Isolation, Identification Of Aromatic Compounds-degrading Bacteria And Characterization Of Its Degrading Properties

Aromatic hydrocarbons are compounds with one or more benzene rings. With anincrease in molecular weight, their solubility in water decreases; melting and boilingpoint increase and vapour pressure decreases. The common sources of aromatichydrocarbon in environment include natural as well as anthropogenic. They areubiquitously present contaminants which are toxic, mutagenic. They do not degradeeasily under natural conditions. Because of their presence in all components ofenvironment, aromatic hydrocarbons are easily to be bio-accumulated. In the generalregulation of aromatic polluants, human beings can make use of physical, chemicaland biological means, but the widespread degradation of these methods was notthorough and easy to cause secondary pollution. Microbial treatment was a method oflow cost, high efficiency, no secondary pollution and easy operation. Therefore,screened the efficient degradation bacteria, studied the degradation characteristics andits related enzymes, and applied into the environment had an important theoretical andpractical significance. In this study, strain M7and strain M8were isolated byenrichment culture and screening from the activated sludge sample of a chemicalplant in Hubei. Both the strains possess the ability to effectively degrade aromatichydrocarbon.Based on the results of phenotypic features, physiological-biochemicalproperties, and phylogenetic similarity of16S rRNA gene sequences, strain M7andstrain M8were designated as Acinetobacter and Ochrobactrum. Strain M7is resistantto tetracycline, ampicillin, chloromycetin and spectinomycin, and sensitive to otherantibiotics. And strain M8is resistant to any of the7antibiotics. Both the two strainscan’t grow and ferment with saccharides. Degrading bacterial M7and M8could usevarious types of aromatic compounds as the sole carbon source to grow. The broadsubstrate range of strains M7and M8indicates their high potential in bioremediationof contaminated environment.The optimal conditions of the two strains for both growth and degradationprocess are as follows: temperature30℃, pH7.0, shaking speed200r/min andinoculation rate5%. Under the optimal conditions, strain M7could decompose over99%of phenol (500mg/L) and80%naphthalene (500mg/L). The degradation rate ofpyridine (500mg/L) is almost61.2%after8day culture. The effects of environmental factors on the growth and naphthalene degrading were investigated. Theresults indicated that externally added carbon source influenced the degradation rateobviously. Peptone and ammonium oxalate accelerate the naphthalene degradation ofstrain M7, and ammonium oxalate, pyrogallo, m-dihydroxybenzene, yeast extract,peptone, salicylic acid generally could increase the degradation rate of naphthalenefor strain M8. The strains M7and M8were also applied to the degradation of cokingwastewater, brewery wastewater and swine wastewater. The COD removal efficiencyof strain M7was39.61%、83.68%、95.64%, respectively. And the COD removalefficiency of strain M8was21.43%、75.00%、72.38%, respectively. After treated bystrain M7and strain M8within72h, the coking-plant wastewater effluent and thebrewery wastewater effluent can meet the third grade national discharge requirements;the pig manure water not only can reach the national second-degree wastewaterdischarge standard but also the first grade national discharge requirements; thebrewery wastewater effluent can meet the first grade national discharge requirementsin terms of TN. Specially, both the strains are highly effective to remove TotalPhosphorus (TP) with the degrading rate of more than80%from brewery wastewater.CatA gene, nahH gene, and nahAc gene, encoding catechol1,2-dioxygenase,catechol2,3-dioxygenase, naphthalene dioxygenase respectively, play an importantrole in the degradation pathway. Cloned from the strain M7, nahH gene was clonedfrom strain M8, and nahAc gene was cloned from both the strains. Sequencinginformation showed that the CatA gene from strain M7was high homologous tocatechol2,3-dioxygenase of Pseudomonas sp. PH7. Both strains have nahAc genewith the homologous to Pseudomonas sp.. Considering the products GC-MS analysisand the clone of degrading enzyme gene, it suggests that strain M7possesses the metapathway, and strain M8possesses the ortho pathway.