Cucaita, A. | bioRxiv Science

bioRxiv2021

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.

microbiology↗

bioRxiv2017

Denitrifying metabolism of the methylotrophic marine bacterium Methylophaga nitratireducenticrescens strain JAM1

Methylophaga nitratireducenticrescens strain JAM1 is a methylotrophic, marine bacterium that was isolated from a denitrification reactor treating a closed-circuit seawater aquarium. It can sustain growth under anoxic conditions by reducing nitrate (NO3-) to nitrite (NO2-), which accumulates in the medium. These physiological traits are attributed to gene clusters that encode two dissimilatory nitrate reductases (NarGHJI). M. nitratireducenticrescens strain JAM1 also contains gene clusters encoding two putative nitric oxide reductase (NO) reductases and one putative nitrous oxide (N2O) reductase, suggesting that NO and N2O can be reduced by strain JAM1. In this study, we show that strain JAM1 can reduce NO to N2O and N2O to N2 and can sustain growth under anoxic conditions by reducing N2O as the sole electron acceptor. Although strain JAM1 lacks a gene encoding a dissimilatory copper-(NirK) or cytochrome cd1-type (NirS) NO2- reductase, NO3--amended strain JAM1 cultures produce N2O, representing up to 6% of the N-input. NO2- was shown to be the key intermediate of this production process. In NO3-amended cultures, we analyzed denitrification genes in succession of net N2O-production and -consumption phases at the gene expression level. These phases were found to correlate with changes in the expression levels of the NO reductase gene cnorB1 and nnrS, which indicated NO production in the cultures.\n\nImportanceBy showing that all the three denitrification reductases are active, this demonstrates that Methylophaga nitratireducenticrescens JAM1 is one of many bacteria species that maintain genes associated primarily with denitrification, but not necessarily related to the maintenance of the entire pathway. The reason to maintain such incomplete pathway could be related to the specific role of strain JAM1 in the denitrifying biofilm of a denitrification reactor from which it originates. The small production of N2O via NO in strain JAM1 did not involve Nar contrary to what was demonstrated in Escherichia coli. M. nitratireducenticrescens JAM1 is the only reported Methylophaga species that has the capacity to grow under anoxic conditions by using NO3- and N2O as sole electron acceptors for its growth. It is also one of a few marine methylotrophs that is studied at the physiological and genetic levels in relation to its capacity to perform denitrifying activities.

microbiology↗