Nitrogen Removal Of Water And Sediment In Grass Carp(Ctenopharyngodon Idella) Aquaculture Ponds By Mixed Niteifying And Denitrifying Bacteria And Its Effects On Bacterial Community

Pond culture is one of the important freshwater culture modes in China.With the development of intensive culture,excessive feeding of high protein feed has become a normal situation.The increase of pond nitrogen load because of the accumulation of excess nitrogen in pond water and sediment.The change of nitrogen form in pond was mainly affected by nitrification and denitrification.Nitrifying and denitrifying bacteria can specifically change the presence of nitrogen in ponds.In this study,mixed nitrifying and denitrifying bacteria were used as research objects to explore the changes of nitrogen and microbial communities in pond water and sediment after the addition of mixed nitrifying and denitrifying bacteria.1.In this study,an acrylic transparent tube with a diameter of 0.5 m,a height of 2m and a thickness of 7 mm were set up in grass carp（Ctenopharyngodon Idella）pond to build a small enclosed experimental environment.The mixed nitrifying and denitrifying bacteria were added into the tube with the concentrations of 0,0.15,0.30and 0.60 mg/L.The change of nitrogen in pond water and sediment and the change of nitrifying and denitrifying bacterial community in the sediment in acrylic transparent tube were investigated through 15d experiment.The experimental results are as follows:The addition of mixed nitrifying and denitrifying bacteria at different concentrations had a significant effect on the change of nitrogen in the acrylic tube of pond.The nitrogen removal rate of nitrifying and denitrifying bacteria in each concentration group was the fastest from day 4-7,and the nitrogen removal effect was not obvious from day 10-15.At the concentration 0.15 and 0.30 mg/L groups,NO₃^--N had an upward trend from day 7-10,and the rate of nitrification was higher than that of denitrification.At the concentration of 0.60mg/L,NO₃^--N remained stable from day 7-15,and the rate of nitrification was equal to the rate of denitrification.The optimum relative concentration of nitrifying and denitrifying bacteria was 0.60 mg/L.Ammonia nitrogen（NH₄⁺-N）,nitrite（NO₂^--N）,nitrate（NO₃^--N）and total nitrogen（TN）decreased by 75.83%,93.09%,38.02%and 45.16%in 0.60 mg/L concentration group.Within 15days,the C/N ratio（C/N）of water and sediment increased significantly,but the C/N ratio of pond water increased more significantly.Water C/N ratio increased by 76.00%and sediment C/N ratio increased by 51.96%in 0.60 mg/L concentration group.The amplicon sequencing results showed that the abundance of nitrifying and denitrifying bacteria in the sediments was correlated with water quality.At the genus level,the species composition of nitrifying bacteria in different concentration groups was similar,including Nitrosomonas,Nitrosovibrio and Nitrosospira,which accounted for a relatively high proportion,and the proportion of each treatment group was significantly different.The species composition of denitrifying bacteria in different concentration groups was similar,including Thauera,Azoarcus and Magnetospirillum,which accounted for not a high proportion,the proportion of Azospira and Idiomarina was not high,and There was no significant difference between groups with different concentration of bacteria.Correlation analysis showed that the pond sediments had aerobic nitrification and facultative anaerobic denitrification.Species composition and species level heat map analysis showed that the species composition of nitrifying and denitrifying bacteria did not change significantly at the genus level,but changed significantly at the species level.With the addition of nitrifying and denitrifying bacteria,dominant landmark species played a key role in sediment purification.2.In this study,the obtained 0.60 mg/L mixed nitrifying and denitrifying bacteria were used as external additives to add in the pond big environment.The changes of nitrogen in pond water and sediment and pond microbial community were investigated by 15 d experiment.The experimental results are as follows:In the experimental group,0.6 mg/L nitrifying and denitrifying bacteria had obvious purification effect on pond water and sediment.The nitrogen removal rate in pond was the highest from day 4-7,and the nitrogen removal effect was not obvious from day 10-15.In this concentration group,NH₄⁺-N decreased by 78.29%,NO₂^--N decreased by 89.29%,NO₃^--N decreased by 46.24%,TN decreased by 46.27%.During one cycle,the C/N ratios of pond water and sediment increased significantly.The C/N ratio of pond water increased by 67.41%,and the C/N ratio of sediment increased by66.67%.16S rRNA sequencing was performed on pond and sediment samples at the 15day.The results showed that the microbial diversity in pond sediments was significantly higher than that in water.At the genus level,the composition of dominant species in pond water was similar,including Cyanobium＿PCC-6307,hgc I＿clade and CL500-29＿marine＿group,which accounted for a relatively high proportion.The composition of dominant species in pond sediments is similar,including Thiobacillus,SC-I-84,Sva0485,Candidatus＿Competibacter and Desulfatiglans,which accounted for not a high proportion.The changes of species in pond water and sediment were similar,and the proportion of dominant species in pond changed obviously after adding nitrifying and denitrifying bacteria.The proportion of hgc I＿clade in pond water increased when the cyanobium＿PCC-6307 decreased,that of Thiobacillus and SC-I-84 in sediment increased when the Candidatus＿Competibacter decreased.The analysis of genus level species composition and species level heat map showed that the community structure of pond microorganisms did not change significantly at genus level,but changed significantly at species level.The addition of mixed nitrifying and denitrifying bacteria promoted the transformation of the dominant species of microorganisms in the pond.In conclusion,the input of exogenous nitrifying and denitrifying bacteria can effectively reduce the nitrogen load of pond water and sediment.The abundance of nitrifying and denitrifying bacteria,the dominant landmark species of nitrifying and denitrifying bacteria and the dominant landmark species of the pond worked together to purify nitrogen in the pond and improve the C/N ratio in the pond.The experimental study showed that when the concentration was 0.6 mg/L,the nitrogen load of pond water and sediment decreased most obviously,and the pond purification effect was good.