Research On Nitrogen Transformation In The Sediment Of Integrated Multitrophic Aquaculture System Of Chinese Shrimp + Hard Clam + Mullet

Integrated multi-trophic level aquaculture（IMTA）is a highly efficient,healthy and sustainable aquaculture model that achieves complementary ecological niches by scientifically matching aquatic plants and animals at different trophic levels,thus effectively promoting the transformation and recycling of materials within the aquaculture system.As one of the most important biogenic elements in aquaculture ponds,the transformation and transport of nitrogen not only affects the growth and development of cultured organisms,but is also closely related to a series of environmental issues such as the changes in the growth and decline of harmful nitrogenous compounds such as ammonia nitrogen and nitrite in the aquaculture environment,as well as eutrophication and the emission of the greenhouse gas N₂O.In order to gain insight into the nitrogen cycling process of Chinese shrimp+hard clam+mullet multi-trophic level integrated aquaculture ponds,this study examined and analysed the structure and diversity of microbial communities,expression of functional genes and potential rates of denitrification reactions in pond sediments using high-throughput sequencing,real-time fluorescence quantitative PCR,¹⁵N isotope labeling tracing,etc.Meanwhile,a simulated experimental system was constructed to verify and investigate the effect of bioturbation of Mercenaria mercenaria on the nitrogen cycling process in the sediment.The results of the study are as follows:1.The composition,structure and diversity of microbial communities at the sediment-water interface of Chinese shrimp+hard clam+mullet integrated multi-trophic level culture ponds were studied and analysed using high-throughput sequencing technology.The results showed that a total of 887455 bacterial 16S r RNA gene optimized sequences were obtained from all samples,and a total of 66 phyla,189orders,430 orders,662 families and 1151 genera of bacteria were identified by comparison with the database.At the phylum taxonomic level,the dominant groups were Gamma proteobacteria,Anaerolineae and Bacteroidia.The diversity of microorganisms in pond sediments varied during July,August and September,with greater similarity between samples at the same time.By constructing a simulated experimental system with a control group and a test group with different densities of hard-shelled clams,we found that the similarity of microbial community structure in the sediments of the control and test groups differed significantly,confirming that the bioturbation effect of hard-shelled clams can alter community structure and diversity.The community structure was not significantly affected by single environmental factors,with ammonia and nitrate having a greater impact on the community structure.2.The expression of bacterial 16S rRNA genes and nitrogen cycle-related genes at the sediment-water interface of Chinese shrimp+hard clam+mullet integrated multi-trophic level culture ponds was examined using real-time quantitative PCR（q PCR）technique.The results showed that the copy number of bacterial 16S r RNA genes ranged from 1.42×10⁹～2.20×10⁹ Copies-μL^-1 and the expression did not differ significantly at different times;the expression of nitrogen-fixing nif H genes ranged from 3.18×10⁶～7.03×10⁶ Copies-μL^-1,and gradually increased with the culture time,with significant differences between August samples and June（P<0.05）and significantly correlated with temperature,DO and NO₂^--N（P<0.05）;ammonia oxidation AOB gene expression was 8.64×10³～6.56×10⁵ Copies-μL^-1 and significantly different in pond sediment in July,August and September（P<0.05）and significantly positively correlated with DO（P<0.05）;denitrification nos Z gene expression was 1.55×10⁵～8.64×10⁵ Copies-μL^-1,with significantly higher copy numbers in pond sediment in July than in June（P<0.05）,slightly higher than in August but not significantly different（P≥0.05）,significantly negatively correlated with p H and DO（P<0.05）,and highly positively correlated with temperature and nitrate（P<0.05）.The expression of the anaerobic ammonia oxidation hzo gene in the pond sediment was lower than that of the denitrification nos Z gene overall,ranging from 5.96×10⁴ to1.56×10⁵Copies/μL^-1,with similar trends to the nos Z gene,and the expression in the pond sediment in July was significantly higher than that in June and August（P<0.05）,a significant positive correlation with nitrate and nitrite（P<0.05）,and a highly significant negative correlation with DO（P<0.01）.In the simulations with different densities of Mercenaria mercenaria,it was found that the M.mercenaria altered the depositional environment through bioturbation,which in turn affected the abundance of bacteria associated with the nitrogen cycle:nitrogen-fixing bacteria were inhibited and the abundance of nitrifying,denitrifying and anaerobic ammonia-oxidising bacteria was significantly increased,with the most significant effect in the medium density test group（120 clams/m²）.3.The potential rates of denitrification reactions associated with the sediment-water interface of Chinese shrimp+hard clam+mullet integrated multi-trophic level culture ponds were measured using¹⁵N isotope labeling tracer method.The results showed that the denitrification rate was 8.09μmol·kg^-1·h^-1 and the anaerobic ammonia oxidation rate was 3.50μmol·kg^-1·h^-1.The contribution of denitrification to nitrogen removal was 69.8%and that of anaerobic ammonia oxidation was 29.2%.The bioturbation effect of the M.mercenaria was able to increase the denitrification and anaerobic ammonia oxidation rates in the sediment,with the highest denitrification rate in the medium density test group（120 grains/m²）,which was 38.8%higher compared to the control group,which was in close agreement with the functional genetic results,which also indicated that the denitrification and anaerobic ammonia oxidation rates were related to the abundance of the relevant bacteria,but the contribution of the M.mercenaria to denitrification and anaerobic ammonia oxidation denitrification was not A significant effect was found in this study.4.A preliminary characterization of the greenhouse gas N₂O fluxes at the water-gas interface of Chinese shrimp+hard clam+mullet integrated multi-trophic level culture ponds was carried out using the static box method.₂The results showed that the N₂O fluxes at the pond water-air interface ranged from 0.012-0.073 mg·（m²·h）^-1between July and September and showed a gradual increase,with significant correlations（P<0.05）with DO,salinity,NH₄⁺,and NO₃^-.The results of this study provide a theoretical basis for further revealing the ecological mechanisms of nitrogen nutrient cycling and utilization in the integrated multi-trophic level aquaculture of marine ponds,improving the existing IMTA model and further improving the nutrient utilization efficiency of the aquaculture system,which is of great significance for the healthy and sustainable development of marine pond aquaculture.