Screening,Identification And Immobilization Of Aerobic Denitrifying Bacteria And Their Denitrification Performance

In order to improve the problems of high operating cost and low denitrification efficiency in the traditional biological denitrification process where nitrification and denitrification processes need to be carried out independently,aerobic denitrifying bacteria were screened and isolated from soil and activated sludge samples,and their physiological characteristics,strain genera,optimum denitrification conditions,and denitrification pathways were investigated in this study.In addition,the synergistic denitrification ability of the composite strains was explored by optimizing the combination of multiple aerobic denitrifying bacteria.Based on this,the immobilized microbial technology was used to immobilize the composite strains with the best denitrification effect.Meanwhile,the denitrification performance of the immobilized composite strains was investigated under different initial p H,rotational speed,temperature and salinity,and the free and immobilized composite strains were injected into the actual water samples to examine their practical application potential.（1）An aerobic denitrifying bacterium named 5-2 was screened from the soil of Shanghai paddy field,and its physiological characteristics and species were clarified by morphological observation,drug sensitivity test and 16S r DNA sequencing,and the strain 5-2 was finally identified as Acinetobacter junii.A strain of aerobic denitrifying bacteria was screened from the activated sludge of Shanghai Songjiang sewage treatment plant and named 15-2,and its physiological characteristics and species were clarified through the above analysis,and finally identified as Stenotrophomonas maltophilia.（2）The denitrification performance of strains 5-2 and 15-2 under different environmental conditions was investigated separately using a single-factor effect test to determine their optimal denitrification conditions.The optimum denitrification conditions for strain 5-2 is:initial p H value of 10.0,sodium acetate as carbon source,C/N=12,90 r/min,35℃,and the removal rates of nitrate and total nitrogen were 97.83%and 65.85%respectively,after 120 h of incubation under these conditions;the optimum denitrification conditions for strain 15-2 is:initial p H value of 7.0,sodium acetate as carbon source,C/N=12,90 r/min,and 35℃.After 120 h of incubation under these conditions,the removal rates of nitrate and total nitrogen were 98.87%and 59.86%,respectively.In addition,the aerobic denitrification-related enzyme activities and functional genes of strains 5-2 and 15-2 were examined,and the denitrification pathways of strains 5-2 and15-2 were hypothesized as follows:nitrate nitrogen was first reduced to nitrite nitrogen by nitrate reductase（NAR）,and then nitrite nitrogen was reduced to nitrogenous gas by nitrite reductase（NIR）to achieve the purpose of denitrification.（3）The combination of strain 5-2,15-2 and strain 15-1,which had good denitrification ability in the primary screening test,were combined in different volume ratios to obtain a total of 13 strain combinations.After 72 h incubation under the same conditions,it was found that the combination of strain 5-2 and strain 15-2（2:1 volume ratio）had the highest nitrogen removal efficiency,with 98.01%and 62.46%of nitrate and total nitrogen removal,respectively.In addition,the performance of the immobilized pellets made according to the orthogonal test table was evaluated,The best performance of immobilized pellets was found when the concentration of polyvinyl alcohol（PVA）was 100 mg/m L,sodium alginate（SA）was 10 mg/m L,activated carbon（AC）was 30 mg/m L,and saturated calcium chloride（Ca Cl₂）borate solution was 50mg/m L.（4）The three immobilization methods of PVA+SA+AC,PVA+SA,and polyurethane foam（PUF）were used to immobilize the composite strains under the above optimal immobilization conditions to investigate their nitrogen removal ability under different initial p H value,rotational speed,temperature and salt concentration,and it was found that the immobilized composite strains were more adaptable to the environment compared with the single strains.Then the free composite strains and three immobilized composite strains were injected into the actual wastewater,and their nitrate nitrogen removal rates were measured to be 45.74%,91.49%,90.39%and 96.39%after 48 h,respectively.Its denitrification capacity is as follows:PUF immobilized composite strains>PVA+SA+AC immobilized composite strains>PVA+SA immobilized composite strains>free composite strains.