On The Stoichiometry Of Carbon And Nitrogen During Nitrification And Its N₂O Yield In The Pearl River Estuary

Nitrification is a critical process in nitrogen cycle,in which the ammonia oxidation is a key step of nitrification driven by two different functional microorganisms,ammonia-oxidizing bacteria(AOB)and ammonia-oxidizing archaea(AOA)under the aerobic condition.Both AOB and AOA use the energy generated during ammonia oxidation to fix the inorganic carbon for the synthesis of their own cell components.Due to the diverse physiological characteristics and pathways of carbon fixation,the stoichiometry of utilized carbon and nitrogen during ammonia oxidation by AOB and AOA may be different.However,this issue is still not constrained to date.On the other hand,nitrous oxide(N2O),one of the greenhouse gases,is produced as a by-product during ammonia oxidation.Unlike AOB,the AOA metabolic pathway for N2O production is still under debate.Moreover,the potentially distinct N2O yields of AOB and AOA need to be further resolved.Characterized by mixing between freshwater and seawater with salinity and nutrient gradients,the estuarine ecosystem is an ideal habitat to elucidate the responses of different microbial communities including nitrifiers to changing physicochemical settings.Strong nitrification and N2O supersaturation have been observed in the upper Pearl River estuary(PRE),which is likely attributed to exacerbation of the eutrophication by human activities.However,the controlling mechanisms of N2O production and yield in the upper PRE are still not clear.Aiming to reveal the stoichiometry of carbon and nitrogen in ammonia oxidation and the relative contributions of AOB and AOA to N2O production,we investigated the distribution and bioactivity of AOB and AOA in response to environmental factors in the PRE.Furthermore,the utilization and isotope fractionation effects of carbon and nitrogen of AOB vs.AOA during ammonia oxidation was compared through both pure strain incubations and in situ incubation experiments.The major findings of this study are summarized as follows:1.During two cruises to the PRE in July 2014 and July 2015,bacterial 16S rRNA,archaeal 16S rRNA,?-amoA,arch-amoA and nirS genes were used as biomarkers to study the distribution and activity of bacteria,archaea,AOB,AOA and denitrifiers through fluorescence qPCR and reverse transcription of cDNA.The environmental factors affecting on the distribution and activity of AOB and AOA were also discussed.The microbial community structure displayed significant spatial variability in the PRE,which is mainly influenced by water mass mixing.In the upper estuary,bacteria predominated over archaea.While the abundances of AOB and AOA were overall on the same order of magnitude in this region,cDNA characterizing the activity of AOB(AOB-cDNA)was comparable in magnitude to that for AOA(AOA-cDNA).The abundances of bacteria and AOB decreased downstream to the mid-estuary,whereas archaea and AOA increased.AOA-cDNA was slightly higher than AOB-cDNA in this region.In the lower estuary,AOA became the dominant ammoxiders,and AOA-cDNA significantly exceeded AOB-cDNA.The individual cell activity of AOB characterized by the cDNA/DNA ratio was higher than that of AOA along the whole PRE.The distribution of nirS gene showed insignificant regional differences.We infer that AOB was more adapted the environment of lower salinity,higher nutrients,more NH3/NH4+substrate,lower DO and higher TSM.AOA was more adapted the marine environment with higher salinity and lower NH3/NH4+ substrate(<1 ?M).2.During the cruise in July 2014,we used stable carbon isotopic ratios(?13C)to trace the source of particles in the PRE.Base on the size difference of a single cell between AOB and AOA,their in situ distribution and activity were distinguished by the size fractionated experiments.We also applied an endmember mixing model to analyze the C/N and ?13C of size fractionated particles.In addition,we quantified the utilization of carbon and nitrogen and isotopic fractionation effects(?)of carbon and nitrogen during the ammonia oxidation driven by AOA via incubation experiments using the AOA pure strain Nitrosopumilus maritimus SCM1.The results were compared with those obtained in previous laboratory culture studies using the AOB pure strains,both of which were further discussed relative to the field measurements.While AOB was the dominant functional microorganism in the freshwater(terrestrial)region,AOA dominated in the marine environment.AOB was mainly associated with particles showing higher activity.In contrast,more proportion of AOA was free-living and expressing higher activity the sea-water with high salinity and low TSM.Although the total abundance of AOB-cDNA was lower than that of AOA-cDNA,the individual cell activity of particulate AOB was higher relative to particulate AOA.Using the ratios of amoA/16S rRNA genes as end-members to estimate the ?13C of size fractionated particulate matters dominated by AOB and AOA,respectively,we found that ?13CAOB was more negative than ?13CAOA.This may be due to the distinct environmental DIC-?13C substrate or the different inorganic carbon assimilation pathways between AOB and AOA.In the late stationary state of a closed incubation system,of AOA cultures(N.maritimus SCM1),the average C/Nbulk was 6.0,which is higher than that of 3.1-5.0 for AOB pure strains.The average C isotope fractionation(?bulk/DIC)of N.maritimus SCM1 was estimated to be 19.4‰ using a Raleigh fractionation model,which is smaller than the ?bulk/DIC of terrestrial ?-AOB(Nitrosomonas europaea,20.0-30.0‰).The N isotope fractionation 15?NH4+ of N.maritimus SCM1 obtained in our incubations was 25.2‰,which is also lower than the 15?NH4+ of terrestrial ?-AOB(N.europaea,32.8-38.2‰).Laboratory culture studies imply that the difference in the carbon and nitrogen composition of biomass(C/Nbulk),carbon and nitrogen isotopic fractionation(?bulk/DIC and 15?NH4+)of AOB and AOA may be dependent on nitrifier species diversity,distinct environmental inorganic carbon and nitrogen substrate,or the availability of substrate.Using the steady-state fractionation model for an open system in the PRE,the?bulk/DIC in the AOB-dominated fresh water region was estimated to be 23.7-31.0‰and the ?bulk/DIC in the AOA-dominated marine region was estimated to be 20.6-23.3‰.Both values agreed well with the lab culture results.3.During the PRE cruise in July 2015,transformation of inorganic nitrogen species and the N2O yield during nitrification were evaluated via the in situ incubation experiments conducted in the hypoxic upper reach.In addition,the relative contributions of AOB and AOA to the N2O production in the PRE were estimated.Nitrification is the dominant process and primary contributor for N2O production in the upper Humem.N2O yield associated with individual cell of AOB is higher than that of AOA.Oxygen conditions and cell abundances of AOB and AOA could co-impact on N2O yield.Whereas in the Guangzhou upper reach with hypoxic to anaerobic conditions,nitrification and denitrification processes potentially coupling or co-occurring.N2O yield contributed by AOB and AOA was not balanced with the field N2O output,indicating other processes may contribute to N2O production.To conclude,this study reveals the influence of various environmental factors on the microbial structures and activities in the PRE.AOB and AOA showed difference in utilization of carbon and nitrogen,and isotope fractionation effects(?)during ammonia oxidation.N2O produced through nitrification in the hypoxic upper estuary was verified,the relative contribution of AOB vs.AOA to N2O yield was primarily revealed.