113 Unraveling nitrogen, sulfur and carbon microbial bioreactor responses to stress were above the threshold of 30 cycles. RT-qPCR units were expressed in 2-ΔΔCT by subtracting, first, normalizer with target functional gene CT values, and second, R0 and R3 from R1 and R4 CT values, respectively. Replicates were discarded if CT values were above 30 cycles. RESULTS Diverse microbial community members cycled methane, nitrate, nitrite, ammonium, and sulfide For over one year since the combination of the two parent enrichment cultures, the bioreactor ran under stable conditions (Figure 1, box 1) (Arshad et al., 2017). During the ammonium removal experiment, the bioreactor received the same daily quantities of nitrate (3 mmol), methane (488 mmol) and sulfide (0.67 mmol) as previously described (Arshad et al., 2017). A comprehensive investigation of daily substrate consumption was carried out in the aforementioned study (corresponding to the zero Figure 1, box 1), which showed that sulfide was completely consumed with a nitrate removal rate of 2.6 mmol per day. The residual nitrate amount fluctuated around 0.4 mmol/day, while no nitrite could be detected in the bioreactor (Arshad et al., 2017). In the present investigation, ammonium was removed from the medium (Figure 1, box 1), and nitrate, nitrite and ammonium substrate consumption was monitored for a period of 12 days prior to metatranscriptomics biomass (T2) collection (10 weeks without ammonium). Nitrate had a residual concentration of 3 mM, while ammonium, nitrite and sulfide concentrations remained below the detection limit. The overall nitrate and nitrite concentrations were consistent with the previously determined concentrations (Arshad et al., 2017). During the sulfide and NO toxicity stress experiment (Figure 1, box 5), methane, nitrate and ammonium were not provided to the reactor. Only sulfide and NO were added as substrates in order to enrich sulfide-oxidizing nitric oxide reducers. While sulfide concentrations remained below detection limit for the entire experiment, indicating continuous complete sulfide removal, nitrite accumulated to 870 μM during the second week of the bioreactor receiving only sulfide and NO, and remained between 100-400 μM during the remainder of the experiment. This 4
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