Studies On Microbial Degradation Of Tetrahydrofuran

Tetrahydrofuran, as a low toxic but capable of causing cancer, abnormal, variation pollutant, is entering environments increasingly day by day because of its wide usage in industry, agriculture, medicine, daily staples and so on. The dissertation is aimed to microbial degradation of tetrahydrofuran and composed of the following four parts: isolation of THF degrading microorganisms, optimization of the degrading conditions, cloning of THF degrading genes and studies on application of THF degrading strains in bioreactor.( 1 ) Isolation of THF degrading microbial strainsA strain, designated as YYL, was isolated from the mixture of four environment samples collected from several different environments after nearly one year enrichment and identified as Rhodococcus sp.YYL. Strain YYL could tolerate THF of high concentration up to 200mM, far higher than the previous reported strains. Total 20mM THF could be degraded in 102h. It was worth to be further studied and utilized in THF degradation. And at same time, two Bacillus strains were isolated and identified as Bacillus cereus and Bacillus aquimaris, respectively.( 2 ) Optimization of THF degrading conditionsTHF degrading and cell growth conditions were optimized based on Plackett-Burman experiment design and response surface methodology. Three metal ions, which had positive effect on THF degradation, were identified and the main components in the base mineral medium were taken into consideration of the main and interactive effect on THF degradation. The optimized medium was composed of: NH₄Cl 1.80g/L, K₂HPO₄·3H₂O 0.81g/L, yeast extraction 0.06g/L, MgSO₄·7H₂O 0.40g/L, ZnSO₄·7H₂O 0.006g/L, FeSO₄·7H₂O 0.024g/L, initial pH 8.26. Under the optimized condition, the maximum THF degradation rate increased to 137.60 mg THF·h^-1·g dry weight by Rhodococcus sp.YYL, which was nearly five times of that by the previously described THF degrading Rhodococcus strain. The optimized medium was much simpler than that reported.( 3 ) Cloning of the THF degrading monooxygenase gene clusterThe genus Rhodococcus was the first reported one capable of degrading THF, however, nothing about the related genes has been reported by now. In this research, COnsensus-DEgenerate Hybrid oligonucleotide Primer (CODEHOP) PCR and genomic walking method (TAIL PCR) were first used for isolating the complete THF degrading monooxygenase alpha-subunit gene in Rhodococcus sp.YYL, referencing the related genes from Pseudonocardia sp.K1. Further consensus primers was designed for isolating the monooxygenase gene cluster, according to the amplified sequence and the THF degrading multiple component monooxygenase gene cluster in Pseudonocardia sp.K1. It was found that the gene structure and expression direct in Rhodococcus sp.YYL were the same as in Pseudonocardia sp.K1, and further more the gene of each subunit had a similarity higher than 94% with that in Pseudonocardia sp.K1. There existed only one difference that a transposase gene was inserted between the monooxygenase and aldehyde dehydrogenase genes. The amino acid sequence analysis showed that the monooxygenase alpha-subunit was a transmember protein, which might be an important reason that the monooxygenase activity could not be detected in vitro. Polygenetic analysis of the amino acid sequence of each subunit and the 16S rDNA sequences of related strains demonstrated that an evolutionary distance was nearly 0 between THF degrading monooxygenases in Rhodococccus sp.YYL and Pseudonocardia sp. K1, but the two stains had a relative long distance in classification status. The result further proved that the THF degrading multiple component monooxygenase genes might occur by horizontal gene transfer.(4) Application of the isolated THF degrading strain YYL in SBR reactorThe result of acute toxicity test illustrated that the activities of catalase, urease, dehydrogenase, phosphatase and protease in activated sludge were all significantly inhibited and the microbial diversity was also lowered clarified by PCR-DGGE profiles when THF concentration was higher than 160mM. THF was much more toxic to culturable bacteria and fungi than actinomycete pointed out by the tradition spread plate method. The inhibition of enzyme activities and decrease of microbial diversity led to the decrease of the efficiency of activated sludge in waste water treatment. Detecting of the enzyme activities in activated sludge during the running of the reactor illustrated that the activities of catalase, urease, dehydrogenase, phosphatase and protease were significantly inhibited, especially the dehydrogenase, even though the artifical waster water contained THF of only 10mM. Shannon index of microbial diversity in activated sludge decreased from 1.424 to 0.823. All the tests illustrated that release of THF have negative effect on activated sludge and more attention needed to be paid to THF treatment.It was known that there were only a few strains capable of degrading THF and it was difficult to enrich mixture cultures. Bioargumentation was an effective way to solve this problem. In this research, the activated sludge collected from aerating pool in Zhenhai Refined and Chemical Company was used as the carrier in SBR for Rhodococcus sp.YYL planting. According to the study, successful planting of Rhodococcus sp.YYL in the reactor needed co-inoculation of other two bacilli, Bacillus cereus and Bacillus aquimaris. The result implied that Rhodococcus sp.YYL needs the existence of other two bacilli to create a suitable micro-environment for its application in activated sludge.Total 20mM THF could be completely degraded with Hydraulic Retention Time of 20h after the successfully planting of the Rhodococcus sp.YYL and optimization of the running conditions. The planting of Rhodococcus sp.YYL in activated sludge also elevated its resistance to pH variation, resulting that the THF degrading efficiency was not significantly affected under solution pH value of 5.0, while pure strain alone could be significantly inhibited.