Preparation Of PAHs-degrading Complex Microbial Inoculants And Its Applicational Study In Contaminated Soil

11 strains of PAHs degradation bacteria were isolated from soil sampled from coking factories.Degradational bacteria flora were formed in consideration of the culture conditions,characteristics and the degradation capacity for PAHs.The PAHs degradation capacity was enhanced by adding C,N,P,metal ions and surfactant and the optimization of cultivation conditions.The reinforcement roles of nutrient,surfactant and soil-microbes-plant system were inestigated through pot culture experiment.In addition,the actual capacity of PAHs degradation was also checked in polluted sewage irrigation area with the aim of obtaining high efficient degradation bacteria as well as the corresponding applicational technologies for remediation of PAHs contaminated farmland.The main results are listed as follows:(1)11 strains of PAHs degradation bacteria,which survied on the plates with PAHs as the only carbon source after enrichment culture,were isolated from PAHs polluted soil.Through morphological observation,physiological and biochemical experiments and 16 s r RNA sequencing,DJ-3 was classified into pseudomonas sp..DJ-10 into bacillus sp.,and the rest 9 strains into klebsiella sp..The PAHs degradation rate of DJ-3(49.1%),DJ-8(44.5%),DJ-10(53.9%)was significantly higher than other strains.(2)The above 3 strains with the highest PAHs degradation capacity,DJ-3,DJ-10 and DJ-8 were selected for further combination research.The results showed that i)any combinations,2 or 3,did not showed antagonism relationship,ii)for each strain,the degradation ability for phenanthrene was much higher than for pyrene,and the PKB group(DJ-3+DJ-8+DJ-10)showed the highest degradation capacity for phenanthrene and pyrene,which was significantly higher than any single or any other combination of bacterial strains,iii)metal ions had significant effect both on the growth of bacteria and their ability of PAHs degradation.The optimal culture system for degradation bacteria containing 0.15 g/L,0.25 g/L,0.20g/L,0.4 g/L of Mg SO4·7H2O,Co Cl2,Fe SO4·7H2O,Mn SO4·H2O,respectively.iv),the additional C,N,P could significantly facilitate the growth of degrading bacteria,and the optimal culture system for PKB flora were 37℃,initial p H7,with 1%-2% inoculation volume,1/3-2/3 loading volume for fermentation,Under above optimal conditions,OD600 of PKB group was the highest and reached to 0.50;the degradation rate for phenanthrene and pyrene were highest,reached 95.1%,75.4%,respectively,and degradation rate for PAHs was 85.3%.(3)Adding suitable amount of C,N,P source material to contaminated soil-microbe bioreactor system were beneficial to the growth of bacteria and PAHs degradation capacity.Chicken manure could effectively increase the bacteria count and the degradation efficiency instead of chemical nutrients and trace elements.The optimum ratio of polluted soil to chicken manure in biological reaction system was 5:1.The optimal inoculation quantity,culture temperature,humidity were 10%,37℃ and 50%.Under this condition,PAHs degradation rate reached to the highest value of 50.11% after 60 days.(4)Ryegrass and rhamnolipid could effectively enhance the PAHs degradation ability of PKB combination in the soil.Adding “ryegrass + degradation bacteria + rhamnolipid” into the reaction system,soil PAHs degradation was more apparent,mainly improved the degradation of tetracyclic,pentacyclic.The degradation rate for soil PAHs was 61.1% after 60 d of planting ryegrass in BRH treatment,in which the average degradation rate of tetracyclic PAHs,and pentacyclic and total PAHs reached to 60.36% and35.39%,respectively.(5)The PKB agent were prepared by mixing PKB,peat,chicken manure with a ratio of 1:8:1,then adding 0.5% biosurfactant rhamnolipid,finally the water content of the agent was 15%,and effective number of bacteria was more than 108 CFU/g.Before use,chicken manure were rotten and treated with peat In PAHs contaminated soil-microbial-plant system,PAHs degradation bacteria could significantly increase the degradation rate of acenaphthene,fluoranthene and benzo(a)pyrene in the planting and chicken manure ferttilized field.The contents of acenaphthene,fluoranthene and benzo(a)pyrene in soil reduced37.76%、19.41%、and 15.66%,respectively,in the MFL treatment after three years of cultivation.The degradation rate of benzo(a)pyrene was 12.46% in the MFC treatment planting corn,which was 11.7 times of that of CKC treatment.The degradation rate of benzo(a)pyrene was 15.6% in the MFL treatment planting ryegrass,which was 3.7 times of CKL treatment,the efficiency was more significant,especially for epipodium PAHs.