Screening Of Toluene Degrading Strains And Preliminary Study On Degradation Characteristics Of The Strains

With the development of economy,environmental problems occur frequently,the pollution of Volatile Organic Compounds(VOCs)to the environment becomes more and more serious.As one of the common VOCs in the pharmaceutical industry,toluene is a serious threat to human health and ecological environment due to its toxicity and carcinogenic effects.Among many treatment methods for VOCs,the biodegradation method has become the focus of current research because of its low energy consumption,high efficiency,and no pollution.Functional microorganisms are the direct degraders of pollutants.Therefore,screening efficient degrading bacteria is the core of biodegradation method.In this study,toluene was used as the sole carbon source to screen and identify the strains of activated sludge.The following research results were obtained:1.Toluene(50-1000 mg/L)was used as the sole carbon source to acclimate the activated sludge of a chemical plant in Nanjing for four cycles.Nine isolated strains of functional microorganisms degrading toluene were obtained by using "pour plate method" and "streak plate method" to screen and rescreen the domesticated culture medium.By morphological observation and 16 S r DNA molecular identification,it was determined that strain 125 W was Ensifer adhaerens,strain 134 W was Klebsiella sp.,strain 214 W was Raoultella planticola,strain 224 R was Serratia marcescens,strain 316 Y was Bacillus megaterium,strain 326 Y was Paenibacillus sp.,strain 424 W was Delftia sp.,strain 524 W was Sphingobacterium multivorum,and strain 536 W was Pseudomonas putida.2.Under the condition of 30°C,150 rpm,10% of the inoculated quantity and 236.18 mg/L toluene concentration,the single strain degradation experiment was carried out to evaluate the biodegradation ability of functional microorganisms to toluene.Compared with other strains,strains 125W(Ensifer adhaerens MT431909)and 536W(Pseudomonas putida MT431910)had stronger degradation ability,with the removal efficiency of 99.85% and 99.90% respectively in 24 hours(p < 0.001).The removal efficiency of toluene by the other 7 strains was about 20% in 24 hours.Strains 125 W and 536 W were used as the test strains for subsequent tests.Through the literature review,no research about the degradation of toluene by E.adhaerenshas been found.3.Our results indicated that the degradation ability of toluene was affected by temperature,p H,inoculated quantity and initial concentration of toluene.The optimum degradation temperature of strains 125 W and 536 W were 30°C and 35°C,respectively.The optimum degradation p H range was 7.0~8.0,the optimum degradation inoculated quantity was 5%,and the removal efficiency of toluene with initial concentration less than 577.3 mg/L in 24 hours was more than 99%.4.Strains 125 W and 536 W grew well in 10 volatile organic compounds selective media with final concentration of 100 mg/L.10 volatile organic compounds were ethanol,phenylethanol,toluene,methanol,benzene,xylene,tween 80,phenol,DMSO and 2-mercaptoethanol.The results showed that the two strains had good broad spectrum of volatile organic compounds utilization.5.The crude enzyme of strain 536 W was extracted and the key enzyme of toluene degradation was determined.The activities of toluene dioxygenase(TDO)and catechol 2,3-dioxygenase(C23O)were detected.It is preliminarily determined that the degradation pathway of strain 536 W was that toluene dioxygenase oxidized toluene to 3-methylcatechol,and then catechol 2,3-dioxygenase oxidized the intermediate position to open the ring,and finally enterd the tricarboxylic acid cycle.6.The chemotaxis of strains 125 W and 536 W were studied by “agarose plug assays”.It was found that the two strains could sense the changes of toluene and catechol concentration in the growing environment,and produced positive chemotaxis movement,then selectively grow according to the concentration gradient of substrate distribution.In conclusion,E.adhaerens strain 125 W and P.putida strain 536 W have good biodegradation ability for toluene and broad-spectrum utilization for various volatile organic compounds.Both of the two strains can sense the change of chemical substances in the growing environment,which is beneficial to the bioavailability and biodegradation of target pollutants.Both of the two strains are ideal toluene degrading bacteria.