| N2O is not only an important greenhouse gas,and also contribute to ozone destruction in the stratosphere,and it has become the major issue influencing the natural ecosystem and threatening the survival of mankind.Wastewater treatment process potentially contributes to N2O emission,which can be produced in the process of nitrification and denitrification as well.Constructed wetlands(CWs) is a mosaic of aerobic and anaerobic microsites that nitrification and denitrification could be occurring at the same time.Therefore,it is likely that multiple processes are contributing simultaneously to N2O formation.CWs are widely used for wastewater treatment with many advantages,including low cost,easy operation and maintenance. Therefore,active study on production mechanism and control of N2O in CWs is of important practical significance.The nitrous oxide fluxes in two typical CWs,i.e.subsurface flow(SF) and free water surface(FWS) were studied by the method of static chamber-gas chromatography.They were investigated that the correlations between N2O emission and its influential factors,including CW structure,plant species,influent concentration,C/N ratio,water quality,wet-dry alternation and temperature.Based on this,the measures were put forward for controlling N2O emission.Colony libraries were constructed and the gene diversity were analyzed by the denaturing gradient gel electrophoresis(DGGE) of functional gene in close relationship with N2O emission, which provided fundamental data for the regulation and control of N2O release in the way of microbial population structure.The main contents and results are following:(1) The nitrous oxide fluxes in two typical CWs were studied,and the results showed that the mean N2O fluxes were 296.5,28.2,249.2 and 10.8μg·(m2·h)-1 in SF and FWS systems with and without Phragmites australis,respectively.The two typical wetlands were all the sources of atmosphere nitrous oxide as a whole.SF wetland with Phragmites australis exhibited a higher risk of N2O emission,and the mean N2O flux in this system was higher than the values reported in the literature for ecosystems e.g.farmland,forest,grassland and marsh.The mean N2O flux in FWS wetland with Phragmites australis was higher than the values reported in the literature for forest and grassland marsh,but lower than other ecosystems.The nitrous oxide fluxes in test wetlands presented obvious seasonal and diurnal variation,and the highest N2O emission flux was in July.The highest flux was(762.9±239.3)μg·(m2·h)-1 and(91.9±20.3)μg·(m2·h)-1 in SF and FWS wetlands with Phragmites australis,respectively.The peak flux mostly occurred around midday,whereas the minimum flux likely occurred in the early morning.The results indicated that the growth of Phragmites australis and temperature were the key factor controlling the variation of N2O fluxes.The average N2O emission from the microsites above the inflow zones was higher than that above the outflow microsites.High influent strength promoted nitrification and denitrification,and high fluxes were obtained.(2) It was found that N2O emission of microcosm wetlands were significantly affected by CW structure,plant species,influent concentration,C/N ratio,water quality,wet-dry alternation and temperature.SF wetland exhibited a higher risk of N2O emissions,and the mean N2O flux in this system was higher than FWS wetland. Phragmites australis participate in the N2O emission directly or indirectly.The results showed that N2O fluxes had obvious differences in vegetation systems ranging from -130.2μg N2O m-2 h-1 to 1847.8μg N2O m-2 h-1,and the highest flux of N2O was observed in the TL systems.The higher risk of N2O emissions was observed in CWs planted with Typha latifolia and Scirpus validus,while the lowest N2O emission was in Phragmites australis systems.The proportion of loaded N emitted as N2O-N in this work was 0.33%-0.65%,or 0.0033-0.0065 kg N2O-N produced per kg of N input. The seasonal variation of N2O fluxes was different in six vegetation systems,but the lowest N2O fluxes were all in winter.The mean N2O flux increased with influent concentration,but they had no significant difference.It was found that N2O emission and the performance of microcosm wetlands were significantly affected by COD/N ratio of wastewater influent.A large quantity of N2O emission was detected from lower and higher COD/N ratio systems.The total N2O emission at a COD/N ratio of 20 was 10 times greater than that at a COD/N ratio of 10 and 5.During the operation of the microcosm wetland at a COD/N ratio of 5,both successful treatment performance and N2O emission control were obtained.There was no organic carbon input in the systems with an influent COD/N ratio of 0 and nitrification was the dominant process for N2O emission.The accumulation of NO2- and low pH value stimulate N2O emission.N2O emission increased after discharge in the system with wet-dry alternation.Temperature was the key factor controlling the variation of N2O fluxes.Therefore,using FWS structure,stabilization of C/N ratio and pH,avoiding wet-dry alternation and reducing accumulation of NO2- would be very important measures for controlling the greenhouse-effect gas emission of N2O.Phragmites australis should be given priority to adopt for the lower risk of N2O emissions and successful treatment performance.(3) The clone result showed the microbial diversity in subsurface constructed wetlands of reeds is very abundance,which including rhizophere bacterium, nitrogen-removing bacterium and nitrogen-fixing bacterium.According to the data obtained in this study,ammonia-oxidizing bacterial community showed little diversity in the system.Nitrosomonas sp.and Nitrosospira sp.were the dominant bacterium containing amoA functional gene which may play a leading role in contributing to N2O production in constructed wetlands.(4) The bacterial community in the substrate was analyzed by PCR-DGGE,and the results showed that bacterial communities were significantly affected by plant species, influent concentration and wet-dry alternation.The similarity of communities between each other was low in the DGGE profiles.In order to realize effect of different sets of universal primers on the analysis of microbial community based on targeted sequence of 16S rDNA,16S rDNA fragments were amplified with two primer sets(F357/R518 and F968/R1401).Separated patterns of the targeted sequence of primer F357/R518 were better than of F968/R1401.Thereby,while the sample of CWs was analyzed by DGGE,primer F357/R518 was more effective than F968/R1401.For the CWs with Phragmites australis and wet-dry alternation,the band in nosZ DGGE profiles was abundant in some region with 40%-42%denaturant concentration.These microorganisms were important for reducing N2O to N2,and the N2O emission was lower in Phragmites australis and wet-dry alternation systems.
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