Microbial Nitrogen Incorporation Identified By Stable Isotope Probing In Euphotic Zone Of South China Sea

Primary production in the marine euphotic zone is limited by the availability of nutrients.Nitrogen compounds are some of the most limited nutrients in the euphotic zone.Beside uptake of dissolved organic nitrogen(DON),heterotrophic bacteria also assimilate dissolved inorganic nitrogen(DIN),which is reported by increasing works.Competition for nitrogen-based nutrients between heterotrophic bacteria and phytoplankton directly influences the productivity of phytoplankton.Incorporation of NH4+ and NO3-by heterotrophic bacteria also complicates new and regenerated productivity.Archaeal communities,especially ammonia-oxidizing archaea,also play a crucial role in controlling marine productivity.Ammonia-oxidizing archaea decrease available NH4+ and thus increase the amount of NO3-available for phytoplankton.In the euphotic zone,light has the greatest effect on the uptake of nitrogen-based nutrients by phytoplankton and the archaeal ammonia oxidation process,whereas the availability of organic matter mainly influences the uptake of nitrogen-based nutrients by heterotrophic bacteria and archaea.In this study,seawater from 20%and 1%surface photosynthetically active radiation(sPAR)water depths at a mesotrophic site(I1)and an oligotrophic site(SEATS)in the South China Sea were incubated at simulated in situ conditions.Following incubation,DNA-stable isotope probing combined with nitrogen uptake rates,qPCR and high-throughput sequencing were performed to characterize the uptake of typical DIN species(NH4+,NO3-and NO2-)and DON compounds(urea)by bacteria and archaea in the<20 ?m size fraction(3-20 ?m phytoplankton included)and in the<3 ?m size fraction(3-20 ?m phytoplankton removed),respectively.Microbial communities,especially the bacterial communities,in our study slightly preferred NH4+ over urea,but much preferred it over NO3-and NO2-.Uptake rates of nitrogen-based slightly changed with the distinct weakening of light radiation where phytoplankton provided organic energy.In contrast,uptake rates of nitrogen-based obviously decreased with the distinct weakening of light radiation in the absence of phytoplankton to provide organic energy.These results indicated that microbial nitrogen-based nutrient uptake was influenced by organic matter and light radiation at the same time,and the influence of light radiation was more pronounced in oligotrophic environments.In oligotrophic site SEATS,percentages of eukaryotic phytoplankton and archaeal 15N-DNA at 20%sPAR were significantly higher than at 1%sPAR,while percentages of bacterial 15N-DNA between 20%and 1%sPAR were not significantly different,indicated that the uptake of nitrogen-based nutrient by eukaryotic phytoplankton and archaea was more affected by light.Further,the abilities of nitrogen-based nutrient incorporation,especially the NH4+ incorporation abilities of archaeal communities at 20%sPAR were significantly higher than at 1%sPAR,indicating that the major sink of NH4+ at 20%sPAR was absorption by the microbial community.In contrast,NH4+ was slowly absorbed by the microbial community at 1%sPAR;therefore,ammonia-oxidizing archaea might have more available NH4+ for ammoxidation at 1%sPAR than 20%sPAR.This deduction was proofed by DNA-SIP combined with high-throughput sequencing of archaeal 16S rRNA gene which showed that MGI was mainly found in 1%sPAR,and archaea MGII nitrogen substrates incorporation mainly occurred at 20%sPAR.Bacterial communities in the microbial assemblages of the sites were the major nitrogen substrate incorporators.The phytoplankton in 3-20 ?m size fraction made up approximately 10%of the microbial community at both depths of site I1.The percentages of bacterial 15N-DNA significantly decreased when the phytoplankton fraction was removed.However,the low abundance of phytoplankton(3-20 ?m size fraction)at the SEATS site had an insignificant effect on the percentages of bacterial 15N-DNA.These results indicated that bacterial nitrogen-based nutrient incorporation benefited from the presence of phytoplankton.It is likely that phytoplankton supply organic matter for bacterial communities to facilitate nitrogen-based nutrient incorporation.High-throughout sequencing of bacterial and archaeal 16S rRNA genes showed that the bacterial community structure between 20%and 1%sPAR was significantly different.Additionally,the bacterial community structure in the<20 ?m and<3 ?m size fractions from the same depth also showed significant differences.The bacterial communities from samples supplemented with different nitrogen-based nutrients in the same treatment group had a more similar structure compared with samples in other treatment groups.These results indicated that the differences in bacterial community structure were mainly caused by environmental variations,and bacterial communities showed diverse selectivity for nitrogen-based nutrients in the same environment.Photosynthetic bacteria were the major incorporator of nitrogen substrates at 20%sPAR,while heterotrophic bacteria incorporated nitrogen substrates at both 20%and 1%sPAR;they also competed for nitrogen substrates with photosynthetic bacteria at 20%sPAR.The diversity of photosynthetic bacteria that incorporated nitrogen substrates was low.The nitrogen-based nutrients at 20%sPAR at both sites were primarily incorporated by species of one Prochlorococcus clade.The heterotrophic bacteria that incorporated nitrogen-based nutrients were more diverse,including those related to a-Proteobacteria and y-Proteobacteria.The relative abundances of ?-Proteobacteria were higher at mesotrophic site I1,while the relative abundances of Prochlorococcus were higher at the oligotrophic site SEATS.This indicated that heterotrophic bacteria have some advantages in nitrogen uptake in relatively eutrophic environments.The characteristics of the archaeal nitrogen-based nutrient incorporation between the two depths were also significantly different.Archaea MGI,which exhibit strong environment adaptiveness,was mainly found in 1%sPAR samples,but was unstimulated by the supplementation of 2 ?M of NH4+ and urea.In contrast,aArchaea MG II nitrogen substrates incorporation mainly occurred at 20%sPAR depth.