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 (μ) and doubling times (td) of each methanogen. The μ and td based on methane accumulation in the headspace showed values consistent with literature values for M. barkeri and M. soehngenii. However, μ and td 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 μ and td 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.

Effect of model methanogens on the electrochemical activity, stability, and microbial community structure of Geobacter spp. dominated biofilm anodes

“…The Methanothrix soehngenii GP6 (DSM 3671) strain was kindly provided by the microbiology laboratory at the department for agrotechnology and food sciences of Wageningen University and Research, Wageningen, The Netherlands. All three methanogens were cultured in the simplified methanogen medium BFS01 containing per liter: 0.348 g K 2 HPO 4 , 0.227 g KH 2 PO 4 , 0.5 g NH 4 Cl, 0.406 g MgCl 2 × 6 H 2 O, 0.25 g CaCl 2 × 2 H 2 O, 2.25 g NaCl, 1.42 mg FeCl 2 × 4H 2 O, 0.85 g NaHCO 3 , 0.3 g L-Cysteine hydrochloride monohydrate, 1 mL trace element solution SL-10, and 1 mL Wolin’s vitamin solution-10 71 . Before each inoculation, the BFS01 medium was supplemented with carbon sources and electron donors as indicated in Table 1 .…”

“…Nearly stable CH 4 concentration in the culture headspaces over several days was used as an indicator of the maximum cell density achieved for each methanogenic culture. Due to their cell morphology: 1) M. barkeri , which aggregated as irregular-sized, interconnected cell clumps 71 , 2) M. formicicum , which formed cell colony resembling a sponge-like structure 71 , and 3) M. soehngenii , which had a rod-shaped filament morphology 71 , optical density measurement (using OD 600 ) and cell counting (using Multisitzer 3 Coulter Counter, Beckmann Coulter TM) were attempted, but regarded as inappropriate. 5 mL of each methanogenic culture were used for process monitoring and the remaining 125 mL were used for the electrochemical experiments, to ensure comparable methanogenic biomass in the replicates of each single experiment (e.g., M. barkeri + Geobacter spp.…”

“…biofilm). See specifications and additional information on the BFS01 medium used, growth monitoring and physiological parameters for each methanogen in Dzofou et al 71 .…”

“…The bacterial and archaeal diversity of the samples was determined by metabarcode sequencing, as previously described 71 .…”

Syntrophy between bacteria and archaea enhances methane production in an EGSB bioreactor fed by cheese whey wastewater

“…The physicochemical characteristics of AnGS, such as pH, EC, ORP, TS, TVS, TVS/TS ratio, color, and odor, were analyzed according to APHA (2005). The sludge volume index (SVI), specific methanogenic activity (SMA), and density were determined according to Cruz-Salomón et al (2017b), Wongburi and Park (2022), and van den Berg et al (2023), respectively. All the parameters were measured during the next 24 h.…”