Ecology Study On Activity,abundance And Diversity Of Nitrite-oxidizing Bacteria In Seawater Of The Northern South China Sea

Nitrification is a critical component of marine nitrogen cycle,which could be the most important nitrogen transformation process in nutrient-deficient and oxic seawater environments in the South China Sea.Nitrifiers including ammonia-oxidizing bacteria(AOB),ammonia-oxidizing archaea(AOA)and nitrite-oxidizing bacteria(NOB),are chemolithoautotrophic microorganisms and they dominate the nitrification process,oxidizing NH3 via NO2-to NO3-,conserving energy and fixing carbon dioxide.They can directly couple the nitrogen cycling process and the carbon cycling process.The second step of nitrification-nitrite oxidation is carried out by NOB.To a certain extent,NOB controls the direction and efficiency of nitrogen cycling(NO2-?NO3-)in the South China Sea.Carbon fixation by NOB,as important autotrophic microorganisms,may be very important part of primary productivity and carbon storage in the South China Sea.NOB may play an essential role in both marine nitrogen cycle and carbon cycle.So far,the study of marine NOB mainly focuses on nitrite oxidation rate,however,the study on ecology of NOB,such as their abundance,diversity,community structure,spatial distribution and controlling factors in marine environment is rarely conducted.Moreover,most of the previously studied environments are oxygen minimum zones(OMZs)or other special environments.The studies of NOB were much less frequently conducted in the generally oxic seawater environments that are common in the ocean.Lots of scientific issues about marine NOB still remain unknown,such as their in situ activities,abundance,diversity,community structure,spatial distribution,and controlling factors in marine environments.The understanding of marine NOB is still not comprehensive and systematic.Therefore,it is necessary to study the activity,abundance,community structure,spatial distribution and controlling environmental factors of NOB in the ocean.In this thesis,the nitrification rate was determined by stable isotope 15N tracer method in the northern South China Sea(nSCS).Two key functional genes,nxrB(encoding the nitrite oxidoreductase ? subunit)and aclA(encoding the ATP citrate lyase a subunit),were used as molecular markers to design PCR primers,construct functional gene DNA libraries(gene library)and cDNA libraries(gene transcript library),and quantify the different NOB groups and their active communities.We also collected and determined various environmental factors and analyzed the activity,nitrification rate,diversity,community structure,gene and transcript abundance,spatial distribution of different NOB groups,and the correlations between environmental factors,community structure and gene/transcript abundance of NOB groups by a diversity analyses,phylogenetic analysis,principal coordinates analysis,Jackknife clustering analysis,canonical correspondence analysis,and pearson correlation analyses.There is a difference in the spatial distribution of nitrification rate in the seawater of nSCS.For vertical distribution,the nitrifiers in the surface seawater may be inhibited by light or by substrate competion from phytoplankton so the in situ nitrification rate is very low or negligible.The rates of ammonia oxidation and nitrite oxidation at the bottom of shallow sea station or subsurface of deep sea stations are the highest,and gradually decreased below 200 m at deep sea stations.For horizontal distribution,nitrification rate in offshore was higher than inshore stations.Nitrite oxidation rate is generally higher than ammonia oxidation rate,and Pearson correlation analysis result shows that there is no significant correlation between ammonia oxidation rate and nitrite oxidation rate,indicating that they are not completely coupled.We thus hypothesize that,in addition to the nitrite provided by ammonia oxidation for nitrite oxidation,NO3-reduction in seawater organic aggregates or in sediments may also provide NO2-for nitrite oxidation.The four pairs of primers designed in this study are highly specific.DNA and cDNA clone libraries of functional genes nxrB and aclA of Nitrospinaceae and Nitrospira showed that all functional genes can be constructed for the DNA and cDNA clone libraries except that the transcript sequences of the Nitrospira aclA gene can not be detected at all sampling sites,so no Nitrospira aclA cDNA libraries were constructed.There is almost no PCR product in the surface water of nSCS,which is consistent with the nitrite oxidation rate determination results.However,we can construct both DNA and cDNA clone libraries below surface water.The proportion of Nitrospinaceae is much higher than that of Nitrospira in seawater of nSCS,accounting for about 98%of marine NOB in nSCS.The aclA gene sequences are more variable than nxrB of NOB in nSCS,indicating that the nxrB gene sequences of marine NOB are more conservative,which is more favorable to marine NOB that need the functional stability of the critical NXR enzymes to conserve energy from nitrite oxidation.Nitrospira in the seawater of nSCS belongs to Nitrospira lineage ?,and all obtained nSCS nxrB sequences in the Nitrospinaceae lineage are closest relatives to Nitrospina nxrB sequences previously obtained from the eastern tropical southern Pacific oxygen minimum zone or several Nitrospinaceae strains obtained from other marine environments.Nitrospinaceae and Nitrospira have obvious dominant species,and the functional genes of dominant species have transcriptional activity,widely distributed in the nSCS.In order to determine which NOB groups exist in the seawater of nSCS,we quantified several NOB groups(including Nitrospinaceae,Nitrospira,Nitrobacter,Nitrotoga,and Nitrococcus)that may be present in the ocean.Only Nitrospinaceae and Nitrospira were detected,and Nitrospinaceae dominate in nSCS.Therefore,Nitrospinaceae are the main participants and contributors to nitrite oxidation,which can account for about 3.9%of the total microbial community in nSCS.The spatial distribution of nxrB gene abundance and transcript abundance of two NOB groups are consistent with aclA abundance.Functional genes abundance were almost undetectable in surface of ocean,while the abundance in subsurface of deep sea and bottom of shallow sea is the highest,consistent with nitrite oxidation rate.The abundance of nxrB gene was slightly higher than aclA gene,but the abundance of nxrB transcripts was much higher than aclA transcripts,indicating that the expression of the NXR gene is higher.Pearson correlation analysis result showed that the gene abundance of Nitrospinaceae nxrB,Nitrospinaceae+Nitrospira nxrB,Nitrospinaceae aclA,Nitrospira aclA and the transcript abundance of Nitrospinaceae+Nitrospira nxrB,Nitrospinaceae aclA all were significantly positively correlated with the nitrite oxidation rate(P<0.05).The transcript abundance of Nitrospinaceae nxrB had a marginally significant positive correlation with the nitrite oxidation rate(P=0.066).The results of PCoA and Jackknife clustering analysis showed that the community structure and activity of NOB were related to seawater depth.The community structure of NOB was similar in 5 m,200 m and below 200 m,where the in situ nitrite oxidation activity was low.The community structure of NOB in subsurface is similar,with much higher activity.The structure and activity of NOB in the subsurface seawater are different from those of NOB in the surface,middle and deep seawater.The result showed that dissolved oxygen concentration(DO)was significantly correlated with the community structure and spatial distribution of NOB in seawater of nSCS by CCA analysis of Nitrospinaceae nxrB gene,Nitrospinaceae+Nitrospira nxrB gene and Nitrospinaceae aclA gene transcripts,which suggests that DO has a significant effect on marine NOB,and lots of studies have shown that both Nitrospinaceae and Nitrospira are microaerobic bacteria,so high concentrations of DO may inhibit these NOB groups.The result showed that salinity was also significantly correlated with the community structure and spatial distribution of NOB in seawater of nSCS by CCA analysis of Nitrospinaceae+Nitrospira nxrB gene sequences.The result showed that NO3-was significantly correlated with the community structure and spatial distribution of NOB in seawater of nSCS by CCA analysis of Nitrospira aclA gene,Nitrospinaceae nxrB gene transcripts and Nitrospinaceae+Nitrospira nxrB gene transcripts.NO3-is the product of nitrite oxidation,which also means that the NOB in seawater of nSCS is active,consistent with higher nitrite oxidation rate.The result showed that seawater depth was significantly correlated with the community structure and spatial distribution of Nitrospinaceae in seawater of nSCS by CCA analysis of Nitrospinaceae aclA gene sequences.The light intensity,temperature and substrate concentration in different water depths are different,which may have a greater impact on marine NOB.Pearson correlation analysis showed that there was a significant positive correlation between ammonia oxidation rate and NH4+,NO2-,and Chl-?.Nitrite oxidation rate had significant positive correlation with DIC.The gene abundance of Nitrospinaceae nxrB,Nitrospinaceae+Nitrospira nxrB,Nitrospinaceae aclA and Nitrospira aclA had a significant positive correlation with DIC,which is the substrate for carbon dioxide fixation of NOB,indicating that NOB in seawater of the nSCS has activity of carbon sequestration.The gene transcript abundance of Nitrospinaceae nxrB,Nitrospinaceae+Nitrospira nxrB,Nitrospinaceae aclA had a significant positive correlation with salinity.The relative abundance of the three functional genes of Nitrospinaceae nxrB,Nitrospinaceae+Nitrospira nxrB and Nitrospinaceae aclA had significant positive correlations with water depth,Eh,SiO32-,PO43-,NO3-,and DIN,and had significant negative correlations with temperature,DO,pH,and DOC.The relative abundance of Nitrospira aclA gene has significant positive correlations with Eh,NO3-,and DIN,and significant negative correlations with temperature,pH,and DOC.Both CCA and Pearson correlation analyses indicate that the environmental factors NO3-,salinity,DO,water depth and DIC were correlated significanly with the community structure,spatial distribution,abundance and activity of NOB in seawater of the nSCS.