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

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.

. Schematic of the conditions used to test the denitrification capacity of the Ref300N-23C biofilm cultures -The original biofilm was thawed, scraped from the carriers and distributed to vials containing the ASW medium supplemented with 300 mg NaNO 3 -N/L and 0.15% methanol and 20 free carriers. These carriers were transferred 5 times in fresh medium. At the 5 th , 10 th , 15 th and 20 th carrier-transfers, series of three colonized carriers were distributed into other vials containing fresh culture medium and 17 new carriers. The vials were cultivated and the carriers transferred as before.
-At the 10 th , 12 th , 15 th and 20 th carrier-transfers, series of 15 freshly colonized carriers were transferred into vials containing the prescribed medium to assess the impact in varying the NO 3 -, NO 2 and methanol concentrations or the oxic conditions on the denitrifying activities.
-At the 25 th transfer, series of 15 freshly colonized carriers were transferred into vials containing the prescribed medium to assess the impact in varying the pH, the temperature and the NaCl concentrations on the denitrifying activities. The biofilm was cultivated for two days in these prescribed conditions before the carriers to be transferred in fresh prescribed medium. -Kinetics of NO 3 and NO 2 were monitored at regular intervals. Protein and DNA extractions were then performed from the biofilm and the suspended biomass.  The original biofilm was thawed, scraped from the carriers and distributed to vials containing the ASW medium supplemented with prescribed concentrations of NO 3 -, NO 2 -, methanol and NaCl, or containing the IO medium (Table 2), and 20 free carriers. The vials were incubated at the prescribed temperatures and at pH 8 under anoxic or oxic conditions (Table  2). In average once a week, the carriers were transferred five times into fresh prescribed medium and incubated in the same conditions. For the 2.75-5%NaCl assay, the first five transfers were incubated in the same conditions of Ref300N-23C cultures, then the three subsequent transfers were incubated in ASW medium with 5% NaCl. NO 3 and NO 2 concentrations were measured in each one or two days. Methanol and NaNO 3 were added when needed if NO 3 was completely depleted during the week. DNA was extracted from the biomass for PCR-DGGE and qPCR assays. During the 5 th carrier-transfer cultures (or the 8 th for the 2.75-5%NaCl and the 200/200N biofilm cultures), 15 carriers from each vial were transferred into their respective fresh medium, and concentrations of NO 3 and NO 2 were measured at regular intervals. Protein content in the whole vials was then measured.  2.75-5%NaCl (ASW, 300 mg-NO 3 --N/L, 23°C for 5 transfers at 2.75% NaCl, then 3 transfers same conditions but at 5% NaCl) * * OB Tr1 Tr2 Tr3 Tr4 Tr5s Tr5B   JAM1/GP59   NL23   OB Tr1 Tr2 Tr3 Tr4 Tr5s Tr5B OB Tr1 Tr2 Tr3 Tr4 Tr5s Tr5B   0% 0.5% 1.0% NaCl Figure S4.

Bacterial community profiles derived by PCR-DGGE of the biofilm cultures cultivated in ASW with different NaCl concentrations
The original biofilm (OB) was cultivated in the ASW medium composed of 0%, 0.5% and 1.0% NaCl. Carriers were transferred 5 times. DNA extraction was performed at the end of the carrier-transfer cultures. PCR-DGGE migration profiles (8% polyacrylamide DGGE with a 30% to 70% denaturant gradient) of 16S rRNA gene sequences were derived from the OB, the suspended biomass of the 1 st to the 5 th carrier-transfers (Tr1 to Tr5S), and the carrier biofilm after the 5 th carrier-transfer (Tr5B). Triplicates produced the same profiles.
OB Tr1 Tr3 Tr5s Tr5B   JAM1/GP59   NL23   OB Tr1 Tr3 Tr5s Tr5B Ref300N-23C IO Figure S5. Bacterial community profiles of the Ref300N-23C biofilm cultures and the IO biofilm cultures by PCR-DGGE The original biofilm (OB) was cultivated in the IO medium under the same conditions of the Ref300N-23C biofilm cultures (ASW). DNA was extracted from the OB, from the suspended biomass after the 1 st , 3 rd and 5 th carrier-transfers, and from the carrier biofilm after the 5 th carrier-transfers (Tr5B). 16S rRNA gene sequences were PCR amplified and segregated on PCR-DGGE (8% polyacrylamide DGGE with a 30% to 70% denaturant gradient). Triplicates produced the same profiles.