| The influx and enrichment of nitrogen is one of the most important factors responsible for the eutrophication of inland freshwater.The nitrification coupled with denitrification is a pathway of removing nitrogen in the form of gas,and the nitrification process is obligatory for the denitrification,both of which are of great importance for the balance of nitrogen in the freshwater ecosystem.The process of nitrification is mainly accomplished by ammonia-oxidizing microorganism(AOM)and nitrite-oxidizing bacteria(NOB),both of which are ubiquitous in nature and widely distributed in terrestrial,marine and freshwater ecosystems and so on.However,the knowledge of the population distribution,community structure,functional activity and their relative environmental factors still remain unclear now.In this study,the overlying freshwater and sediment have been collected from different eutrophicated areas for the investigation of Taihu Lake ecosystem.15N stable isotope tracing,13C stable isotope probing and high-throughput pyrosequencing techniques have been combined to reveal the effect of eutrophication levels,oxygen contents,temperature and nitrogen inputs on the nitrification process and nitrifying microorganism in Taihu Lake sediment,and the major results are as follows:(1)The surface sediment(0～-2 cm)from meso-eutrophic Meiliang Bay with higher nitrogen input in Taihu Lake was collected,and the culture experiment under different temperatures(10℃,20℃ and 30℃)was constructed based on the 15NH4NO3 and NH415NO3 double labeling tracing technique and FLUAZ model.The results showed that gross nitrification rates were 3.83,4.46 and 12.46 mg Kg-1 d.w.s d-1,gross denitrification rates were 4.14,14.93 and 15.23 mg Kg-1 d.w.s d-1,and the coupled nitrogen removal rates were 10.10%,35.55%and 37.21%respectively under the different culture temperatures.Temperature exhibited the significant impacts on the both gross nitrification(P<0.001)and denitrification rates(P<0.001)and nitrogen removal rates(P<0.001).There were significant correlations between gross nitrification rates and NH4+-N and TOC contents(P<0.05)as well as between gross denitrification rates and PNR and NO3-values(P<0.05).Furthermore,36%-84%of NH4+ was consumed by nitrification in Meiliang Bay sediment and more than 90%of nitrification product NO3-was consumed by denitrification and transformed to gas to the atmosphere under the temperature gradients.33%-81%of NH4+ in Meiliang Bay sediments was removed by nitrification.Therefore,nitrification plays an important role in the nitrogen removal in Meiliang Bay sediment.(2)qPCR,PCR-DGGE and high-throughput pyrosequencing technologies based on the amoA and 16S rRNA genes were applied to investigate the abundance of ammonia-oxidizers and community structure of nitrifying microorganism in water and surface sediment(0～-2cm)from meso-eutrophic Meiliang Bay and light-eutrophic central lake.Results demonstrated that the different eutrophication levels didn’t affect the the community compositions of nitrifying microorganism in Taihu Lake ecosystem but significantly affected the abundance of ammonia-oxidizers in water and sediment.The community structures of AOA in overlying water from both eutrophic areas were similar,and the community compositions of AOA in surface sediments from both sampling sites were similar too.However,the community structure differences of AOA between water and sediments were detected.The Nitrosopumilus and Nitrososphaera clusters are the shared AOA groups in both water and sediments,while the Nitrosotalea cluster is the distinct AOA cluster in water.AOB in both eutrophic areas were affiliated with Nitrosospira and Nitrosomonas clusters,with the Nitrosospira cluster(97.54%)as the dominant group in the surface sediment of meso-eutrophic area and the Nitrosomonas cluster(51.43%)as the the dominant group in the surface sediment of light-eutrophic area.NOB in surface sediments from two eutrophic areas was predominated by the Nitrospira cluster.Typical eutrophic indicators(TN,TP,ammonium and nitrate)and eutrophication related indicators(pH and DO)are the important environmental factors for affecting the abundance of ammonia oxidizers and community structure and abundance of nitrifiers.(3)13C-labeled DNA-SIP sediment microcosm experiment was applied to investigate the active nitrifying microorganism,and results showed that the active AOA in meso-eutrophic sediments with higher ammonium concentration were mainly affiliated with the Nitrosopumilus,Nitrosotalea and Nitrososphaera clusters,and the active AOA in light-eutrophic sediments with lower ammonium concentration mainly included the Nitrosopumilus and Nitrososphaera clusters,with the Nitrosopumilus as the predominant cluster in both eutrophic sediment.The active AOB in both eutrophic areas were mainly affiliated with the Nitrosomonas and Nitrosospira clusters,and there was the proportional variations from predominant Nitrosomonas cluster in the surface sediment to the Nitrosospira cluster in the deeper sediment.The active NOB was mainly affiliated with the Nitrospira cluster in all sediment,the abundance of which declined with the elevation of the ammonium concentration and sediment depth.The lower autotrophic activities of AOA and AOB were detected in the deeper sediment of meso-eutrophic Meiliang Bay and surface sediment of light-eutrophic central lake respectively.Results above demonstrated that the different eutrophication indexes(mainly referred to ammonium)and oxygen statuses could alter the contribution of ammonia oxidizers to the nitrification process and the community structures and abundance of active nitrifying microorganism.(4)The surface sediment was collected from both eutrophic areas,and 13C-labeled DNA-SIP sediment microcosm experiment was constructed based on the different nitrogen inputs and temperatures(10℃,20℃ and 30℃).Results showed that the autotrophic activity of AOB,which were mainly affiliated with the unknown Nitrosomonas-like and Nitrosomonas Is79A3 lineages,were the predominant ammonia-oxidizer in meso-eutrophic Meiliang Bay sediment.The relative abundance of the Nitrosomonas-like lineage increased with the elevation of temperature and the decline of nitrogen inputs,while the Nitrosomonas Is79A3 lineage exhibited the opposite tendency.Both AOA and AOB were not autotrophically active in both eutrophic sediment at 10℃ with normal and high nitrogen inputs but active at 20℃and 30℃.The active AOB in the light-eutrophic sediment was predominated by the Nitrosomonas-like lineage,and the active AOA were mainly affiliated with Nitrososphaera and Nitrosopumilus clusters,and the relative abundance of the Nitrosopumilus cluster decreased with the elevation of temperature and the decline of nitrogen inputs,while the Nitrososphaera cluster showed the opposite tendency.The active NOB in both eutrophic sediment was predominated by the Nitrospira cluster.The temperature significantly affected the relative abundance of the Nitrosopumilus,Nitrososphaera,Nitrosomonas-like,Nitrosomonas Is79A3 and Nitrotoga lineages(P<0.05),and the nitrogen inputs significantly affected the Nitrosomonas Is79A3 and Nitrotoga lineages(P<0.01).Results demonstrated that the different temperatures and nitrogen inputs could not only alter the roles of ammonia-oxidizers in the sediment nitrification process but the community compositions and abundance of nitrifying microorganism in both eutrophic sediment. |
PDF Full Text Request