Strain-level genetic diversity ofMethylophaga nitratireducenticrescensconfers plasticity to denitrification capacity in a methylotrophic marine denitrifying biofilm
Authors: Geoffroy Valérie , Payette Geneviève , Mauffrey Florian , Lestin Livie , Constant Philippe , Villemur Richard 
Published: April 2018

BackgroundThe biofilm of a methanol-fed, fluidized denitrification system treating a marine effluent is composed of multi-species microorganisms, among whichHyphomicrobium nitrativoransNL23 andMethylophaga nitratireducenticrescensJAM1 are the principal bacteria involved in the denitrifying activities. Strain NL23 can carry complete nitrate (NO${}_{3}^{-}$) reduction to N2, whereas strain JAM1 can perform 3 out of the 4 reduction steps. A small proportion of other denitrifiers exists in the biofilm, suggesting the potential plasticity of the biofilm in adapting to environmental changes. Here, we report the acclimation of the denitrifying biofilm from continuous operating mode to batch operating mode, and the isolation and characterization from the acclimated biofilm of a new denitrifying bacterial strain, named GP59.MethodsThe denitrifying biofilm was batch-cultured under anoxic conditions. The acclimated biofilm was plated onMethylophagaspecific medium to isolate denitrifyingMethylophagaisolates. Planktonic cultures of strains GP59 and JAM1 were performed, and the growth and the dynamics of NO${}_{3}^{-}$, nitrite (NO${}_{2}^{-}$) and N2O were determined. The genomes of strains GP59 and JAM1 were sequenced and compared. The transcriptomes of strains GP59 and JAM1 were derived from anoxic cultures.ResultsDuring batch cultures of the biofilm, we observed the disappearance ofH. nitrativoransNL23 without affecting the denitrification performance. From the acclimated biofilm, we isolated strain GP59 that can perform, likeH. nitrativoransNL23, the complete denitrification pathway. The GP59 cell concentration in the acclimated biofilm was 2–3 orders of magnitude higher thanM. nitratireducenticrescensJAM1 andH. nitrativoransNL23. Genome analyses revealed that strain GP59 belongs to the speciesM. nitratireducenticrescens. The GP59 genome shares more than 85% of its coding sequences with those of strain JAM1. Based on transcriptomic analyses of anoxic cultures, most of these common genes in strain GP59 were expressed at similar level than their counterparts in strain JAM1. In contrast to strain JAM1, strain GP59 cannot reduce NO${}_{3}^{-}$ under oxic culture conditions, and has a 24-h lag time before growth and NO${}_{3}^{-}$ reduction start to occur in anoxic cultures, suggesting that both strains regulate differently the expression of their denitrification genes. Strain GP59 has the ability to reduce NO${}_{2}^{-}$ as it carries a gene encoding a NirK-type NO${}_{2}^{-}$ reductase. Based on the CRISPR sequences, strain GP59 did not emerge from strain JAM1 during the biofilm batch cultures but rather was present in the original biofilm and was enriched during this process.DiscussionThese results reinforce the unique trait of the speciesM. nitratireducenticrescensamong theMethylophagagenus as facultative anaerobic bacterium. These findings also showed the plasticity of denitrifying population of the biofilm in adapting to anoxic marine environments of the bioreactor.