Screening,identification And Denitrification Performance Of Aerobic Denitrifying Bacteria In Grass Carp Ponds

Ctenopharyngodon idellus,commonly known as squid,grass roots,black herring,is one of the four major fish in China.In 2016,the output of grass carp in China was589,800 tons.With the rapid development of China's aquaculture industry,the farming method has changed from the traditional extensive type to the modern scale and intensification.The feed input in the breeding process cannot be fully utilized by the fish body,and the fish body's own excrement is deposited in the In the pond,the endogenous nitrogen pollution in the pond culture is serious,which not only deteriorates the water quality,but also causes the disease of the farmed animals to erupt,the quality and output of the cultured products are declining,and the discharge of the tailwater from the serious nitrogen exceeding the standard will lead to the pollution of the river water environment.Therefore,how to reduce nitrogen pollution in pond culture water has become a hot issue in the aquaculture field.Biological denitrification is considered to be one of the effective ways to remove nitrogen from water by its advantages of no pollution and high efficiency.The traditional biological nitrogen removal theory believes that denitrification is a strictly anaerobic process.Oxygen inhibits the expression of the reverse digestion reductase gene and the activity of denitrifying reductase,and cannot play the role of microbial denitrification.The discovery of aerobic denitrifying bacteria has broken through the bottleneck of traditional biological nitrogen removal.The occurrence of aerobic denitrification is due to the aerobic denitrification enzyme system present in the cells.Aerobic denitrifying bacteria can reduce nitrogen and nitrous oxide to nitrogen in an aerobic environment,which provides a new idea for biological nitrogen removal.Since then,more and more aerobic denitrifying bacteria have been separated from different environments.The discovery of aerobic denitrifying bacteria provides a new research direction for the removal process of nitrogen in existing aquaculture waters.The grasshopper?Ctenopharyngodon idellus?located in Zhongshan City uses a new wheel-recycling process for the zero-emission breeding pond.The whole process of farming does not change water and zero emissions for three consecutive years.In this study,the“zero-emission”culture pond of grass carp was used as the research object.The traditional microbial separation and purification method was used to isolate and identify the aerobic denitrifying bacteria in the water and sediment of grass carp,and to select high-efficiency aerobic denitrifying bacteria.Optimize the high-efficiency denitrification culture conditions of this strain,explore the denitrification performance,put the strain in the nitrogen-rich water to carry out the denitrification test,and evaluate the practical application effect of the strain by the change of nitrogen indexes in the water.This study provides a theoretical basis for the practical application of in situ remediation of grass carp culture ponds while enriching the resources of aerobic denitrifying bacteria.The main experimental contents are summarized as follows:1.Separation and screening of aerobic denitrifying bacteria from the water of grass carp culture ponds in Zhongshan City,Guangdong Province.First,89 strains of bacteria were initially screened using the acid-base indicator method,and then quantitatively calculated using denitrification medium.Oxygen denitrifying bacteria.2.After 16S rDNA gene sequence analysis,the 25 strains belonged to Pseudomonas,Enterobacter,and Acinetobacter.Two strains with the strongest nitrogen removal ability were selected,named ZS1 and ZS2,respectively,and the two strains were identified.Combined with the morphological observation,physiological and biochemical characteristics and 16S rDNA gene sequence analysis,the strain ZS1 was Pseudomonas furukawaii,Gram-negative bacteria,and the cells were rod-shaped and had flagellar hairs.The 16S rDNA sequence of strain ZS1 with a length of 1333 bp was uploaded to NCBI with accession number MK615112;strain ZS2 was Citrobacter portucalensis,Gram-negative bacteria,the colonies were smooth,moist,and the edges were neat.The16S rDNA sequence of strain ZS2 with a length of 1339 bp was uploaded to NCBI under accession number MK641482.3.The optimum conditions for aerobic denitrification of strain ZS1 were as follows:temperature was 30?,pH was 8.0,carbon source was sodium acetate,C/N was 15,and rotation speed was 100 r/min.4.The optimum conditions for aerobic denitrification of strain ZS2 were as follows:temperature was 30?,pH was 7.0,carbon source was glucose,C/N was 20,and rotation speed was 200 r/min.5,strain ZS1 after 48h culture,the content of NO₃^--N decreased from 48.93mg/L to1.27mg/L,the removal rate was 97.40%,the removal rate reached 0.993 mg/?L·h?;TN content from 52.04 The mg/L was reduced to 8.40 mg/L,the removal rate was83.86%,the removal rate was 0.909 mg/?L·h?,and there was no nitrous accumulation.6.After 48h culture,the content of NO₃^--N decreased from 43.21mg/L to 6.32mg/L,the removal rate was 85.37%,the removal rate reached 0.7685 mg/?L·h?;the content of TN was from 46.72.The mg/L was reduced to 10.13 mg/L,the removal rate was78.32%,and the removal rate was 0.7623 mg/?L·h?.7.The denitrification mechanism of strain ZS1 was studied at the molecular level.As a result,it was confirmed that the strain ZS1 was present in the nitrate reductase NarG.8.When strain ZS1 was added to grass carp pond culture wastewater,the experimental group with strain ZS1 was reduced from the initial NO₃^--N concentration of 48.089 mg/L to 7.628 mg/L during the entire 10 day experimental period.The removal rate of NO₃^--N was 84.14%.The concentration of NO₂^--N in the experimental group decreased from 1.639mg/L to 1.393mg/L,and the removal rate of NO₂^--N was15.01%.The bacteria played a good role in regulating and improving the aquaculture water environment.Compared with the control group,the content of NH₄⁺-N,NO₃^--N and NO₂^--N in the experimental group supplemented with strain ZS1 was significantly lower than that in the control group.