【作者】 王电站；

【导师】 周立祥；

【作者基本信息】 南京农业大学 ， 生态学， 2009， 博士

【摘要】 硝基苯/苯胺类化合物是一类具有稳定化学性质、高毒性和易在生物体内积累的优先控制污染物,硝基苯和苯胺是其中最为简单的化合物,是重要的基础化工原料之一。随着化工工业的发展,对硝基苯和苯胺的需求呈明显上升趋势,因此进入环境的量也会增多。微生物降解在硝基苯/苯胺废水和污染环境修复等方面具有明显优势,自然界中原来并不存在利用硝基苯/苯胺的微生物资源,经过长期演化,微生物通过产生诱导酶来利用硝基苯/苯胺作为碳源、氮源或能源,进行生长和繁殖,从而使硝基苯/苯胺得以降解或转化。由于苯环的存在,在环境中苯环难以开环矿化,硝基苯、苯胺在环境中表现出结构稳定,对外界氧的作用有较强的抵抗力,芳环不容易被氧化,即在通常情况下,利用氧不容易使苯环裂解矿化；另一方面,苯环结构的对称性和稳定性使其不易发生生化反应,生物降解性差,且具有较大的生物毒性。本文从降解菌的驯化筛选、降解途径、降解机理、共代谢、分子遗传学角度,阐述了硝基苯/微生物降解研究的最新进展,明确了应进一步加强工程菌的构建及其应用开发研究,综述了在硝基苯类化合物污染环境的微生物修复方面,共代谢和混合菌株的协同作用具有重要的应用前景,并介绍了颗粒污泥法在城市污水处理中的应用,借此阐述了本研究的目的。本研究采用三角瓶在摇床上好氧振荡的方法,用硝基苯废水处理厂的好氧污泥驯化。首先培养能够降解硝基苯/苯胺的混合菌群,然后从此混合菌群中筛选纯化高效降解硝基苯/苯胺的优势菌种。在驯化过程中,发现混合菌群形成颗粒化,即为颗粒污泥,采用此颗粒污泥(混合菌群)进行降解硝基苯/苯胺的研究；然后分别从降解硝基苯的混合菌中筛选、纯化出三株菌——细菌NB1、杆菌NB2和丝状菌NB3,从降解苯胺的混合菌中也筛选纯化出三株菌——酵母菌AN1、杆菌AN2和丝状菌AN3,分别研究了NB1-NB3对硝基苯的降解效果和AN1-AN3对苯胺的降解效果；并分别在NB1、NB2、NB3之间、在AN1、AN2、AN3之间相互复配成几种复合菌,研究了复合菌对硝基苯和苯胺的降解效果。结果表明,混合菌群在以NB为唯一碳源和氮源的情况下降解NB的效果最好,该混合菌群降解NB时最适宜的温度为28℃,能够适宜于pH9.0以下的弱碱性环境,且最佳的pH值为7.0,当NB的起始浓度为600mg·L-1时,混合菌群适应期较短,在6h以下,混合菌群在24h内能够完全降解NB,降解速率最大,达到28.8mg·(L·h)-同样地,以苯胺为污染物培养出来的混合菌群在以苯胺为唯一源碳和氮源的情况下,具有较强的降解苯胺的能力,最适宜的温度为28℃,适于pH6.0-8.0的弱酸弱碱环境,且最佳的pH值为7.0,摇床的转速宜为180rpm,盐度宜为0.4%-0.6%,且当苯胺的起始浓度为600mg/L时,在18h内被完全降解,混合菌群降解苯胺的速度达到33.6mg/(L. h)。相比而言,苯胺较硝基苯容易被生物降解掉。采用纯菌进行的实验结果还表明,在降解硝基苯的三株纯菌株NB1-NB3中,细菌NB1对硝基苯的降解能力较强,经过36h对硝基苯的转化率达到56%,矿化率达到41%,是降解硝基苯混合菌中的优势菌种,经鉴定,NB1属于解鸟氨酸克雷伯氏菌；而在AN1-AN3中,酵母菌AN1则对苯胺的降解能力较高,经过36h,对苯胺的转化率达到68%,矿化率达到59%,是降解苯胺的混合菌中的优势菌种,经鉴定,AN1属于假丝酵母菌。与混合菌相比,纯菌的降解效果稍差些。纯菌的复合实验结果表明,硝基苯降解菌NB1-NB3之间、苯胺降解菌AN1-AN3之间都存在明显的协同效应；在降解硝基苯的过程中,细菌NB1与酵母菌AN1之间存在相互协同效应；而在苯胺的降解过程中,没有表现出明显的协同或拮抗效应。来自降解硝基苯的三类菌(NB1、NB2、NB3)中,细菌NB1为优势菌,且这几种菌复配后,尚没有发现颗粒化的形成,而来自降解苯胺的三类菌(AN1、AN2、AN3)中,以酵母菌AN1为主体,且复配成的A13和A123复合菌形成了颗粒污泥,且A123复合菌在36h的OD600值达到了0.94,苯胺的转化率达到了89%。细菌、杆菌、丝状菌复配成的N123复合菌,36h的OD600值达到0.43,硝基苯的转化率达到了84%。由此说明苯胺生物处理过程中污泥颗粒化的主要原因在于丝状菌的大量繁殖,而降解硝基苯的优势菌为细菌,降解苯胺的优势菌为酵母菌,且苯胺生物处理过程中较硝基苯更易形成颗粒污泥,且与酵母菌分泌的胞外多聚物EPS可能存在密切的关系,有待今后深入探讨。好氧颗粒污泥法用于含硝基苯类化工废水的处理是一种新的尝试,具有很大的实际应用价值。更多还原

【Abstract】 Nitrobenzene and aniline, widely used industrial chemicals, are two important nitroaromatic compounds that have been listed as priority pollutants for their nature of chemical stability, high toxicity, resistant to degradation and potential of accumulation in organism.With the development of chemical industry, China’s demand for nitrobenzene and aniline will continuously increase, and their wrongly handled and disposed of endanger both human and environmental health. Thus, the degradation of nitrobenzene and aniline is of great concern. Microbial degradation has obvious advantages in treatment of wastewater, gases and soil that containing nitrobenzene and aniline. These microorganism can utilize nitrobenzene or aniline as carbon source, nitrogen source or energe source for their growth and reproduce.Due to the chemical stability, however, benzene ring is hard to be opened by oxidation of oxygen. Thus, nitrobenzene and aniline are difficult to be mineralized. On the other hand, symmetry and stability of benzene ring also makes them hard to be degraded by biochemical reactions. Besides, nitrobenzene and aniline have great biological toxicity.This paper presents the latest research reports about the biodegradation of nitrobenzene and aniline, which include the screening and domestication of degrading microbes, the degradation pathways and degradation mechanism. Furthurmore, this paper emphasizes the importance of the development and application of engineering strains, and introduce the mineralization of nitrobenzene and aniline by mixed microbes and the application of granular sludge in the treatment of municipal wastewater.The highly efficient nitrobenzene/anline-degrading aerobic microbes were screened and domesticated from activated sludge and sediments from a nitrobenzene wastewater treatment plant. During the domestication processes, the mixed microbes formed granular sludge. Based on these mixed microbes, we studied the aerobic biodegradation of benzene and aniline.Bacteria(NB1), bacillus(NB2) and filamentous Bacteria(NB3) as well as yeast(AN1), bacillus(AN2) and filamentous Bacteria(AN3) are screened and purified from the mixed nitrobenzene-degrading bacteria and aniline-degrading bacteria, respectively. Besides the degradation effect of nitrobenzene or anline by each microorganism, their combined degradation effect in the treatment of corresponding nitroaromatic compounds has also been studied.The result show that the mixed nitrobenzene-degrading microbes degraded nitrobenzene most efficiently in the conditions with nitrobenzene as sole carbon and nitrogen source. The optimum condition for growth of the mixed microbes and degradation of nitrobenzene was 28℃and pH 7.0, and weak alkaline environment with pH below 9.0 is suitable for their growth.When nitrobenzene concentration was 600 mg/L, the mixed microbes can quickly adapt the environment in 6h, and they can completely degraded nitrobenzene in 24h, the maximum degradation rate could reach as high as 28.8 mg·(L·h)-1Similarly, the mixed anline-degrading microbes that cultivated in the environment with aniline can degraded aniline more efficiently in the conditions with aniline as sole carbon and nitrogen source than in the conditions with other carbon source and nitrogen source. The optimum condition for growth of the mixed microbes and degradation of aniline was 28℃and pH 7.0, and the environment with pH between 6.0 and 9.0 is suitable for their growth. When aniline concentration was 600 mg/L, the degradation rate could reach 33.6 mg (L·h)-1, and aniline can be completely degraded within 18 h.The results obtained from the experiments undergoing with pure microbes show that Bacteria(NB1) is the dominant bacteria among the nitrobenzene-degrading microbes. The conversion rate and mineralization rate of nitrobenzene by NB1 within 36 h can reach 56% and 41%, respectively.Among the aniline-degrading microbes, yeast(AN1) was the dominant bacteria. The conversion rate and mineralization rate of aniline by ANl within 36 h can reach 68% and 59%, respectively.The biodegradation results show that the mixed microbes degraded corresponding nitroaromatic compounds more efficiently than pure bacteria. It was suggested that there was an obvious synergistic effect among nitrobenzene-degrading bacteria as well as aniline-degrading bacteria, but no obvious synergistic effect or antagonism exists between nitrobenzene-degrading bacteria and aniline-degrading bacteria.No granular sludge formed in the mixed nitrobenzene-degrading microbes, which, however, appeared in the mixed aniline-degrading microbes in which the dominant bacteria was yeast(AN1). The OD600 of the mixed nitrobenzene-degrading microbes(N123) that consisted of Bacteria(NB1), bacillus(NB2) and filamentous Bacteria(NB3) was 0.43 within 36 h, and the degradation rate of nitrobenzene then was 84%.Similarly, The mixed aniline-degrading microbes(A123) that consisted of yeast(AN1), bacillus(AN2) and filamentous Bacteria(AN3) shows high aniline degradation rate. The OD600 of them within 36 h was 0.94, and the degradation rate of aniline reached 89%.The main reason for the formation of sluge granulation in biodegradation of nitrobenzene and aniline was the filamentous Bacteria multiply, and the secretion of extracellular polymer substances by yeast can explain why it is easier for the formation of sluge granulation in aniline degradation process than in nitrobenzene degradation process. Aerobic granular sludge is a new attempt to treat nitroaromatic compounds wastewater, and has very important practical value.更多还原