109 Unraveling nitrogen, sulfur and carbon microbial bioreactor responses to stress During regular and experimental conditions, the reactor was checked daily for general parameters including pH with an Applisense electrode (Applikon, Delft, The Netherlands) and nitrate and nitrite concentrations with MQuantTM colorimetric test strips (Merck, Darmstadt, Germany). During experimental conditions, nitrate and nitrite were also measured with the Griess assay as previously described (Arshad et al., 2017), and ammonium was measured fluorometrically after reaction with 10% ortho-phthaldialdehyde as previously described (Taylor et al., 1974). NO in headspace samples was measured with a Sievers Nitric Oxide analyzer (NOA280i; GE Power Water & Process Technologies, Boulder, CO), and sulfide in liquid samples was measured with the methylene blue assay (Moest, 1975) (HACH, Loveland, CO, USA). Sulfate was determined via the barium precipitation method using the Sulfate Assay Kit following manufacturer’s instructions (Sigma Aldrich, Saint Louis, MI, USA). Sample measurements were carried out in technical triplicates. Nucleic acid extractions and sequencing Biomass samples for metagenomic sequencing were collected during regular and experimental periods (Figure 1) and stored at -20°C. All DNA extractions were performed using the DNAeasy Power Soil Kit (Qiagen, Hilden, Germany) following manufacturer’s instructions with two modifications: bead beating was performed in a TissueLyzer (Qiagen, Hilden, Germany) for 10 min and autoclaved MilliQ water was used instead of the kit’s buffer in the last elution step. RNA extractions were initially performed (for T1 and T2) using the RiboPure Bacteria Kit (Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer’s instructions. During the stress experiment (T4 and T5), this method no longer resulted in sufficient extracted RNA for sequencing, potentially due to extensive extracellular polymeric substance production, and thus the method was changed to RNAeasy Power Soil Kit (Qiagen, Hilden, Germany), which was also used for two additional samples (T0 and T3). All RNA samples were treated with DNAase I at 37°C for 30 min from the RiboPure Bacteria Kit (Thermo Fisher Scientific, Waltham, MA, USA). RNA extractions were performed in three biological replicates. Both RNA extraction for rt-qPCR and metatranscriptomics were subjected to the same DNAase treatment. After this treatment, rt-qPCR RNA extractions were tested for DNA contamination. As explained below, RNA samples detected background DNA contamination, equivalent to CT values of RNA extractions of negative controls (water). In addition, 4
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