| The Eutrophication problem caused by various reasons such as excessive nitrogen level in aquaculture wastewater are the most critical obstacle to the sustainability of aquaculture industry in China at present.Tidal shellfish,a kind of filter-feeding organism,can not only filter the microalgae and suspended organic particles from the water body,but also exchange materials in sediment/water interface through their bioturbation activity,which in turn changes water ecosystem environment.In the present study,we firstly focused on the farming areas of four different clam species including Ruditapes philippinarum,Mercenaria Mercenaria,Meretrix lusoria and Sinonovacula constricta,in the tidal shellfish ecological purification pond.The microbial community structure in sediments were investigated using high-throughput sequencing technology,and changes in abundance of key functional genes during the nitrogen cycle process at the sediment/water interface were also analyzed using quantitative PCR technology.Second,the nitrogen cycle reaction rate of denitrification（Denitrification）and anaerobic ammonia oxidation（Anammox）at sediment/water interface were measured using the nitrogen stable isotope tracer technology.At last,a mesocosm for tidal shellfish culture was established to analyze the effects of different clam species and stock density on microbial community composition and the main nitrogen cycle reaction processes at sediment/water interface.This study systematically revealed the ecological purification function of filter-feeding clams and their influence on the nitrogen cycle in pond mariculture.The results are as follows:（1）Effects of four buried-dwelling shellfish on water quality and bacterial community structure in sediments.In 2019,an ecological purification pond with tidal shellfish culture was constructed,in which R.philippinarum area,S.constricta area,M.Mercenaria area and M.lusoria area were set up and the corresponding clams with similar biomass were stocked,respectively,and the rest area without any clams were set as the control area.The microbial community composition of each area sediment in July,August,September and October were investigated by using the high-throughput sequencing techneque.The results showed that the dominant bacterial genera in the M.Mercenaria area,R.philippinarum area,S.constricta area and M.lusoria area were Woeseia,Bacillus,Salinimicrobium and Woeseia,respectively.The results of quantitative PCR analysis revealed that Nitrogenase（nif H）gene abundance in the shell culture areas in August were significantly lower than that in control area（p<0.05）,Nitrogenase（nos Z）gene abundance in July and August was significantly higher（p<0.05）,and Ammonia-oxidizing bacteria（AOB）gene abundance in July was significantly lower（p<0.05）;In addition,the results of porewater quality analysis showed that the concentration of ammonia nitrogen,nitrite,nitrate and phosphate of sediment porewater in the control area was higher than that in the clam culture areas,and R.philippinarum culture has been found a significant effect on removal of ammonia nitrogen.The concentration of ammonia nitrogen,nitrite and nitrate in the R.philippinarum culture area decreased gradually along with the clam cultivation time extension.（2）Effects of Ruditapes Philippinarum on Denitrification and Anaerobic Ammonia Oxidation Rates of the Sediments/water Interface in Seawater Aquaculture Pond.In 2020,an ecological purification pond with R.philippinarum culture was constructed,in which only R.philippinarum was stocked in the clam culture area while the area without any clams were set as the control.The change of main nitrogen cycle reaction rate at the sediment/water interface was analyzed in the ecological purification ponds.The sediment samples of the clam culture area and control area were collected in September,October,November and December,respectively,and the slurry culture experiments were performed,and the denitrification and anaerobic ammonia oxidation reaction rates were detected by using the nitrogen stable isotope tracer technology.The results showed that the anammox reaction was found in the R.philippinarum ecological purification pond both in October and November,and there were a coupling reaction of denitrification and Anammox;The denitrification rate of sediments in the four months in the clam culture area was higher than that in the control area,and it had been found the highest in September,reaching 0.005833μmol kg-1 h-1;There was a significant positive correlation between temperature and denitrification reaction（p<0.01）,and between NH4+and Anammox reaction as well（p<0.01）.（3）A medium-scale simulation experiment on the influence of shellfish on bacterial community structure and nitrogen cycle.In 2020,a medium-scale mesocosm clam culture system was constructed to systematically analyze the effects of different shellfish species and different culture stocking densities on the microbial community composition and nitrogen cycle process in pond sediments.The impact of different shellfish on the microbial community structure in sediments were analyzed by using the high-throughput sequencing technology,the results showed that the dominant bacterial genera in the M.Mercenaria group,the S.constricta group and the R.philippinarum group were Halomonas,Paenisporosarcina and Halomonas,among which Halomonas is a kind of denitrifying bacteria;The abundance of nif H gene in S.constricta group was significantly higher than that of the control,R.philippinarum and M.Mercenaria group at day28（p<0.05）;the nos Z gene abundance of the R.philippinarum group was significantly higher than that of the control at day14（p<0.05）;The AOB gene abundance of the R.philippinarum group was significantly higher than that of the control and the S.constricta group at day7（p<0.05）.The results of further experiment with different culture densities of R.philippinarum（4.5±0.9g）（low density 62.5 grains/m2,medium density 125 grains/m2,high density 250 grains/m2）showed that the dominant genus of the R.philippinarum in each density group is Gramella,and the genus Halomonas and Salinimicrobium account for a relatively high proportion of the bacterial community structure,which belong to denitrifying bacteria;the nif H gene abundance of each group was higher than that of the control at day28（p<0.05）,and the nos Z gene abundance of the medium-density group was higher than that of the control except for day14（p<0.05）.The abundance of nos Z gene in the medium-density group was significantly higher than that in low-density group at day21（p<0.05）.The present study found that culture of R.philippinarum area,S.constricta area and M.Mercenaria in the ecological purification ponds can significantly affect microbial community composition in pond sediments,which was conducive to the occurrence of reactions at the sediment/water interface.Nitrification and anaerobic ammonia oxidation can effectively promote the denitrification process of pond sediments.The results of this research could not only enrich the nitrogen cycle theory for aquatic ecosystems,but also provide new ideas for the research and practice of biological purification of marine aquaculture wastewater that are currently vigorously carried out in China. |
