Modulation of carbon-to-nitrogen ratio shapes the microbial ecology in a methanol-fed recirculating marine denitrifying reactor

Background Nitrate (NO3−) can accumulate in closed-circuit ecosystems to a toxic level. Adding heterotrophic denitrification process to the water treatment is a strategy to reduce this level. This type of process usually requires the addition of a carbon source. Carbon-to-nitrogen ratio (C/N) is a key parameter known to influence both the function and the activity of microbial communities in bioprocesses. Few studies have examined the influence of C/N on denitrification systems operated under methylotrophic and marine conditions. Here we assessed the influence of C/N (methanol and NO3−) on the performance of a laboratory-scale, recirculating denitrifying reactor operated under marine conditions. We monitored the evolution of the bacterial community in the biofilm to assess its stability during the operating conditions. Finally, the relative gene expression profiles of Methylophaga nitratireducenticrescens strain GP59, the main denitrifier in the denitrifying biofilm, were determined during the operating conditions and compared with those of GP59 planktonic pure cultures. Methodology A 500-mL methanol-fed recirculating denitrification reactor operated under marine conditions and colonized by a naturally occurring multispecies denitrifying biofilm was subjected to eight different C/N. We monitored several physico-chemical parameters (denitrifying activities, methanol consumption, CO2 production) throughout the operating conditions. The evolution of the bacterial community in the biofilm during these conditions was determined by 16S rRNA gene amplicon sequencing. Metatranscriptomes were derived from the biofilm to determine (1) the relative gene expression profiles of strain GP59, and (2) the functional diversity of the active microorganisms in the biofilm. Results Changes in C/N did not correlate with the denitrification dynamics (NO3− and NO2− reduction rates, NO2− and N2O dynamics), but did correlate with the methanol consumption rates, and the CO2 production rates. Throughout the operating conditions, nitrite and N2O appeared transiently, and ammonium was not observed. The bacterial community in the reactor increased in diversity with biofilm aging, especially among heterotrophic bacteria, at the expense of methylotrophic bacteria. The relative expression profiles of strain GP59 in the biofilm are distinct from those of planktonic pure cultures of strain GP59, and that the expression of several riboswitches and xoxF would be involved in these differences. Conclusions When the biofilm community is well established in the reactor, it can withstand changes in C/N with limited impact on the denitrification performance. The increase in the proportion of heterotrophs would allow the reactor to be more flexible regarding carbon sources. This knowledge can be useful for improving the efficiency of denitrification system treating close circuit systems such as marine recirculating aquaculture wastewater or seawater aquarium.