107 Unraveling nitrogen, sulfur and carbon microbial bioreactor responses to stress MATERIALS AND METHODS Bioreactor operation under regular and experimental conditions In this study we used a previously described complex co-culture (Arshad et al., 2017). Briefly, this co-culture was created by combining biomass from a marine enrichment culture containing sulfide-oxidizing and anammox bacteria (Russ et al., 2014) together with a freshwater nitrate/nitrite-dependent anaerobic oxidation of methane (N-AOM) enrichment culture dominated by “Ca. Methanoperedens” archaea and “Ca. Methyloramibilis” bacteria (Ettwig et al., 2016). After approximately one year, the stable co-culture was characterized for substrate consumption and community composition (Arshad et al., 2017). This same stable co-culture was subjected to a 10-week substrate starvation (ammonium removal) experiment (Figure 1, box 1), and later, after regular operation conditions were re-established, to a 7-week sulfide and nitric oxide stress experiment (Figure 1, box 5). Briefly, under regular operation, the reactor culture was kept under anoxic conditions and fed daily with 0.67 mmol of sulfide, 1.4 mmol of ammonium, 3 mmol of nitrate, and 488 mmol of methane. The medium contained a solution of salts, trace elements, minerals and vitamins as previously described (Arshad et al., 2017), and the pH was kept at 7.1 (Figure 1, box 0 and 4). Under experimental operation, the following conditions were modified. During the ammonium removal experiment, the ammonium concentration in the medium was gradually decreased from 7 to 0 mM over one month (Figure 1, box 1). Two more weeks passed without ammonium addition to the reactor in order to ensure that no residual ammonium was present, and the first biomass sample for metatranscriptomic sequencing (T1) was collected and stored at -80°C. After 10 more weeks without ammonium, another biomass sample for metatranscriptomic sequencing was collected (T2) and ammonium was fully re-introduced into the medium. For exact dates, see Supplementary Table 1. 4
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