Multiple anaerobic processes are responsible for carbon mineralization in eutrophic nitrate-rich marine environments (e.g., upwelling areas, estuaries, and aquacultures), involving electron acceptors from both the nitrogen and sulfur cycle. The interaction of these processes is less understood. Our aim was to investigate the functional interaction of nitrate reduction, denitrification and sulfate reduction in an anaerobic marine sludge. We hypothesize that sulfide (from sulfate reduction) (i) causes incomplete denitrification, and (ii) directs nitrate conversion from denitrification to dissimilatory nitrate-reduction to ammonium (DNRA). In situ microsensor profiling in stagnant sludge revealed the typical stratification of nitrate reduction on top of sulfate reduction. Increasing the bulk nitrate concentration lead to a downward shift of the nitrate-sulfide transition zone; within this zone, N2O accumulated to 260 µM. Batch incubations of sludge with sulfide and 15N-nitrate showed the formation of ammonium at the expense of N2 with increasing sulfide concentrations; an indication of the predicted shift from denitrification to DNRA. However, at the highest sulfide concentration tested (1.6 mM), only 22% and 20% of the nitrate added were converted to N2 and ammonium, respectively, while N2O became the main end product (56%). These findings were corroborated by pure culture experiments with nitrate-reducing and denitrifying isolates from the sludge. Our results show that the presence of sulfide generally decreased growth rates but increased N2O production. We conclude that sulfide plays a key role in causing incomplete denitrification, presumably by inhibiting the N2O reductase, and enhancing DNRA compared to denitrification.
Multiple anaerobic processes are responsible for carbon mineralization in eutrophic nitrate-rich marine environments (e.g., upwelling areas, estuaries, and aquacultures), involving electron acceptors from both the nitrogen and sulfur cycle. The interaction of these processes is less understood. Our aim was to investigate the functional interaction of nitrate reduction, denitrification and sulfate reduction in an anaerobic marine sludge. We hypothesize that sulfide (from sulfate reduction) (i) causes incomplete denitrification, and (ii) directs nitrate conversion from denitrification to dissimilatory nitrate-reduction to ammonium (DNRA). In situ microsensor profiling in stagnant sludge revealed the typical stratification of nitrate reduction on top of sulfate reduction. Increasing the bulk nitrate concentration lead to a downward shift of the nitrate-sulfide transition zone; within this zone, N2O accumulated to 260 µM. Batch incubations of sludge with sulfide and 15N-nitrate showed the formation of ammonium at the expense of N2 with increasing sulfide concentrations; an indication of the predicted shift from denitrification to DNRA. However, at the highest sulfide concentration tested (1.6 mM), only 22% and 20% of the nitrate added were converted to N2 and ammonium, respectively, while N2O became the main end product (56%). These findings were corroborated by pure culture experiments with nitrate-reducing and denitrifying isolates from the sludge. Our results show that the presence of sulfide generally decreased growth rates but increased N2O production. We conclude that sulfide plays a key role in causing incomplete denitrification, presumably by inhibiting the N2O reductase, and enhancing DNRA compared to denitrification.