144 Chapter 5 13C-CH 4 • nitrate/nitrite (daily) • ammonium (during ) 0 days 36 18 54 T0 hours T1 T2 hours T3 Long-term exposure: Na2S 0.25 mM Medium T0 T1 T2 T3 • • metagenomics • metagenomics granule • metagenomics planktonic (only T2/T3) • metatranscriptomics • PHA liquid Na2S 0.5 mM Na2S addition control toxicity exposure 74 Figure 1. Bioreactor sulfide toxicity experiment workflow. Whole bioreactor activity assays with labelled 13C-CH4 are indicated in brown (4-5 days). A duration of 4-5 hours passed between T0-T1 and T2-T3. Sulfide additions (0.5 mM Na2S x 3 H2O) are indicated by an arrow (spike) or green rectangle (acclimation period of 6.5 weeks). Bioreactor DNA and RNA sampling To track microbial community and activity over time, DNA and RNA samples were collected at four different time points for downstream metagenomics and metatranscriptomics, respectively (Figure 1). The first two time points, T0-T1, were collected immediately before and 2 hours after the first 0.5 mM sulfide spike. The latter two points, T2-T3, were collected after the longer sulfide exposure period (~ 6.5 weeks), immediately before and after 2 hours of 0.5 mM sulfide addition (Figure 1). To characterize the granular vs free-living morphotype of “Ca. Methanoperedens”, biomass was vacuum filtered at time points T0, T1, T2 and T3 using hydrophilic polycarbonate membranes (5.0 µm and 47 mm diameter) (TMTP04700) (Millipore, Darmstadt, Germany). DNA was extracted once, whereas triplicate extractions were performed per time point for RNA. Biomass was immediately stabilized upon sampling by mixing 2 ml biomass with 6 ml PowerProtect DNA/RNA solution (1:3) (Qiagen Benelux B.V., Venlo, The Netherlands). The stabilized mixture was spun down and the remaining pellet was freeze-dried overnight and stored at -70ºC. DNA extractions were performed using the DNeasy PowerSoil Kit (Qiagen, Hilden, Germany) and RNA was extracted with the RNeasy PowerSoil Kit (Qiagen, Hilden,
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