Metabolic Mechanism Of Ammonium And Nitrite Nitrogen Conversion By Salt-tolerant Pseudomonas Stutzeri F2 And Its Application In Spotted Seabass Rearing

Ammonia nitrogen and nitrite exceeds the normal limit is one of the culprits of algae bloom and water eutrophication in aquaculture,and also affects the health of aquatic animals,causing death and significant economic loss in serious situations.Heterotrophic nitrification-aerobic denitrification（HNAD）microorganisms metabolize ammonium and nitrite nitrogen to non-toxic nitrate,gaseous and biological nitrogen by enzymatic conversion through nitrification,denitrification and assimilation.In this paper,a strain of heterotrophic-aerobic denitrifying bacteria Pseudomonas stutzeri F2 with the ability to remove ammonium and nitrite nitrogen efficiently was screened from aquaculture environment to target the problem of excessive ammonia and nitrite nitrogen in aquaculture.The research was conducted on the nitrogen conversion ability,functional gene screening,nitrogen metabolism pathway and application experiments in aquaculture of strain F2.The main research contents and results of this paper are as follows:（1）A strain F2 was isolated and screened from the substrate of aquaculture ponds in Fujian Province,and was identified as P.stutzeri by 16S r DNA sequencing and Blast comparison analysis.The tolerance concentration of nitrite nitrogen was increased to 300.0mg/L after the domestication of strain F2.The nitrogen balance analysis calculated that 71.29%of nitrite nitrogen was converted to gaseous nitrogen through the aerobic denitrification pathway.The NO₂^--N removal rate was reached 98.67%at 300.0 mg/L nitrite nitrogen addition concentration with sodium succinate as carbon source,C/N of 18,incubation temperature of 28℃,initial p H 8.0,and shaker speed of 210 rpm.In addition,it was found that the strain not only used nitrite nitrogen,but also ammonium nitrogen as a sole nitrogen source with excellent growth performance and ammonium nitrogen removal rate.With ammonium nitrogen and nitrite nitrogen as the compound nitrogen source,it was found that strain F2 preferentially used ammonium nitrogen before nitrite nitrogen and could promote the conversion of nitrite nitrogen by the strain.（2）Genome-wide information analysis revealed that strain F2 contains a circular chromosome of 4,979,911 bp genes and a plasmid of 86,675 bp in length.It also contains 45S r RNAs,4 16S r RNAs,4 23S r RNAs,58 t RNAs,2 prophages and 10 gene islands.The functional gene annotation into COG,GO and KEGG databases revealed that the functional gene annotation in COG database focused on amino acid conversion metabolism,signal transduction and energy production and transport;the functional gene annotation in GO database focused on catalytic activity and linkage of enzymes,cells and components,cellular processes and metabolic processes;the functional gene annotation in KEGG database focused on carbohydrate metabolism,amino acid metabolic synthesis,signaling and transmembrane movement.The analysis of functional genes related to nitrogen metabolism revealed that strain F2 had a complete nitrate denitrification pathway,assimilation pathway,allotropic pathway and ammonium nitrogen assimilation pathway,and also identified functional genes related to ammonium ion transport across the membrane,regulation of amino acid synthesis and nitrogen fixation.Genome-wide co-linearity analysis revealed that strain F2 was related to Pseudomonas stutzeri T13,A1501,SDU10 and 28a24.（3）After studying the growth characteristics and nitrogen removal rate of strain F2 under different NH₄⁺-N and NO₂^--N concentrations,transcriptomic analysis of the differences in the transcript levels of functional genes between the high and low ammonium nitrogen groups Amo500 and Amo100 revealed that 459 functional genes were up-regulated and 556functional genes were down-regulated when the added concentration of ammonium nitrogen was increased..The differential genes were annotated into GO and KEGG databases and found to be focused on the metabolic synthesis of nitrogen,transmembrane transport of nutrients,metabolic utilization of carbon sources and gene synthesis.In contrast,421functional genes were up-regulated and 433 functional genes were down-regulated at the transcriptional level in the high nitrite nitrogen group Nti500 and the low nitrite nitrogen group Nti300.The differential genes were mainly focused on the metabolic synthesis of nitrogen,transmembrane transport of nutrients and denitrification pathway of nitrite.The P.stutzeri-Δamt B strain was constructed using knockout technology,and the growth performance and ammonium removal rate of the mutant strain were found to be significantly reduced at high ammonium nitrogen concentrations.Combined with the transcriptome data,the nitrogen removal rate was found to be 94.92%with sodium pyruvate as the carbon source,shaker speed of 210 rpm and ammonium nitrogen addition concentration of 500.0 mg/L.（4）The study evaluated the supplementation of strain F2 in rearing water and found significant effects on growth characteristics,non-specific immunity,gut and liver morphology and gut microflora of juvenile flowering bass（Lateolabrax maculatus）.Different from the control group（T0）,where ammonia nitrogen levels were reduced by water exchange in the circulation system,the T1 and T2 groups supplemented the rearing water with low（3.0×10³CFU/m L）and high（3.0×10⁵ CFU/m L）concentrations of P.stutzeri F2 with the ability to remove ammonia nitrogen and nitrite,respectively.After 33 d of rearing,the weight gain rate,specific growth rate,feed utilization and survival rate of the experimental group of spotted seabass were significantly improved（p<0.05）.P.stutzeri F2 activated superoxide dismutase（SOD）,lysozyme（LZM）and catalase（CAT）activities in the serum of the experimental group of spotted seabass;increased glutathione peroxidase（GSH-Px）activity in the liver while decreasing malondialdehyde（MDA）levels.Microscopic observation of tissue sections revealed significantly improved muscle thickness and mucosal fold height in the T2 group of the midgut.The study of changes in the gut microbiota of spotted seabass by 16S r RNA gene sequencing revealed a significant decline in the relative abundance of Proteobacteria（phylum）and Photobacterium（genus）in the P.stutzeri F2 supplemented groups.Aeromonas veronii challenge test to assess the disease resistance of spotted seabass found that the survival rate of the T2 group was the highest.Our study showed that the supplementation of P.stutzeri F2with 3.0×10⁵ CFU/m L in the rearing water increased the growth performance of spotted seabass,activated innate immunity,improved the relative abundance of gut microbiota,and enhanced resistance to A.veronii,providing a new idea for the application of probiotics.