【作者】 郑春莉；

【导师】 周集体；

【作者基本信息】 大连理工大学 ， 环境工程， 2007， 博士

【摘要】 本文报道了3株新分离的在好氧条件下以硝基苯为唯一碳、氮、能源的菌株，对其进行了生理生化特性研究和rDNA分子序列分析，考察了3株菌的游离态细胞和固定化细胞生长及降解硝基苯的特性，并推测出菌株降解硝基苯的途径，以期为实际硝基苯工业废水的处理提供理论依据通过形态特征、生理生化特性以及rDNA分子序列分析，3株菌分别鉴定为胶红酵母菌（Rhodotorula mucilaginosa Z1）、微白黄链霉菌（Streptomyces albidoflavus Z2）和藤黄微球菌（Micrococcus luteus Z3）。菌株Z1、Z2、Z3在GenBank的登录号分别为DQ778627、DQ855477、DQ855476。菌株Z1、Z2、Z3已作为专利菌种保存于中国普通微生物菌种保藏中心，注册编号分别为CGMCC No.1758、CGMCC No.1759、CGMCCNo.1760。菌株Z1的最适生长与降解条件为：温度30℃，pH=7.0，摇床转速180 r／min，接种量9mg／L（干重）。菌株Z2的最适生长与降解条件为：温度30℃，pH=7.0，摇床转速150r／min，接种量12mg／L（干重）。菌株Z3的最适生长与降解条件为：温度25℃，pH=7.0，摇床转速150 r／min，接种量9mg／L（干重）。在以硝基苯为唯一碳、氮、能源的液体培养基中，最佳降解条件下，当硝基苯初始浓度为200mg／L时，菌株Z1、Z2、Z3完全降解硝基苯的时间分别为60h、72h、120h，TOC去除率均在98％以上，该实验结果表明硝基苯最终被矿化为无害的二氧化碳和水。在以硝基苯为唯一碳、氮、能源的液体培养基中，菌株Z1、Z2和Z3对硝基苯的最大耐受浓度分别为450、400和250mg／L。最佳降解条件下，菌株Z1、Z2和Z3对硝基苯的降解动力学均符合典型的底物抑制模型，菌株Z1的模型参数为：q_max=4.11（1／h），K_s=151.42mg／L，K_i=32.569mg／L；菌株Z2的模型参数为：q_max=3.19（1／h），K_s=106.73mg／L，K_i=38.03mg／L；菌株Z3的模型参数为：q_max=2.32（1／h），K_s=67.13mg／L，K_i=40.29mg／L。进一步考察了高盐度下以及其它有机物质和硝基苯共存时3株菌对硝基苯的降解。在以硝基苯为唯一碳、氮、能源的液体培养基中，最佳降解条件下，当硝基苯初始浓度为200mg／L时，3株菌均能在盐度（以氯化钠质量浓度计）≤5％的条件下有效降解硝基苯。最佳降解条件下，当150mg／L的苯酚和200mg／L的硝基苯共存或75mg／L的苯胺与200mg／L的硝基苯共存时，菌株Z1能有效降解硝基苯；当200mg／L的苯酚和200mg／L的硝基苯共存或50mg／L的苯胺与200mg／L的硝基苯共存时，菌株Z2能有效降解硝基苯；当25mg／L的苯酚和200mg／L的硝基苯共存或25mg／L的苯胺和200mg／L的硝基苯共存，菌株Z3几乎不降解硝基苯。采用表面吸附固定化技术，选取大孔网状载体DW-22为固定化材料分别考察了菌株Z1、Z2和Z3在DW-22型载体上的生长与降解硝基苯的最适条件。菌株Z1在DW-22型载体上的最适生长与降解条件为：温度30℃，pH=7.0，摇床转速160r／min，接种量6mg／L（干重）。菌株Z2在DW-22型载体上的最适生长与降解条件为：温度30℃，pH=7.0，摇床转速130r／min，接种量10mg／L（干重）。菌株Z3在DW-22型载体上的最适生长与降解条件为：温度25℃，pH=7.0，摇床转速130r／min，接种量6mg／L（干重）。在以硝基苯为唯一碳、氮、能源的液体培养基中，最适生长与降解条件下，当硝基苯初始浓度为200mg／L时，固定化Z1、Z2、Z3细胞完全降解硝基苯的时间分别为30h、40h、96h。固定化细胞对硝基苯的降解动力学均符合经典的底物抑制模型，固定化Z1细胞的动力学参数为：q_max=8.38（1／h），K_s=185.59mg／L，K_i=132.39mg／L；固定化Z2细胞的动力学参数为：q_max=6.34（1／h），K_s=153.63mg／L，K_i=126.73mg／L；固定化Z3细胞的动力学参数为：q_max=4.31（1／h），K_s=110.39mg／L，K_i=54.95mg／L。与游离态细胞相比，固定化细胞的耐热性、耐盐性和耐毒性均得到提高。本研究同时进行了固定化Z1细胞降解硝基苯的半连续流试验，实验结果表明：与游离态细胞相比，固定化细胞具有良好的耐水力负荷和冲击负荷的能力。推测了菌株Z1降解硝基苯的途径：硝基苯经部分还原生成2-氨基酚，2-氨基酚再开环降解，进一步矿化；同时在2-氨基酚的开环过程中，副产物吡啶甲酸生成，Z1进一步降解吡啶甲酸。本研究同时推测了菌株Z1降解吡啶甲酸的途径：吡啶甲酸经羟基化反应生成6-羟基吡啶甲酸，6-羟基吡啶甲酸再开环降解，最终矿化为无害的二氧化碳和水。综上所述，本研究分离的胶红酵母菌（Rhodotorula mucilaginosa Z1）、微白黄链霉菌（Streptomyces albidoflavus Z2）和藤黄微球菌（Micrococcus luteus Z3）在实际硝基苯工业废水的处理中有着广阔的应用前景。更多还原

【Abstract】 This dissertation reports three newly isolated strains, which are able to utilize nitrobenzene as the sole source of carbon, nitrogen and energy under aerobic condition. The physiological biochemical properties of the three strains were studied and the rDNA molecular identifications were carried out. The strain growth and nitrobenzene degradation characteristics of the free cells and irmnobilized cells were investigated. Metabolic pathway of the nitrobenzene degradation was proposed. The purpose of this dissertaion is to provide an academic basis for the treatment of practical nitrobenzene-containing wastewaters.According to the morphological characteristics, physiological biochemical properties and rDNA sequences, the three strains are identified as Rhodotorula mucilaginosaZ1, Streptomyces albidoflavusZ2 and Micrococcus luteusZ3, respectively. The rDNA sequences of Z1, Z2. and Z3 have been submitted to GenBank with the accession numbers DQ778627, DQ855477 and DQ855476, respectively. Z1, Z2 and Z3 are deposited as patent strains in China General Microorganism Culture Center with the accession numbers CGMCC 1758, CGMCC 1759 and CGMCC 1760, respectively.The optimal conditions for both strain Z1 growth and nitrobenzene degradation are: temperature 30℃, pH=7.0, shaking velocity 180r/min and inoculation size 9mg/L （dry weight）; the optimal conditions for both Z2 growth and nitrobenzene degradation are: temperature 30℃, pH=7.0, shaking velocity 150r/min and inoculation size 12mg/L （dry weight）; the optimal conditions for both Z3 growth and nitrobenzene degradation are: 25℃, pH=7.0, 150r/min, inoculation size 9mg/L （dry weight）. When nitrobenzene serves as the sole source of carbon, nitrogen and energy and the initial concentration is 200mg/L, strain Z1 degrades nitrobenzene completely after 60h under the optimized conditions, Z2 72h and Z3 120h. TOC removing rates are over 98% for all the three strains, which demonstrate that nitrobenzene is mineralized to CO₂ and H₂O. Strain Z1 can tolerate nitrobenzene up to a concentration of 450mg/L, Z2 400mg/L and Z3 250mg/L. Under the optimized conditions, the degradation kinetics of nitrobenzene by the three stains can be expressed by Andrew equation. The kinetic parameters of strain Zl are: q_max=4.11（1/h）, K_s=151.42mg/L, K_i=32.569mg/L; the kinetic parameters of Z2 are: q_max=3.19（1/h）, K_s=106.73mg/L, K_i=38.03mg/L; the kinetic parameters of Z3 are: q_max=2.32（1/h）, K_s=67.13mg/L, K_i=40.29mg/L.The nitrobenzene degradation by the three strains in high salinity was studied. When nitrobenzene serves as the sole source of carbon, nitrogen and energy and the initial concentration is 200mg/L, the three strains still degrade nitrobenzene effectively in 5% salinity （NaCl, mass concentration） under the optimized conditions. Even 150mg/L phenol or 75mg/L aniline mixed into 200mg/L nitrobenzene, strain Z1 degrades nitrobenzene effectively under the optimized conditions. 200mg/L phenol or 50mg/L aniline mixed into 200mg/L nitrobenzene, Z2 degrades nitrobenzene effectively under the optimized conditions. However, Z3 degrades nitrobenzene little when 25mg/L phenol or 25mg/L aniline is mixed into 200mg/L nitrobenzene under the optimized conditions.The surface-adsorption immobilization method was adopted. Macro-pore carrier of DW-22 was selected as the immobilized material. The optimal conditons for the growth of strain Z1 on DW-22 and nitrobenzene degradation are: temperature 30℃, pH=7.0, shaking velocity 160r/min and inoculation size 6mg/L （dry weight）; the optimal conditons for the growth of Z2 on DW-22 and nitrobenzene degradation are: temperature 30℃, pH=7.0, shaking velocity 130r/min and inoculation size 10mg/L （dry weight）; the optimal conditons for the growth of Z3 on DW-22 and nitrobenzene degradation are: temperature 25℃, pH=7.0, shaking velocity 130r/min and inoculation size 6mg/L （dry weight）. When nitrobenzene serves as the sole source of carbon, nitrogen and energy and the initial concentration is 200mg/L, immobilized Z1 degrades nitrobenzene completely after 30h under the optimized conditions, Z2 40h and Z3 96h. Under the optimized conditions, the degradation kinetics of nitrobenzene by immobilized Zl, Z2 and Z3 can be expressed by Andrew equation. The kinetic parameters of immobilized Zl are: q_max=8.38（1/h）, K_s=185.59mg/L, K_i=132.39mg/L; the kinetic parameters of immobilized Z2 are: q_max=6.34（1/h）, K_s=153.63mg/L, K_i=126.73mg/L; the kinetic parameters of immobilized Z3 are: q_max=4.31（1/h）, K_s=110.39mg/L, K_i=54.95mg/L. Compared to free cells, immobilized cells exhibit strong capacity to endure more heat, salinity and toxicity. Immobilized Z1 is selected for further study on nitrobenzene degradation under semi-continuous flowing modes and the experimental results indicate that immobilized cells can tolerate higher nitrobenzene shock-load than free cells.The metabolic pathway of nitrobenzene degradation by strain Z1 was investigated. The proposed pathway is: nitrobenzene undergoes partially reduction to form 2-aminophenol, and then 2-aminophenol is cleaved and mineralized. Meantime, in the cleavage of 2-aminophenol, picolinic acid emerges as a byproduct. Z1 also degrades picolinic acid. The proposed pathway is: picolinic acid undergoes hydroxylation to form 6-hydroxy picolinic acid and 6-hydroxy picolinic acid is cleaved and mineralized.In conclusion, Rhodotorula mucilaginosaZ1, Streptomyces albidoflavusZ2 and Micrococcus luteusZ3 have higher potential for being applied to nitrobenzene industrial wastewater treatment.更多还原