Lestin, L.; Villemur, R.

Background. Carbon-to-nitrogen ratio (C/N) is an essential parameter known to influence both the function and the activity of microbial communities in bioprocesses. The right balance between the contribution of each of these resources are essential for sustainable and cost-efficiency bioprocess. Here we aimed to assess the influence of C/N (here methanol and nitrate) on the performance of a recirculating denitrifying reactor for 31 weeks under marine conditions. We also monitored the evolution of its microbial community during the operating conditions. 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 over 31 weeks under anoxic conditions. We monitored several physico-chemical parameters (denitrifying activities, methanol consumption, CO2 production) throughout the operating conditions. Evolution of the bacterial community in the biofilm during the operating conditions was determined by 16S rRNA gene amplicon sequencing. Metatranscriptomes of representative conditions were performed to derive (1) the relative gene expression profiles of Methylophaga nitratireducenticrescens strain GP59, the main denitrifier, and (2) the functional diversity of the biofilm. Results. Changes in C/N did not impact the denitrifying activities of the recirculating reactor but did impact the carbon dynamics. Throughout the operating time, nitrite and N2O appeared transiently, and ammonium was not observed. The bacterial community in the reactor increased in diversity as the biofilm aged, especially in heterotrophic bacteria, at the expense of methylotrophic bacteria. The functional diversity suggests that heterotrophs could use formaldehyde, which may have been released by methylotrophs in the biofilm, as energy and carbon source. The relative expression profiles of strain GP59 in the biofilm is distinct of those of GP59 planktonic pure cultures, and that the expression of several riboswitches and xoxF would be involved in such differences. Conclusions. When the biofilm community is well established in the reactor, it can sustain changes in C/N with limited impact on the denitrification performance. Increase in the proportion of heterotrophs would allow the reactor to be more flexible for carbon sources. Such knowledge can be useful in the optimisation of denitrification process.