methane accumulation in the headspace differed from literature data for M. formicicum but still allowed sufficient growth. The lowered salt concentration and the omission of chemically complex organic components in the medium may have led to the observed deviation from $\mu$ and $t_d$ for M. formicicum as well as the changed morphology. 16S rRNA gene-based amplicon sequencing and whole genome nanopore sequencing further confirmed purity and species identity.

Keywords: bioelectrochemical systems; doubling time; medium adaptation; methanogenic archaea; microbial electrochemical technology; specific growth rate.

Figure 1

Photographs (week 3, top) and phase-contrast photomicrographs (week 7, bottom) of: (A,B) Methanosarcina barkeri, (C,D) Methanobacterium formicicum, and (E,F) Methanothrix soehngenii, grown in the BFS01 medium, bars indicate 10 μm.

Figure 2

Growth characteristics of Methanosarcina barkeri, Methanobacterium formicicum, and Methanothrix soehngenii in the BFS01 medium: (A) OD₆₀₀, (B) CH₄ concentration in the headspace of the cultures, (C) acetate (blue and black dots) and formate (red dots) consumption, (D) COD removal, error bars indicate CI, n = 3.