108 Chapter 4 Stable NH4 + removal NH4 + (7mM) restored - stable CH4 out 4 Solely H2S (6.7mM) + 10-13% NO 5 3 2 1 0 G0 G: DNAe | metagenomics T: RNAe | metatranscriptomics R: RNAe | rt-qPCR T0 R0 T1 T2 R1 G1 G2 G3 G4 G5 T3 T4 T5 R3 R4 NH4 + (0mM) NH4 + (7mM) CH4 (sat.) 200ml/day NH4 + (7mM) NO3 - (15mM) 100ml/day H2S (6.7mM) CH4 (sat.) back in + 1-5% NO Substrate removal Toxic stresses BR biomass collection for Figure 1. Timeline of experiments. Zero indicates when the bioreactor was stably maintained; 1, period of the ammonium removal experiment; 2, after the ammonium removal experiment and re-stabilization period; 3; reactor moved into a fume hood in preparation for the next experiment; 4, preparatory period for the nitric oxide and sulfide toxicity stresses experiment; 5, nitric oxide and sulfide toxicity stresses experiment. Samples for metagenomics (G0-G5), metatranscriptomics (T0-T5) and RT-qPCR (R0-R1 and R3-R4) were collected as indicated across experimental time points and in between them. The dashed line in the ammonium removal experiment indicates that removal was conducted stepwise (in mM: 7 → 4.5 → 3.5 → 2.5 → 0). Exact dates and experiment durations are provided in Supplementary Table 1. In preparation for the next experiment, the reactor was moved into a laminar flow hood. Methane flow was stopped at the time of reactor moving and reintroduced after 15 days (Figure 1, box 3). Once regular operation conditions and substrate consumption were reestablished, the sulfide and NO toxicity stress experiment began. Initially, the NO concentration in the reactor headspace was gradually increased to 1-5% for 10 weeks so that the community could adapt (Figure 1, box 4). Then, NO concentrations were increased to ~ 10-13% and all substrates except sulfide were removed from the reactor, resulting that only sulfide, NO and medium were provided to the reactor for the following 7 weeks (Figure 1, box 5). A biomass sample for metatranscriptomic sequencing was collected before any NO was added to the reactor (T4) and after the 17 weeks (T5). Originally, the experiment was planned to be conducted in 20 weeks, being that during the 10 last weeks the reactor would be maintained under only sulfide and NO. However, covid-19 lockdown in the Netherlands resulted in restricted access to the laboratory and the timing had to be changed from 20 to 17 weeks. Additional metatranscriptomics samples in between experiments were included in this study: T0, approximately 2 months before the ammonium removal experiment, and T3, 6 months after the ammonium removal experiment and 11 months before the sulfide and NO stress experiment (Figure 1 and Supplementary Table 1).
RkJQdWJsaXNoZXIy MTk4NDMw