Impact of NaCl, nitrate and temperature on the microbial community of a methanol-fed, denitrifying marine biofilm

Background The biofilm of a continuous, methanol-fed, fluidized denitrification system that treated a marine effluent at the Montreal Biodome is composed of a multi-species microbial community, among which Hyphomicrobium nitrativorans NL23 and Methylophaga nitratireducenticrescens JAM1 are the principal bacteria involved in the denitrifying activities. To assess its resilience to environmental changes, the biofilm taken from the denitrification system was cultured at laboratory scale in artificial seawater (ASW) under anoxic conditions and exposed to a range of specific physico-chemical parameters. We previously showed that the seawater formulation and the NaCl concentrations had a strong impact on the H. nitrativorans NL23 population, with its displacement by a new denitrifier, M. nitratireducenticrescens GP59. Here, we report the impact of these cultures conditions on the dynamics of the overall microbial community of the denitrifying biofilm. Methods The original biofilm (OB) taken from the denitrification system was acclimated for five weeks in ASW under anoxic conditions with a range of NaCl concentrations, and with four combinations of nitrate concentrations and temperatures. The OB was also acclimated to the commercial Instant Ocean seawater medium (IO). The bacterial diversity of the biofilm cultures and the OB was determined by 16S ribosomal RNA amplicon metagenome sequencing. Culture-dependent approach was used to isolate other denitrifying bacteria from the biofilm cultures. The metatranscriptomes of some of the biofilm cultures were derived, along with the transcriptomes of planktonic pure cultures of H. nitrativorans NL23 and M. nitratireducentricrescens GP59 cultivated under denitrifying conditions. Results The 16S metagenomic data revealed very high proportions of M. nitratireducenticrescens in the biofilm cultures. H. nitrativorans NL23 was found in high proportion in the OB, both was absent in the biofilm cultures with 2.75% NaCl in the ASW medium. It was found however in low proportions in the biofilm cultures with 0 to 1% NaCl in the ASW medium and in the IO biofilm cultures. Emergence of Marinicella spp. occurred in these biofilm cultures. Denitrifying bacterial isolates affiliated to Marinobacter spp. and Paracoccus spp. were isolated. Up regulation of the denitrification genes in strains GP59 and NL23 occurred in the biofilm cultures compared to the planktonic pure cultures. Denitrifying bacteria affiliated to the Stappia spp. were metabolically active in the biofilm cultures. Conclusions These results illustrate the dynamics of the microbial community in the denitrifying biofilm cultures in adapting to different environmental conditions. The NaCl concentration is an important factor affecting the microbial community in the biofilm cultures. Up regulation of the denitrification genes in strain GP59 and strain NL23 in the biofilm cultures suggests different mechanisms of regulation of the denitrification pathway in the biofilm compared to the planktonic pure cultures. Other denitrifying heterotrophic bacteria are present in low proportions in the biofilm, suggesting that the biofilm has the potential to adapt to heterotrophic, non-methylotrophic environments.