Impact of physico-chemical parameters on the denitrification and the co-occurrence of Methylophaga nitratireducenticrescens and Hyphomicrobium nitrativorans in a methanol-fed marine denitrifying biofilm

Abstract

Background The biofilm of a continuous, methanol-fed, fluidized denitrification system that treated a marine effluent is composed of multi-species microorganisms, among which Hyphomicrobium nitrativorans NL23 and Methylophaga nitratireducenticrescens JAM1 are the principal bacteria involved in the denitrifying activities. This biofilm can be cultured at laboratory-scale under batch mode conditions without losing the denitrifying activities. Here, we report the capacity of the denitrifying biofilm to sustain changes to specific physico-chemical parameters, and the impact of these changes on the denitrification performance and the co-occurrence of H. nitrativorans and M. nitratireducenticrescens. Methods The original biofilm (OB) taken from the denitrification system was acclimated to an artificial seawater (ASW) medium under anoxic conditions to generate the Reference biofilm cultures (300 mg-NO3−-N/L, 23°C). In the first set of assays, the Reference biofilm cultures were subjected to short exposures (1-3 days) of a range of NaCl, methanol, nitrate (NO3−) and nitrite (NO2−) concentrations, and to different pHs and temperatures. In the second set of assays, the OB was acclimated in an ASW-modified medium for five weeks i) to a range of NaCl concentrations (0% to 8%), ii) to four combinations of NO3− concentrations and temperatures, iii) to NO2−, and iv) under oxic conditions. Finally, the OB was acclimated to the commercial Instant Ocean (IO) medium. The growth of the biofilm and the dynamics of NO3− and NO2− were determined. The levels of M. nitratireducenticrescens and H. nitrativorans were measured by qPCR in these cultures. Results The denitrifying capacities of the OB was preserved in the Reference biofilm cultures. In these cultures, however, H. nitrativorans NL23 decreased by three orders of magnitude in concentration with the occurrence of the same magnitude of a new denitrifying bacterial strain (M. nitratireducenticrescens GP59). Results from the first set of assays showed that the Reference biofilm cultures can sustain denitrifying activities in most of the tested conditions. Inhibition occurred when these biofilm cultures were exposed at pH 10 or with 1.5% methanol. Results from the second set of assays showed the persistence of H. nitrativorans NL23 in the biofilm cultures acclimated to low NaCl concentrations (0% to 1.0%). Poor biofilm development occurred in biofilm cultures acclimated to 5% and 8% NaCl. Finally, high proportion of H. nitrativorans NL23 was found in the IO biofilm cultures. Conclusions These results confirm the plasticity of the marine methylotrophic denitrifying biofilm in adapting to different conditions. The NaCl concentration is a crucial factor in the dynamics of H. nitrativorans NL23, for which growth was impaired above 1% NaCl in the ASW-based biofilm cultures in favor of M. nitratireducenticrescens GP59. This study contributes to the understanding on the population dynamics of co-occurring bacteria performing denitrifying activities in biofilm under seawater environment. This could benefit in the development of optimal denitrifying bioprocess under marine conditions.