for 5 min before recording luciferase activity on a LuminoskanTM Ascent microplate luminometer (Thermo Scientific). A standard curve was generated using ATP standards according to the manufacturer’s guidelines. Co-culture of and MC09 R. pomeroyi Methylomonas methanica wild-type and the mutant, (Lidbury et al, 2014), was grown using either R. pomeroyi Δtmm::Gm TMA or TMAO as the sole N source (OD540 ~0.3). Cells were re-suspended in fresh medium containing 1 mM methanol. For each strain, triplicate cultures were set up using either TMA or ammonium chloride as the sole N source (1 mM). was grown using methanol M. methanica as the C source (2 mM) and ammonia (0.5 mM) as the limiting nutrient until the onset of stationary phase. A 5% (v/v) inoculum of (~107 cells) was added to each M. methanica R. culture. Co-cultures were incubated at 25oC on a rotary shaker (150 r.p.m.). For pomeroyi M. cell counts, serial dilutions were generated (n=3) and 10 were spotted (n=3) on methanica μl MAMS plates with methane as the sole C source and incubated at 25oC. Results TMA and TMAO oxidation increases growth yields when grown on glucose R. pomeroyi oxidised TMA and TMAO in the presence of both glucose and ammonia in the R. pomeroyi culture medium (Fig. 2a & 2b). The rate of TMA and TMAO oxidation was greatest through exponential growth but did continue throughout stationary phase when glucose was exhausted
for 5 min before recording luciferase activity on a LuminoskanTM Ascent microplate luminometer (Thermo Scientific). A standard curve was generated using ATP standards according to the manufacturer’s guidelines. Co-culture of R. pomeroyi and Methylomonas methanica MC09 R. pomeroyi wild-type and the mutant, Δtmm::Gm (Lidbury et al, 2014), was grown using either TMA or TMAO as the sole N source (OD540 ~0.3). Cells were re-suspended in fresh medium containing 1 mM methanol. For each strain, triplicate cultures were set up using either TMA or ammonium chloride as the sole N source (1 mM). M. methanica was grown using methanol as the C source (2 mM) and ammonia (0.5 mM) as the limiting nutrient until the onset of stationary phase. A 5% (v/v) inoculum of M. methanica (~107 cells) was added to each R. pomeroyi culture. Co-cultures were incubated at 25oC on a rotary shaker (150 r.p.m.). For M. methanica cell counts, serial dilutions were generated (n=3) and 10 μl were spotted (n=3) on MAMS plates with methane as the sole C source and incubated at 25oC. Results TMA and TMAO oxidation increases R. pomeroyi growth yields when grown on glucose R. pomeroyi oxidised TMA and TMAO in the presence of both glucose and ammonia in the culture medium (Fig. 2a & 2b). The rate of TMA and TMAO oxidation was greatest through exponential growth but did continue throughout stationary phase when glucose was exhausted