Study On Ammonia Nitrogen Removal Characteristic And Its Key Enzymes Of Acinetobacter Sp. Y1

Acinetobacter sp. Y1, with the capability of heterotrophic nitrification and aerobic denitrification, was isolated from a coking wastewater treatment plant located in Taiyuan. By means of ammonia nitrogen and COD removal performance detection, measurement of the activities of the key enzymes in ammonia nitrogen removal process-hydroxylamine oxidoreductase（HAO）, nitrite reductase（NIR）, nitrate reductase（NAR）, and genome sequencing and analysis,results are as follows:1. In 24 h, ammonia nitrogen, total nitrogen, and COD removal rate can reach up to 98%, 94% and 92% respectively, with sodium citrate and ammonium sulfate used as the sole carbon and nitrogen sources. Hydroxylamine, nitrite nitrogen and nitrate nitrogen in nitrification process were detected without accumulation.2. Ultrasonic conditions were optimized using orthogonal experiment, and the optimal conditions were: ultrasonic power 50 W, work/interval time 4,7 s, bacteria density of 1.250 and total working time 20 min. The corresponding specific activities of HAO, NIR and NAR were 0.011, 0.002, 0.018 U/mg protein, respectively. The crude enzymes, obtained by ultrasonic crushing method and osmotic shock method, were compared by SDS-PAGE. The result showed that the former was more suitable to extract HAO resulted in less types and lower concentration of other proteins, as well as higher HAO activity.3. Optimization of HAO production showed that the optimal carbon source, nitrogen source, and C/N ratio were trisodium citrate, ammonium sulfate, and 14, respectively, with an incubation time of 16 h. After extracted crude enzyme by osmotic shock method, DEAE SefinoseTM FF anion-exchange chromatography was used to purify HAO, followed by SefinoseTM CL-6B gel filtration chromatography. A 47 k Da HAO was purified successfully for the first time, with a purification fold of 7.32 and a recovery rate of 19.40%. Enzymatic properties study showed that the optimized enzyme acitivity of purified HAO was tested at p H 8.0 and 30 oC, and kept stable for p H values of 7-8, and temperature below 30 o C; Mg²⁺, Fe²⁺, Fe³⁺, Ag⁺, Pb²⁺, Na⁺ and K+ at 1 mmol/L enhanced the activity seperately, whereas Cu²⁺, Ca²⁺, Ba²⁺ strongly inhibited the activity, and Mn²⁺, Zn²⁺ did little effect on HAO; HAO activity was activated by EDTA-2Na at 0.4 mmol/L, and a negative effect arose as the dose increased; substituting cytochrome C for potassium ferricyanide in reaction mixtures, no HAO activity was observed at all.4. Whole genome of Acinetobacter sp. Y1 was sequencd by whole genome shotgun strategy. It was found that the genome size was 3384069 bp with 39.56% GC content, and the number of scaffolds were 24 based on the result of assembling. 3137 protein-coding genes were anotated in RAST, with total encoded length of 2957373 bp, and coding ratio of 87.39%.5. Functional genes were annotated after comparing with eight major databases, which were PFAM, NR, CDD, Swiss-Prot, COG, Tr EMBL, GO and KEGG. COG database annotation got 2295 genes, corresponding to 20 COG categories. By the way of GO functional classification annotation, 2113 genes were annotated to three terms. In terms of biological process, cellular process and metabiolic process got the two largest number of annotated genes; in cellular component, cell and cell part got the two largest number of annotated genes; in molecular function, binding and catalytic activity got the two largest number of annotated genes. KEGG database annotation results indicated there were 1708 genes involved in 167 metabolic pathways, including nitrogen metabolism, and various pollutants metabolism pathways. In addition, various genes relating to nitrogen metabolism were annotated, like glt B, glt D, nas A, nir B, NRT, GLUD1₂, gud B, cyn T, ncd2, nrt C, gln A, gdh A, nrt B and amo, which provide theoretical basis of ammonia nitrogen removal pathway of Acinetobacter sp. Y1.6. Furthermore, Acinetabacter sp. Y1 was analysised to degrade many types of organic chemicals, such as ethylbenzene, styrene, naphthalene, limonene and pinene, chlorocyclohexane and chlorobenzene, aminobenzoate, ketone bodies, nitrotoluene, polycyclic aromatic hydrocarbon, benzoate, toluene, bisphenol, geraniol, aromatic compounds, dioxin, fluorobenzoate, xylene, chloroalkane and chloroalkene, atrazine, caprolactam, and so on. This was not only to provide the development and application of Acinetobacter sp. Y1, but also to understand various degradation pathways.