Co-culturing Hyphomicrobium nitrativorans strain NL23 and Methylophaga nitratireducenticrescens strain JAM1 allows sustainable denitrifying activities under marine conditions

Hyphomicrobium nitrativorans strain NL23 and Methylophaga nitratireducenticrescens strain JAM1 were the principal bacteria involved in the denitrifying activities of a methanol-fed, fluidized marine denitrification reactor. We believe that a tight relationship has developed between these two strains to achieve denitrification in the reactor under marine conditions. To characterize the potential synergy between strain JAM1 and strain NL23, we compared some of their physiological traits, and performed co-cultures. Pure cultures of strain JAM1 had a readiness to reduce nitrate (NO3-) with no lag phase for growth contrary to pure cultures of strain NL23, which has a 2-3 days lag phase before NO3- starts to be consumed and growth to occur. Compared to strain NL23, strain JAM1 has a higher max for growth and higher specific NO3- reduction rates. Antagonist assays showed no sign of exclusion by both strains. Planktonic co-cultures could only be performed on low NaCl concentrations for strain NL23 to survive. Denitrification rates were twice higher in the planktonic co-cultures than those measured in strain NL23 pure cultures. Biofilm co-cultures were performed for several months in a 500-mL bioreactor filled with Bioflow supports, and operated under fed-batch mode with increasing concentrations of NaCl for strain NL23 to acclimate to marine conditions. Under these conditions, the biofilm co-cultures showed sustained denitrifying activities and surface colonization by both strains. Increase in ectoine concentrations produced by strain JAM1 was observed in the biofilm with increasing NaCl concentrations. These results illustrate the capacity of both strains to act together in performing denitrification under marine environments. Although strain JAM1 did not contribute in better specific denitrifying activities in the biofilm co-cultures, its presence was essential for strain NL23 to survive in a medium with NaCl concentrations > 1.0%. We believe that ectoine is an important factor for the survival of strain NL23 in these environments.