Studies On Denitrifier Bacteria By Protoplast Fusion And It's Utilization In Nitrogen Removal

Nitrogen is still the most important pollution resource of surface water today, the trend of bio-denitrification in wastwater treatment is exploratory research of new denitrification process,new loading material and new denitriifier.With some new bio-denitrification process have been used,the key contrained factor of it's popularization and ultilization is how to improve the effectiveness of denitrifier in it.Some new change is incurred with introduction of biological technology.In order to get a new research field of bio-denitrification in wastewater treatment,or a new method to improve the effectiveness of local denitrifier,this study is ready to screen some high active local denitrifier and cultivate some new higher active denitrifier by protoplast fusion.Therefore, in this paper,some high active local denitrifier such as ammonia oxidation bacteria,nitrifier and denitrifier bacteria were isolated firstly,then their growth curve were made and their characteristic were studied.Then denitrification character of local denitrification combination were researched.After this ,protoplast fusion of the best denitrifier bacteria was performanced and denitrifier character of the best fusant were studied. The resource bactiera of fusant was distinguished by characteristic comparation and 16S rDNA.Lastly,the nitrogen removal efficiency of local denitrifier combination and fusant combination were compared in experiment.The main conclusions are drawn as follows:(1)Three denitrifier bacteria which named FX1,FX2 and FX3 were isolated by limiting dilution and flat crossed separation through special culture medium.FX1 and FX3 was bacillus,FX3 was serratia.By analysing their growth curve and denitrification characteristic,it is indicated that they all had four growth periodic time .Delayed period was in 0-4hours,logarithmic period was in 4-16hours,stabilization period was in 16-20hours and failed period was after 20hours.First,the nitrogen removal efficiency increased obviously in logarithmic period and still increased in stabilization period,but the increasing speed was slown down.In failed period, the nitrogen removal efficiency was weaken visibly and to be steady.By comparation experiments,for these three denitrifier bacteria,FX2 was the best, FX1 was the second and FX3 was the last.It was illustracted by single factor experiment that the nitrogen removal efficiency increased with temperature rising when the temperature was below 30℃,as while as it decreased when the temperature was over 30℃.The resultion of orthogonal experiment indicated that sequence of influencing factors which affected the nitrogen removal efficiency of FX1was: cluture time>>temperature>>pH,for FX2,it was:temperature>>culture time>>pH。The best condition to FX1was:14hours,35℃and pH=7.5;For FX2,it was: 16hours,25℃and pH=7.0.(2)Through colour reaction, ammonia oxidation enriched clutureⅠwas selected to be better in experiments,and A1was isolated.By making it's growth curve,it was found out that it's growth period was longer,it grew slowly in first three days, the removal efficiency of NH3-N also was little;The speed of ammonia oxidation was improved obviously in the forth day to the seventh day.At the same time, nitrifier enriched clutureⅠwas selected to be better in experiments,and three nitrobacteria which named X1,X2 and X3 were isolated.X2 was selected to be the best,and the optimization orthgonal experiment(L9(34)) of nitrifier condition was carried out by X2.The resultion of that sequence of influencing factors which affected the nitrifer efficiency was:culture time>>temperature>>pH,and the best condition to X2 was6days,30℃and pH=7.0.(3)Artifical wastewater was maken to finish the denitrification experiment by combination of local ammonia oxidation bacteria,nitrifier and denitrifier bacteria.The major quality index of the artifical wastewater was: COD=300-350mg/L ,TP=6.9-9.2mg/L,NH3-N=16.2-20.5 mg/L.When there were only ammonia oxidation bacteria or nitrifier bacteria in local combination, the total nitrogen removal efficiency was little,as it was combinated with ammonia oxidation bacteria and nitrifier bacteria,the efficiency increased,but it was still small,and also return-service action was found in it.When ammonia oxidation bacteria,nitrifier and denitrifier bacteria were combinated, the total nitrogen removal efficiency increased sharply,and all were over 90%.CombinationⅠwas better than CombinationⅡ.When ammonia oxidation bacteria,nitrifier and denitrifier bacteria were combinated, group acativition happened apparently.(4)Experiments of protoplast fusion by most effictive denitrifier and denitrification experiment of the best fusant combination.After A1,X2,FX1and FX2 were purificated,they were ultraviolet mutagenized and selected by resistance to drugs.During the ultraviolet mutageniz,the resiatance ability of FX1 and FX2 were stronger,A1 was the second,and A1was the most senstive.Mutant bacteria FXB1and FXB2 were maken by ultraviolet exposure of 80 seconds.In the experiment of resistance to drugs,sodium penicillin G and streptomycin were used to mark the resiatance ability.Direct mutation bacteria were choosed in 40μg/mL,and after five generation cultivation,some with stable heredity were selected to be seed-parent bacteria for protoplast fusion.During the production of protoplast,the concentration of lysozyme was 400μg/mL and the temperature was 30℃,it must be enzymolysised for 10 to 16 hours.The molecular weight of PEG was 6000,and the quality concentration of it was 40%.For this,the time of fusion was 6min,and Ca2+ used in it was 0.015mol/L.Lastly,by sixth generation cultivation and selection of double-resistance flat,there were five fusants which were R1,R2,R3,R4 and R5.(5)R2 was optimizated as the best denitrifier fusant by comparation with R1,R2,R3,R4,R5,and A1,X2,FX1,FX2 by putting them in 250mL artificial wastewater(which was made by dilution the enrichment medium in five times) individually to measure their denitrifier ability.Then 16S rDNA with PCR was used,with comparing the content of same DNA, FX2 was distingulished to be the seed-parent bacteria of R2.(6)Growth curve of R2 was made and compared with FX2.The result showed that the total growth of R2 was improved by protopalst fusion.By denitrification experiment ,it was found out that they had the proximity denitrification effectiveness during first 4 hours.R2 was more effective than FX2 during next 4 hours, this trend continiuned to the 22th hours,and the largest difference was 27.58% in the 16th hours.This declarated that the nitrogen removal efficiency of denitrifier bacteria could be improved about 27%.(7)Comparation of the the nitrogen removal efficiency between local combination bacteria and fusant combination bacteria was carried out.In this ,the better combination of local bacteria was choosed as A1+X2+FX1.It was indicated that the nitrogen removal efficiency of A1+X2+R2 was always better than A1+X2+FX1,especially in the first eight hours,after this,the difference continiuned,but it decreased.It also illustrated that with the jion of best fusant,the effective of bacteria combination can be improved,but with long time,the dominance diminuted.