Krieger, Bärbel; Schwermer, Carsten U.; Rezakhani, Nastaran; Horn, Marcus A.; Gieseke, Armin; van Rijn, Jaap; Drake, Harold L.; Schramm, Andreas

Conventional aquaculture systems release nitrogen compounds and organic matter into marine environments. As an environmentally-friendly alternative, a zero-discharge mariculture system recently was developed containing a 3-stage biofilter for nitrification, denitrification/anaerobic sludge digestion, and sulfide oxidation. Sulfate reduction in the anaerobic part of the system leads to sulfide concentrations exceeding 5 mM, which may affect nitrate reduction and denitrification. Sulfide can inhibit nitrous oxide reductase, trigger a shift from denitrification to dissimilatory nitrate reduction to ammonium (DNRA), or be used as electron donor for nitrate reduction. The goal of this study was to identify and isolate nitrate-reducing and denitrifying bacteria from the biofilter and to investigate their response to sulfide concentrations relevant for the system. Almost 500 nitrate-consuming isolates were screened by 16S rRNA gene-RFLP; for each RFLP pattern representatives were sequenced. In total, 40 different strains were identified, some of them novel species, mostly affiliating with Alphaproteobacteria but also including Beta- and Gammaproteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. The diversity of the isolates was compared to the cultivation-independent diversity of nitrate-reducing and denitrifying bacteria based on narG and nosZ as functional marker genes. Growth experiments revealed great differences in sulfide-tolerance among isolates, ranging from < 50 µM to 5 mM; some strains were also able to oxidize sulfide. Increasing sulfide concentrations generally resulted in increased nitrous oxide production. Batch incubations of anaerobic sludge with 15N-nitrate confirmed the in situ relevance of these results and indicated a sulfide-induced shift from denitrification to DNRA.