Dynamics of a methanol-fed marine denitrifying biofilm: 1-Impact of environmental changes on the denitrification and the co-occurrence of Methylophaga nitratireducenticrescens and Hyphomicrobium nitrativorans
Authors: Payette Geneviève , Geoffroy Valérie , Martineau Christine , Villemur Richard 
Published: August 2019

Background
The biofilm of a methanol-fed denitrification system that treated a marine effluent is composed of multi-species microorganisms, among which Hyphomicrobium nitrativorans strain NL23 and Methylophaga nitratireducenticrescens strain JAM1 are the principal bacteria involved in the denitrifying activities. Here, we report the capacity of the denitrifying biofilm to sustain environmental changes, and the impact of these changes on the co-occurrence of H. nitrativorans and M. nitratireducenticrescens.

Methods
In a first set of assays, the original biofilm (OB) was cultivated in an artificial seawater (ASW) medium under anoxic conditions to colonize new carriers. The new formed biofilm was then subjected to short exposures (1–5 days) of a range of NaCl, methanol, nitrate (NO3−) and nitrite (NO2−) concentrations, and to different pHs and temperatures. In a second set of assays, the OB was cultivated in ASW medium for five weeks with (i) a range of NaCl concentrations, (ii) four combinations of NO3−/methanol concentrations and temperatures, (iii) NO2−, and (iv) under oxic conditions. Finally, the OB was cultivated for five weeks in the commercial Instant Ocean (IO) seawater. 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.

Results
In the first set of assays, the biofilm cultures had the capacity to sustain denitrifying activities in most of the tested conditions. Inhibition occurred when they were exposed to high pH (10) or to high methanol concentration (1.5%). In the second set of assays, the highest specific denitrification rates occurred with the biofilm cultures cultivated at 64.3 mM NO3− and 0.45% methanol, and at 30 °C. Poor biofilm development occurred with the biofilm cultures cultivated at 5% and 8% NaCl. In all biofilm cultures cultivated in ASW at 2.75% NaCl, H. nitrativorans strain NL23 decreased by three orders of magnitude in concentrations compared to that found in OB. This decrease coincided with the increase of the same magnitude of a subpopulation of M. nitratireducenticrescens (strain GP59 as representative). In the biofilm cultures cultivated at low NaCl concentrations (0% to 1.0%), persistence of H. nitrativorans strain NL23 was observed, with the gradual increase in concentrations of M. nitratireducenticrescens strain GP59. High levels of H. nitrativorans strain NL23 were found in the IO biofilm cultures. The concentrations of M. nitratireducenticrescens strain JAM1 were lower in most of the biofilms cultures than in OB.

Conclusions
These results demonstrate the plasticity of the marine methylotrophic denitrifying biofilm in adapting to different environmental changes. The NaCl concentration is a crucial factor in the dynamics of H. nitrativorans strain NL23, for which growth was impaired above 1% NaCl in the ASW-based biofilm cultures in favor of M. nitratireducenticrescens strain GP59.