沼泽红假单胞菌（Rhodopseudomonas palustris）生物降解2-氯苯酚的研究
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摘要
氯苯酚类废水污染环境且难以处理,是当前国内外环境科学与环境工程领域的研究热点与难点。本论文以2-氯苯酚(o-chlorophenol,2-CP)为研究对象,从某农药厂排污口底泥中分离筛选得到一株可降解2-CP的野生菌株,对该菌株进行生化鉴定及16SrDNA系统发育分析,并对其进行紫外诱变,全面探讨了降解菌株对2-CP的毒性效应、降解特性、降解机理等多方面内容,在此基础上进行了固定化菌体细胞处理2-CP的相关试验。通过上述研究,以期为探索2-CP的生物处理方法、污染环境的生物修复及光合细菌的有效利用提供有益的参考。本研究的主要结果如下：
1.从某农药厂排污口下游浅层底泥中富集、驯化、分离、筛选得到1株2-CP降解菌。通过对菌株菌落及细胞形态观察、活细胞紫外光谱扫描、生理生化特征试验、碳源利用试验以及16SrDNA序列同源性分析,鉴定该菌系沼泽红假单胞菌(Rhodopseudomonas palustris),为光合细菌。
2.对降解菌株进行紫外诱变,获得诱变菌株PSB-1D。通过紫外诱变时间与致死率效应试验确定最佳诱变时间为50 s。比较诱变前后菌株对2-CP的降解效果及脱氢酶活性、安全质量浓度和半致死浓度等耐受性考察指标,结果表明,菌株经过紫外诱变处理后对2-CP的降解效果及耐受性能均得到明显改善。
3.不同供氧光照对PSB-1D生长及2-CP降解效果的影响表明,光合细菌PSB-1D在光照厌氧和黑暗好氧两种条件下均能对2-CP共代谢降解。其中：
在光照厌氧条件下,菌株PSB-1D的最佳培养条件为：3.0g/L丙酸钠为共代谢碳源,2.0g/L酵母膏为氮源,初始pH值为7.0,培养温度为30℃,光照度为4000 lx左右,在此条件下培养7d后,PSB-1D对2-CP降解率可达62.08%；
在黑暗好氧条件下,菌株PSB-1D的最佳培养条件为：2.0g/L葡萄糖为共代谢碳源,0.6g/L(NH4)2S04和0.2g/L酵母膏为氮源,初始pH值为7.0,摇床转速130 r/min,在此条件下培养7d后,PSB-1D对2-CP的降解率可达74.2%。
4.采用Andrews方程模拟得到菌株PSB-1 D在光照厌氧和黑暗好氧条件下对2-CP的降解动力学方程,分别为：光照厌氧：黑暗好氧：
5. SDS-PAGE全细胞蛋白电泳结果表明,2-CP的降解酶是菌株PSB-1D在光照厌氧或黑暗好氧条件下,分别利用丙酸钠和葡萄糖作为生长底物提供能源和碳源时,由2-CP作为非生长底物诱导产生的,它们不同于PSB-1D利用生长底物时产生的酶。
6.通过对降解过程中脱氯率及苯甲酸和4-羟基苯甲酸含量的分析,推断出菌株PSB-1D光照厌氧条件下降解2-CP主要是通过脱掉氯离子并生成苯甲酸的代谢途径开环完成的。
7.对降解过程中游离氯离子浓度、菌体细胞提取液中邻苯二酚1,2双加氧酶和邻苯二酚2,3-双加氧酶的酶活性分别进行测定,推断出菌株PSB-1D好氧黑暗条件下降解2-CP的途径主要是先脱掉氯离子,之后再在邻苯二酚1,2双加氧酶的催化作用下将苯环邻位裂解开环进行的。
8.通过不同材料对比试验,确定海藻酸钠为光合细菌PSB-1D的最佳包埋材料。利用向海藻酸钠中添加活性炭的方法可提高固定化微生物小球的性能及其对2-CP的处理效果。以2-CP降解率为考察指标的正交试验确定了固定化PSB-1D菌体细胞的最优方案：活性炭添加量为1%,海藻酸钠浓度为3%,包埋菌体量/包埋材料量为1/20。在此条件下,固定化微生物小球培养7d后对2-CP的降解率为76.5%。
9.采用含固定化微生物小球的SBR反应器对自配2-CP废水进行试验研究,确定最佳工艺条件为：反应时间10h,固定化微生物小球投加量为20g,曝气量为100 L/h,闲置时间为1 h。在此条件下,反应器系统显示出稳定的2-CP去除性能和较好的微生物小球重复利用性。
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.
1.从某农药厂排污口下游浅层底泥中富集、驯化、分离、筛选得到1株2-CP降解菌。通过对菌株菌落及细胞形态观察、活细胞紫外光谱扫描、生理生化特征试验、碳源利用试验以及16SrDNA序列同源性分析,鉴定该菌系沼泽红假单胞菌(Rhodopseudomonas palustris),为光合细菌。
2.对降解菌株进行紫外诱变,获得诱变菌株PSB-1D。通过紫外诱变时间与致死率效应试验确定最佳诱变时间为50 s。比较诱变前后菌株对2-CP的降解效果及脱氢酶活性、安全质量浓度和半致死浓度等耐受性考察指标,结果表明,菌株经过紫外诱变处理后对2-CP的降解效果及耐受性能均得到明显改善。
3.不同供氧光照对PSB-1D生长及2-CP降解效果的影响表明,光合细菌PSB-1D在光照厌氧和黑暗好氧两种条件下均能对2-CP共代谢降解。其中：
在光照厌氧条件下,菌株PSB-1D的最佳培养条件为：3.0g/L丙酸钠为共代谢碳源,2.0g/L酵母膏为氮源,初始pH值为7.0,培养温度为30℃,光照度为4000 lx左右,在此条件下培养7d后,PSB-1D对2-CP降解率可达62.08%；
在黑暗好氧条件下,菌株PSB-1D的最佳培养条件为：2.0g/L葡萄糖为共代谢碳源,0.6g/L(NH4)2S04和0.2g/L酵母膏为氮源,初始pH值为7.0,摇床转速130 r/min,在此条件下培养7d后,PSB-1D对2-CP的降解率可达74.2%。
4.采用Andrews方程模拟得到菌株PSB-1 D在光照厌氧和黑暗好氧条件下对2-CP的降解动力学方程,分别为：光照厌氧：黑暗好氧：
5. SDS-PAGE全细胞蛋白电泳结果表明,2-CP的降解酶是菌株PSB-1D在光照厌氧或黑暗好氧条件下,分别利用丙酸钠和葡萄糖作为生长底物提供能源和碳源时,由2-CP作为非生长底物诱导产生的,它们不同于PSB-1D利用生长底物时产生的酶。
6.通过对降解过程中脱氯率及苯甲酸和4-羟基苯甲酸含量的分析,推断出菌株PSB-1D光照厌氧条件下降解2-CP主要是通过脱掉氯离子并生成苯甲酸的代谢途径开环完成的。
7.对降解过程中游离氯离子浓度、菌体细胞提取液中邻苯二酚1,2双加氧酶和邻苯二酚2,3-双加氧酶的酶活性分别进行测定,推断出菌株PSB-1D好氧黑暗条件下降解2-CP的途径主要是先脱掉氯离子,之后再在邻苯二酚1,2双加氧酶的催化作用下将苯环邻位裂解开环进行的。
8.通过不同材料对比试验,确定海藻酸钠为光合细菌PSB-1D的最佳包埋材料。利用向海藻酸钠中添加活性炭的方法可提高固定化微生物小球的性能及其对2-CP的处理效果。以2-CP降解率为考察指标的正交试验确定了固定化PSB-1D菌体细胞的最优方案：活性炭添加量为1%,海藻酸钠浓度为3%,包埋菌体量/包埋材料量为1/20。在此条件下,固定化微生物小球培养7d后对2-CP的降解率为76.5%。
9.采用含固定化微生物小球的SBR反应器对自配2-CP废水进行试验研究,确定最佳工艺条件为：反应时间10h,固定化微生物小球投加量为20g,曝气量为100 L/h,闲置时间为1 h。在此条件下,反应器系统显示出稳定的2-CP去除性能和较好的微生物小球重复利用性。
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.
引文
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