Properties Of Halomonas Urease And Its Application In High-salt Nitrogen-containing Wastewater

Urea(H2N-CO-NH2)is one of the simplest nitrogen-containing organic compounds.and it is widely used in the production of deicing agents,foaming agents,herbicides,agricultural fertilizers,and automobile exhaust treatment.Agricultural nitrogen fertilizer leaching,industrial production with urea as raw material and human life all produce a large amount of urea-containing wastewater.Furthermore,urea-containing wastewater is often accompanied by high salt(Na Cl > 10 g/L)problems.Urea-containing azodicarbonamide foaming agent industrial sewage(Na Cl = 56 g/L),toilet flushing sewage in coastal cities(Na Cl=10-17g/L)and domestic sewage from ships(Na Cl = 10-20 g/L)were all have high salt content.The resulting high-salt urea-containing wastewater poses a threat to human health and the ecological environment.Therefore,there is an urgent need for an effective method to treat high-salt urea-containing wastewater.In this thesis,a N-removal strain Halomonas sp.H36 with simultaneous urea hydrolysis,simultaneous heterotrophic nitrification-aerobic denitrification(SND)was screened and identified,the enzyme production conditions of the strain urease were optimized,the enzymatic properties of the strain urease were explored,and the N-removal process of the strain with urea as a substrate was investigated under high salinity.And it apply to the treatment of simulated ship’s domestic sewage.(1)Using urea as the sole nitrogen source,13 salt-tolerant growth strains preserved in the laboratory were grown and cultured,and their growth amount and urease activity were determined.After transfer to N-removal medium,the N-removal rate of simultaneous heterotrophic nitrification aerobic denitrification(SND)was determined.A salt-tolerant growth N-removal strain H36 with simultaneous urea hydrolysis-SND was screened out.The16 S r DNA similarity between Halomonas sp.H36 and Halomonas taeanensis NY-3 was99.4% by identification.The growth and urease production of Halomonas sp.H36 under different Na Cl concentrations were investigated.When Na Cl=60 g/L in the medium,the growth amount and urease activity of the strain reached the highest,which were 10.26 g/L and75.28 U,respectively.The total concentration of ectoine synthesized by Halomonas sp.H36 was not affected by the Na Cl concentration in the medium.The average total concentration of ectoine was 1303.02 mg/L.Halomonas sp.H36 is an ectoine-secreting strain.When Na Cl=30g/L in the medium,the strain ectoine secretion rate reached the highest value,78.28%.(2)The production conditions of Halomonas sp.H36 urease were optimized by the method of response surface optimization.The optimized medium for urease production(g/L):glucose 40.26,ammonium sulfate 15.06,Na Cl 34.90,urea 5,monosodium glutamate 20,yeast powder 1,KH2PO4 3,K2HPO4 3H2 O 9,Mg SO4 7H2 O 0.4,Mn SO4·H2O 0.01,mixed trace elements 2 m L.After optimization,the urease activity of Halomonas sp.H36 increased by 47.72%.(3)The enzymatic properties of Halomonas sp.H36 urease were investigated from three aspects: enzyme inducibility,optimal enzymatic hydrolysis conditions and Michaelis-Menten equation.Halomonas sp.H36 urease is inducible.The optimal enzymatic hydrolysis conditions of the strain urease were p H = 8,T = 50 ℃,Na Cl = 0 g/L,and urea concentration was 50 g/L.The relationship between strain urease and substrate(urea)was in accordance with Michaelis-Menten.Km = 16.70 m M,Vmax = 140.85 m M/(mg·min).(4)From the level of molecular biology and enzymology,the N-remove study of Halomonas sp.H36 with urea as substrate was carried out.Halomonas sp.H36 N-removal enzymes genes(or fragments)(ure A,amo A,nir S,nar H)were successfully amplified.Different concentrations of Na Cl in the enzymatic reaction system would affect the N-removal enzymes(urease;ammonia monooxygenase,AMO;nitrite reductase,NIR;nitrate reductase,NAR)of Halomonas sp.H36.Na Cl = 0 g/L,the activities of N-removal enzymes of the strain were the highest.With the increase of Na Cl concentration,the activities of N-removal enzymes of the strain decreased in different degrees.Na Cl = 120 g/L,the activities of urease,AMO,NIR and NAR of the strain still retained 74.25%,22.26%,32.01% and26.82%,respectively.In the study of the N-removal process with urea as the only N source,the CO(NH2)2-N removal rate was 74.77% and the total nitrogen(TN)removal rate was57.67%.Halomonas sp.H36 has the function of simultaneous urea hydrolysis-nitrificationdenitrification with urea as the initial substrate,and can simultaneously remove urea nitrogen(CO(NH2)2-N)and inorganic nitrogen(NH+4-N,NO-2-N,NO-3-N)from high-salt urea-containing wastewater.(5)Halomonas sp.H36 to treat simulated ship’s domestic sewage.After 192 h,the CO(NH2)2-N removal rate was 88.07%,the ammonia nitrogen(NH+4-N)removal rate was91.42%,the TN removal rate was 90.33%,and nitrite nitrogen(NO-2-N)and nitrate nitrogen(NO-3-N)did not accumulate.Halomonas sp.H36 has the ability to degrade total sugar and protein in simulated ship’s domestic sewage,the total sugar removal rate is 89.73%,and the protein removal rate is 97.68%.In this thesis,the genes(or fragments)of ureA,amoA,nir S and nar H were successfully amplified simultaneously from Halomonas.And it was proved that Halomonas has the function of simultaneous urea hydrolysis-nitrification-denitrification with urea as the initial substrate,and can simultaneously remove CO(NH2)2-N and inorganic nitrogen(NH+4-N,NO-2-N,NO-3-N)from high-salt urea-containing wastewater.