Optimization Of Molecular Detection Technology For Phenol-degrading Microorganism And Its Application In Treatment Of Phenolic Industrial Wastewater

As important chemical raw materials and by-products, phenolic compounds widely exist in industrial wastewater. Due to its toxicity, carcinogenicity, teratogenicity and mutagenicity, phenolic compounds have caused gigantic environmental pollution and become one of primary pollutants in water. In order to lessen the effects of the phenolic compounds, considerable attentions have been drawn to the treatment of phenolic industrial wastewate and many methods such as activeated sludge, biofilm, fluidized bioreators, enzyme treatment technology and immobilized biotechnology have been adopted to treat phenolic industrial wastewater.Becaus of high efficiency, low cost, simple operation and not having second pollution, biological treatments have become the main method for phenolic Industrial wastewater treatment. In order to improve the efficiency and degradation rate of biological method, molecular detection technology of phenol-degrading microbes was optimized in this paper. And on this basis phenol-degrading strains with high efficiency were isolated from industrial wastewater containing high concentration of phenol sampled from a Jingdezhen coking plant, Jiangxi province. The characteristics of degradation of the isolated clons and its influence factors were also analyzed in this paper. In order to accurately control the groth of the phenol-degrading microorganism, effects of phenol industrial wastewater on microbial community structure were studied. The major results were summarized as follows:(1) Based on the reported sequences of 16S rDNA gene and phenol hydroxylase gene of phenol-degrading microbes, senven pairs of PCR primers were designed by using bioinformatics. Three pairs of primers were used to amplify the phenol hydroxylase gene, while the other four pairs of primers were used to amplify the 16S rDNA gene of phenol-degrading microbes. The results showed primers M2 and M7 was the optimal primers of phenol hydroxylase gene and 16S rDNA gene respectively, which could be used to analyze and isolate the phenol-degrading microbes.(2) Microbial community structure and its effects by seasons were analyzed by high-throughput sequencing technologies. The results showed chao and ace of microbial richness index was lower, while diversity index was higher. Microorganisms in aerobic activated sludge and anaerobic activated sludge could be divided into 16 phylums, among which proteobacteria was dominant phylum and its percentage was 48%. With temperature increase, the percentage of proteobacteria increased. Diaphorobacter sp. and Ottowia sp. was dominant genus and its percentage was 20.81% and 13.48% respectively. Microbial community structure almost had no change with seasonal change, while the dominant bacteria changed greatly. Diaphorobacter sp. was the dominant bacteria at February and August and its percentage was 29.38%, while Ottowia sp. was the dominant bacteria at May and November, whose percentage was 17.06%.(3) Five phenol-degrading strains with high efficiency named pd-A, pd-B, pd-C, pd-D, and pd-E were isolated from aerobic active sludge sampled from aeration pool of the Jingdezhen coking plant, Jiangxi province. The five isolated clones could grow at the initial phenol concentration higher than 2000 mg L"1. When cultivated in the selective liquid medium with the initial phenol concentration of 1000 mg L-1, pd-D had higher degrading rate than ther other four isolated clones, whose phenol degrading rate was 72.11% at 48 h. The optimal growing conditions of pd-D were 30 ℃, pH 7.0 and 1000 mg L-1 phenol. When cultivated at optimal conditions, pd-D completely degraded the phenol in 96 h and the removal rate of COD was higher than 80%.When the high-effective phenol-degrading microbe pd-D was used in MFC (Microbial Fuel Cell), we found that it had high production capacity of the battery, and comparatively higher efficiency in degradation.(4) A two chamber microbial fuel cell which used phenol as the electronic provider in the anode room and pd-D as the anode catalyst was designed to investigate the power generation and the effects on the degradation efficiency of phenol. The results showed power generation cycle was 7 days. The maximum power of MFC was 0.5 V and COD removal rate was higher than 88%at the initialphenol concentration of 1000 mg L-1. Phenol degradation time shortened 24 h.(5) After ideniified by morphology methods, physiology and biochemical tests and 16S rDNA analysis, pd-D belonged to Clostridium sp..