Studies On Denitrification Characteristics Of Pseudomonas Stutzeri SC221-M And Its Application To Water Quality Control In Grass Carp Aquaculture

In order to reduce the pollutants such as ammonium and nitrite in aquaculture water for grass carp, an isolate of denitrifying bacteria Pseudomonas stutzeri SC221was obtained by directional enrichment. After cobalt-60radiation, the nitrite removal capacity of the strain could be increased from50mg/L to100mg/L. It was named as P. stutzeri SC221-M after such radiation. Following researches on the effect of nitrogen sources, carbon sources, ratio of carbon to nitrogen (C/N) and temperature on the growth, denitrification rate and denitrification gene expression of SC221-M indicated that the best condition for the growth and denitrification of SC221-M was sodium nitrite as nitrogen source, sodium citrate as carbon source, C/N=4-8and at20-35℃. As the results shown, environmental factors could affect the growth and denitrification of SC221-M, and there was certain correlation between denitrification and nirS and nosZ gene expression.Illumina Hiseq2000sequencing platform was used to obtain the complete genome sequence for SC221-M. Based on the assemble result, it was found that the genome size was4,545,027bp with64.01%GC content, the number of scaffold was131with399contig. Comparing with reference strain DSM4166, InDel was not appeared and there were60,641SNPs, including43,396synonymous mutations and10,743non-synonymous mutations in the genome of SC221-M. Through phylogenetic analysis, it was discovered that NirS and NosZ sequences of SC221-M were closely related to those of A1501and DSM4166, indicating that the strains showed a similar denitrification mechanism.The effect of SC221-M and its mixed microbial preparation on hydrochemical indexes of the aquaculture water for grass carp was studied. There were five groups, including addition of0CFU/ml microbial preparation,1×105CFU/ml SC221-M,1×105CFU/ml Bacillus cereus BSC24,1×105CFU/ml mixed preparation (SC221-M: BSC24=1:1) and3×105CFU/ml mixed preparation in15PE buckets containing1400L water. It was found that addition of the microbial preparation could decrease the level of hydrochemical indexes, including TDS, ammonium, nitrite, total inorganic nitrogen, total nitrogen, COD and BOD, and the effect for mixed microbial preparation was better than that for SC221-M or BSC24preparation. The results indicated that the interaction between SC221-M and BSC24played an important role in improving water quality.Water samples were collected from the control and treatment groups after9-day feeding. Bacterial genomic DNA was extracted and the V3region of16S rDNA was amplified by PCR. After the use of Roche454GS FLX Titanium to sequence the PCR products as well as Qiime, Megan and Cytoscapes to analyze DNA sequences, the results revealed that in each group, the most abundant phyla were Proteobacteria. The microbial diversity was increased when adding the mixed preparation and all samples were in different branches in accordance with the treatment through UPGMA cluster analysis. As is shown, microbial community structure in the water was significantly changed after adding microbial preparation, and there was a close relationship between microbial community structure and hydrochemical indexes.