Page 229 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

Braunschweig, Germany with 100 mM acetate and using 0.001% resazurin [w/v] as redox indicator). pH was adjusted to 8.5 with sodium hydroxide, and bottles were made anoxic with a triplicate + 1 bar overpressure gas followed by a 15-minute vacuum cycle using a 9:1 Argon/CO2 gas mixture resulting in a pH of around 7.0 after autoclaving.
Reactor set-up and co-cultivation
Reactor set-up. A 2 L membrane bioreactor (MBR, see Yoon 2016 for details) with an operational volume of 1.5 L, and a settling and bleed cycle to control growth rate, and to select for aggregates, was designed for the co-culturing of methanogens and aerobic methanotrophs (Fig. 1). A general medium was devised that allowed growth of both methanogens and methanotrophs (1.0 mM MgSO4·7H2O, 0.23 mM CaCl·2H2O, 1.7 mM KH2PO4, 5.1 mM NaCl, 7.2 μM FeSO4·7H2O, 3.7 mM NH4Cl, 26.2 mM CH3COOH, 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, Braunschweig, Germany]). pH was adjusted to 7.0. At t = 111 days, Tryptone and yeast extract were removed from the medium to reduce risk of contamination in the bioreactor. Growth tests on Tryptone and yeast extract free medium confirmed that growth of both methanogens and methanotrophs was possible (data not shown). The total medium flow supply was set to 0.5 L day-1 and the volume was kept constant at 1.5 L using a level-controlled effluent pump. The gas flow rate was set to 5 ml min-1. Initial gas mixture composition was set to 1.51 ml Argon/CO2 (9:1) mixture, 2.38 ml air and 0.75 ml methane. After 15 weeks of co-cultivation the gas inflow was increased to 10 ml min-1 to reduce the risk of air diffusion into the reactor. Simultaneously the methane inflow percentage was reduced based on the prevailing methane consumption data. The mixture contained 6.77 ml Argon/CO2, 2.86 ml air and 0.37 ml methane per minute. O2 and pH were monitored using AppliSens probes (AppliSens Z001023551 and Applisens Z010023520, Applikon Biotechnology B.V., Delft, the Netherlands). The pH was controlled at 7.0 ± 0.1 using a probe-linked KHCO3 pump. The MBR was continuously mixed at 150 rpm using a stainless-steel rotor blade controlled by an Applikon stirrer controller (Applikon stirrer controller P100, Applikon Biotechnology B.V., Delft, the Netherlands). The system was operated at room temperature (± 20°C).
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