Study On Biodegradation Of O-chlorophenol By Rhodopseudomonas Palustris

With the known toxicological effects of correlate chlorinated phenols and their derivatives, as well as their environmental contamination with bioaccumulation, study on the treatment of chlorophenols wastewater is the present focus and challenge in research area of environmental science and engineering fields worldwide.In this thesis, the investigation on biodegradation of o-chlorophenol (2-CP) by the photosynthetic bacteria Rhodopseudomonas palustris was carried out. The identification and ultraviolet mutation of 2-CP-degrading bacterial, the characteristics and mechanism of 2-CP degradation and immobilization of photosynthetic bacteria were studied respectively. The results were expected to supply useful references for environmental quality evaluation and bioremediation of halogenated hydrocarbon pollution. The main results are reported as follows:1. A strain of bacterium named 1D with the biodegrability of 2-CP was isolated and screened from shallow substrate sludge in downstream side of the sewage outfall of an insecticide factory. Based on the colony and morphological properties, absorption spectrum analysis of living cells, general physiological biochemical characteristics and 16SrDNA, the strain 1D was identified as photosynthetic bacteria Rhodopseudomonas palustris.2. In order to obtain 2-CP degrading mutant with high efficiency, the strain 1D was mutated by ultraviolet. According to the ultraviolet mutation time and lethality effect curve, the optimizing mutation time was determined as 50 s. The mutant strain was named as PSB-1D. The degradation rate of 2-CP by the mutant strain PSB-1D was up to 67%, which was over 98% higher than that of the original strain 1D. The results of the dehydrogenase activity tests, the lowest observed effect of 2-CP concentrations (LOEC) tests and the 96 h median lethal concentration (96-LCso) tests demonstrated that the mutant strain PSB-1D performed higher tolerance to 2-CP than that of the original strain 1D, being regarded as the better strain in term of the 2-CP degradation.3. Influence on strain PSB-1D growth and 2-CP degradation under various conditions of illumination and oxygen was investigated. The results showed strain PSB-1D was capable of degrading 2-CP, based on cometabolism mechanism, under the illuminated anaerobic condition or dark aerobic condition.Under the illuminated anaerobic condition, the optimizing condition in term of the degradation efficiency was initial 2-CP concentration at 50 mg/L,3.0 g/L sodium propionate as cometabolism carbon substrate,2.0 g/L yeast extract as nitrogen sources, initial pH value at 7.0, culture temperature at 30℃, intensity of illumination at about 4000 lx and culture time at 7 d, with the degradation rate of 2-CP by strain PSB-1D being 62.08%.Under the dark aerobic condition, the optimizing condition in term of the degradation efficiency was initial 2-CP concentration at 50 mg/L,2.0 g/L glucose as cometabolism carbon substrate,0.6 g/L (NH)2SO4 and 0.2 g/L yeast extract as nitrogen sources, initial pH value at 7.0, rotation speed 130 r/min and culture time at 7 d, with the degradation rate of 2-CP by strain PSB-1D being 74.2%.4. Under the illuminated anaerobic condition and dark aerobic condition, the degradation kinetic data fitted the Andrews model well. The biodegradation process of 2-CP can be well described by enzymatic reaction of high concentration inhibition. The 2-CP degradation kinetics equation is: Illuminated anaerobic: Dark aerobic:5. With the total cellular proteins electrophoresis being analyzed by SDS-PAGE, induction mechanism of the key cometabolism degradation enzyme of 2-CP was studied under the illuminated anaerobic condition and dark aerobic condition, which showed that the specific degrading enzyme for 2-CP degradation were induced by 2-CP itself when sodium propionate and glucose were served as the growth substrate respectively.6. Analysis of degradation products of 2-CP testes under the illuminated anaerobic condition showed that benzoic acid was produced in the degradation process. The probable degradation pathway of 2-CP by Rhodopseudomonas palustris PSB-1D taken in the illuminated anaerobic condition was that 2-CP was dechlorinated and passed through benzoic acid routes for aromatic ring cleavage.7. The results of free state chloride ion concentration tests, catechol 1,2-dioxygenase activities and catechol 2,3-dioxygenase assays showed that the probable degradation pathway of 2-CP by Rhodopseudomonas palustris under the aerobic dark condition was 2-CP being dechlorinated initially, and then ortho ring cleavage being catalyzed by catechol 1,2-dioxygenase.8. Rhodopseudomonas palustris PSB-1D being immobilized with sodium alginate as immobilizing carrier and activated carbon as additive material, the optimal conditions of immobilized cell preparation had been determined through orthogonal experiments. The results showed that the optimum conditions for the immobilization were as follows:activated carbon concentration 1%, sodium alginate concentration 3%, immobilized cells/investment materials 1/20. Under the chosen conditions, the biodegradation rate of 2-CP by the immobilized photosynthetic bacteria was 76.5% after 7 days culture.9. The immobilized photosynthetic bacteria were added to the Sequencing Batch Reactor (SBR). The effects of reaction parameters(immobilized cells addition, aeration time, aeration rate, and so on) on 2-CP degradation efficiency in the reactor were studied. The results showed that, under dark aerobic condition, the optimum technological conditions for the bioreactor with effective volume 5 L were as follows:aeration time 10 h, immobilized cells addition 20 g, aeration rate 100 L/h, idle time 1 h. Under this condition, bioreactor system could treat the 2-CP wastewater effectively and steadily, with the removal rate of 60% or so.