84 Chapter 3 a short time (Figure 1A and Supplementary Figure 3). In the eutrophic bioreactor, nitrite accumulated at 80-100 days to about 8 mM, resulting in a temporary stop of nitrate and sulfide addition until all nitrite was converted. From month 3-4 onwards, a biofilm developed on the wall of the eutrophic bioreactor that was separately sampled and sequenced from month 6 onwards (Supplementary Figure 1 and 2). The oligotrophic bioreactor received 400 µM nitrate, 200 µM ammonium, and 200 µM sulfide per day. Here, sulfide was also fully consumed whereas ammonium remained below 200 µM and nitrate was around 200 µM (Supplementary Figure 3). With some exceptions, nitrite did not accumulate (Figure 1B and Supplementary Figure 3). Ammonium showed an increasing trend after 100 days, indicating a permanent shift from denitrification to DNRA (Figure 1B). The oligotrophic bioreactor biofilm developed earlier, at months 1-2. The oligotrophic system had a denser and more uniform biofilm compared to the eutrophic bioreactor (Supplementary Figure 2). Oligotrophic microbiome sustained DNRA while denitrification dominated eutrophic system Sulfide was fully depleted in both bioreactors, suggesting an active (nitratedependent) sulfide-oxidizing microbial community (Supplementary Figure 3). Denitrification dominated nitrate conversion in the eutrophic system as only a minor part was recovered as ammonium (ranging between 0.5 mM and 1 mM) (Figure 1A). In contrast, the oligotrophic bioreactor produced increasing amounts of ammonium over time (Figure 1B): the concentration rose from 200 µM to around 800 µM, indicating that DNRA was more important than denitrification as the nitrate reduction route. To underpin the processes for sulfide conversion and nitrate removal, we investigated the metagenomic read coverage of functional marker genes for these reactions: sqr for sulfide oxidation/detoxification, nrfA for DNRA and six genes (narG, napA, nirS, nirK, norB, nosZ) for denitrification. Coinciding with the higher loading of sulfide (Supplementary Figure 3), the coverage of sqr appeared to be higher in the eutrophic system (Figure 1C). In agreement with the observed ammonium production in the oligotrophic bioreactor, we found increased metabolic potential for DNRA in this system through a particularly high mean coverage of nrfA in
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