Page 228 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

Chapter 10. Co-culturing methanogens and methanotrophs in an MBR
sludge blanket (UASB) reactor to co-cultivate complex methanogenic cultures with the aerobic methanotrophs Methylosinus trichosporium and M. sporium (Miguez et al. 1999). Gerritse and Gottschal were the first to set up defined co-cultures with Methanosarcina barkeri and Methanobacterium formicicum together with aerobic methanotrophic Methylocystis sp. (Gerritse and Gottschal 1993). However, in-depth data on species interactions are lacking. Here, we established a co-culture of the methanogen M. barkeri and aerobic Methylocystaceae methanotrophs in a membrane bioreactor to generate a method to study interspecies interactions between methane cycle microorganisms. Under oxygen-limited conditions, a stable co-culture was monitored over time and several key parameters were determined.
Materials and methods
Strains
Methanosarcina barkeri DSM 800 and Methylosinus sporium DSM 17706 strains were ordered from the DSMZ (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) as actively growing cultures. M. sporium was chosen for high substrate affinity (Murrell, Mcdonald and Gilbert 2000) and co-occurrence in methanogenic-methanotrophic cultures (Shen, Miguez and Bourque 1996; Miguez et al. 1999). Genomic analysis of the M. sporium culture indicated presence of a second strain of another Methylocystaceae species related to Methylocystis rosea, as described in detail in the “Results” section. M. barkeri was chosen for the oxygen-limited reactor set-up due to its relative high oxygen tolerance (up to several hours under atmospheric oxygen levels) and wide substrate range (Kiener and Leisinger 1983; Brioukhanov et al. 2000; Maeder et al. 2006). Furthermore, its reference genome is available (PRJNA230939). M. barkeri can perform acetoclastic methanogenesis via acetate dismutation to methane and CO2. In addition, M. barkeri can be grown on mineral media, a prerequisite for this co-cultivation study (Maestrojuan and Boone 1991). M. sporium was pre-grown on general medium as described at “Reactor set-up & co- cultivation”. M. barkeri was pre-grown on methanogen medium (1.7 mM KH2PO4, 0.9 mM NH4Cl, 0.2 mM MgSO4·7H2O, 0.2 mM CaCl2·2H2O, 0.5 mM NaCl, 0.7 μM FeSO4·7H2O, 0.25 g L-1 Tryptone, 0.25 g L-1 yeast extract, 1 ml 1000x trace elements SL-6 including 81.5 μM CeCl·7H2O [DSMZ, Braunschweig, Germany], and 1 ml 1000x vitamin solution [DSMZ,
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