| Nitrogen cycle is one of the most important material cycle and plays a very important role for the balance of ecosystem. Ammoxidationis a limited-reaction process driven by ammonia-oxidizing microorganisms, which is considered as the most critical reaction step in the nitrogen cycle. Ammonia-oxidizing microorganisms are able to oxidize ammoniato hydroxylaminecatalyzed by ammonia monooxygenase,and then hydroxylamine is oxidized to nitrous acid by nitrite bacteria to be used by plants and animals. Ammonia-oxidizing archaea (AOA)is one of the microbial groups that driven ammoxidation process. A large number of studies have shown that AOA is not only widely distributed in a variety of environmental media, but also is the most abundantammonia-oxidizing microorganism in nature. Therefore, it is important to illuminate the role and function of AOA in the ecosystem to reevaluate the nitrogen cycle.In recent years, AOA have been concentrated in the marine and soil environments, while the study on AOA in wetland ecosystem has Been rarely reported. In this study, three sampling points were set up in the mangrove wetland of Dongzhai Harbour, Hainan Province. S1 sampling point (near mangrove area) is covered with large mangrove plants; S3 sampling point wetland close to the sea(offshore area); and atransition zone S2 sampling point (wetland transition zone) is between S1 and S3. Each sample is set to three depths, namely, 0-2 cm (S), 20?25 cm (M) and 40?45 cm (L). The concentration of polycyclic aromatic hydrocarbons in seawater was determined by solid phase extraction. The concentration of nutritive salt in sediments was determined by spectrophotometry using potassium chloride solution extraction. The determination of nutritive salt concentration in seawater was carried out according to GB 12736.4-2007. In addition, the diversity and distribution of AOA in the sediments of mangrove wetlands were studied by adopting the method of clone library through amoA gene.At the same time, relative abundance of amoA gene in different sampling points was also determined by fluorescence quantitative PCR. Finally, the relationship between AOA gene diversity index and different environmental factors was discussed. This study focused on the diversity,species distribution and its relationship with environmental factors in mangrove wetland ecosystem,with a view to revaluate nitrogen cycling and ammoxidation processes, which provides a reliable scientific basis for the studies on ecological functions and distribution model of mangrove forests in Dongzhai Harbour. It also provides important decision-making reference for the study of ammonia-oxidizing bacteria and their response to environmental stress, as well as protection for the wetland ecosystem.The main findings are as follows:(1) The average concentration of inorganic nitrogen (ammonium salt, nitrite and nitrate) in 15 sampling points at Dongzhai Harbour mangrove wetland was 0.18mg·L-1.The concentration of inorganic nitrogen was decreasing gradually with the sampling point getting closer to the estuary.In accordance with the provisions of the national standard GB 3097-1997, the water quality was known as excellent. The PAHs concentration range was 234.46 ± 114.77 ng·L-1?683.33 ± 157.03 ng · L-1,of which, benzopyrene [a] was not detected, and the water was classified as oil source pollution and have potential ecological risks to aquatic organisms. The results showed that theconcentration of water nutrition in the Dongzhai Harbour protected area reaches the standard, but the polycyclic aromatic hydrocarbon has potential ecological risks to aquatic organisms. This study provided the basic data for the study on the relationship between the diversity of ammonia - oxidizing bacteria and the environmental factors in the mangrove sediments of Dongzhai Harbour.(2) The water content and the concentration trend of three inorganic nitrogens in the sediments of different sampling sites wee: S1> S2> S3. In the same sampling point, the water content and the concentration of ammonium salt in the middle and deep sediments were higher than those in the surface layer, and the three sampling points showed similar laws. The distribution of nitrate nitrogenin the same sampling point didn't show obvious regularity in the vertical direction. The results provided an important reference for the study of material cycling in a mangrove wetland ecosystem.(3) Through the construction of amoA gene cloning library, 298 valid sequences of total cloning library were obtained, which were divided into 34 operating classification units (OTUs) by 98% similarity. For the sub-clone library,the effective clones of sampling point S1 from shallow to deep depth were 40, 40 and 37, respectively. The effective clones of sampling point S2 from shallow to deep depth were 38, 31 and 37, respectively. The effective clones of sampling point S3 from shallow to deep depths were 31 and 39, respectively, where the surface clone failed. Through OTUsclustering analysis, it can be obtained that OTU1, OTU2 and OTU3 were the dominant species of the three sampling points, and the relative abundance was 18%, 11% and 10%, respectively.For the same sampling point, the abundance of species at different depths decreased with depth (S> M> L), and the abundance of species decreases with the sampling point away from the mangrove area (S1> S2> S3). The AO A diversity index of mangrove wetland ecosystem in Dongzhai Harbour was 2.93, which is higher than that of other wetlands in China. Through the construction of phylogenetic tree, 30 OTUsof the 34 OTUs were from the soil cluster, accounting for 88.2%, and 4 come from the ocean cluster. It can be found that most of AOAs in the DongzhaiHarbour mangrove ecosystem come from the soil cluster, which was far from kinship of ocean cluster.(4) The cloning library was constructed by fluorescence quantitative PCR, and the expression of amoA gene of ammoxidation key enzyme - AMO in different sampling points was studied. The distribution law of amoA gene abundance in different sediments and its correlation with environmental factors were also studied.The results show that the expression of AOA amoA gene is the highest in the mangrove area and the lowest in the coastal area, showing the regularity of decreasing expression with the sampling point away from the mangrovearea. There was no significant difference in the abundance between S1 and S3, and the highest expression appears in the middle layer. In order to better evaluate the abundance of ammonia-oxidizing archaea in the environment, the abundance of amoA gene in the environment was also determined. The results show that amoA gene expression in ammonia-oxidized archaea is much higher than that in ammonia-oxidizing archaea,and AOA is more tolerant of oxygen content.The AOA gene copy number was positively correlated with nitrite salt,ammonium salt. The AOA diversity index has no significant correlation with the concentration of nitrite and ammonium salts. Furthermore, The gene abundance and diversity has significant correlation in there sampling sites. |
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