【作者】 金若菲；

【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】 本论文旨在研究基因工程菌Escherichia coli JM109（pGEX-AZR）的生长特性及其对偶氮染料的脱色性能。为了降低基因工程菌应用可能带来的生态风险，将E．coli JM109（pGEX-AZR）与膜生物反应器结合，将微生物有效的截留在反应器之内，以防止其向环境中释放。另外将E．coli JM109（pGEX-AZR）作为高效菌制剂投加到活性污泥体系中进行强化脱色研究，借助现代分子生物技术，研究了其在污泥体系中的生长特性及对污泥群落结构的影响。本研究为基因工程菌在废水处理中的应用提供了一定的理论及技术支持。在以葡萄糖为限制性基质的分批培养中，E．coli JM109（pGEX-AZR）的最佳培养条件为：5g·L^-1葡萄糖，0.3 g·L^-1 NH₄Cl：无机盐培养基(组成为：KH₂PO₄ 2.0 g·L^-1，Na₂HPO₄ 1.3 g·L^-1，MgSO₄ 2.0 g·L^-1，CaCl₂ 0.0364 g·L^-1，FeCl₃ 0.00025 g·L^-1)；初始pH=7.5；接种量10 mL·L^-1；培养温度35℃。IPTG诱导偶氮还原酶表达的最佳条件为：IPTG的投加时机为E．coli JM109（pGEX-AZR）的对数生长后期，即OD₆₆₀为1.7～1.8，最大IPTG的添加浓度以1 mmol L^-1，诱导的持续时间为8 h。可利用乳糖代替IPTG作为偶氮还原酶表达的诱导剂，乳糖的最佳投加浓度为40 mmol·L^-1，乳糖诱导的蛋白含量和偶氮还原酶活性为IPTG诱导时的93.6％和85.7％。E．coli JM109（pGEX-AZR）对酸性大红GR的共代谢脱色动力学包括生长基质的利用动力学、染料的脱色动力学、生物的生长动力学。E．coli JM109（pGEX-AZR）对生长基质-葡萄糖的利用符合Monod方程，其中μ_{max g}为0.07657 g·g^-1·h^-1，K_g为0.3324 g·L^-1；当有酸性大红GR存在时，对葡萄糖的降解存在非竞争性抑制，抑制系数K_ig为0.213g·L^-1。酸性大红GR的脱色动力学符合底物抑制模型Andrews，当存在生长基质时，μ_{max c}为42.45 mg·g^-1·h^-1，K_c为584.93 mg·L^-1，K_ic为556.89 mg·L^-1。E．coli JM109（pGEX-AZR）的产率系数Y_m为0.1 g·g^-1；生物转化能力T_m为121.2 mg·g^-1；内源衰减系数b为0.0378d^-1。E．coli JM109（pGEX-AZR）对多种偶氮染料有较好的脱色效果。供氧条件对偶氮染料脱色的影响很大，厌氧条件有利于酸性大红GR的脱色：脱色最佳的pH值为中性或弱碱性；在25～40℃范围内，随着温度的提高，脱色速率有所提高。当废水中存在浓度为1～5％的NaCl、NaSO₄时，E．coli JM109（pGEX-AZR）对酸性大红GR保持较高的脱色性能，而NaNO₃的存在对酸性大红GR的脱色具有很大影响，随着NaNO₃浓度的增加，脱色率明显降低。用海藻酸钠及聚亚胺酯大孔泡沫对E．coli JM109（pGEX-AZR）进行固定化，固定化细胞对酸性大红GR的脱色都符合底物抑制模型Andrews。比较不同存在形态的E．coliJM109（pGEX-AZR）对酸性大红GR的脱色动力学可知，海藻酸钠固定的E．coli JM109（pGEX-AZR）的最大比脱色速率及半饱和系数都比游离态有所降低，说明由于海藻酸钠固定，底物的传递受到影响，使速率下降；而抑制系数有所增加，说明固定后对细菌有所保护，菌体耐受环境变化的能力提高。而聚亚胺酯大孔泡沫固定菌体的动力学参数全部比游离态的有所提高，说明聚亚胺酯大孔泡沫适合用来固定该菌，实现对酸性大红GR的脱色。将E．coli JM109（pGEX-AZR）投加到浸没式厌氧膜生物反应器（SAnMBR）中，在SAnMBR连续运行过程中，对酸性大红GR具有88％～93％的脱色率。通过膜过滤作用后，出水酸性大红GR的脱色率可以维持在96％左右。膜生物反应器对COD_Cr，的去除率范围为56％～72％。在反应过程中，菌体浓度由初始浓度1.97 g·L^-1升高到2.12 g·L^-1，后逐渐降低，最终维持在1.60 g·L^-1～1.80 g·L^-1之间。将各种不同存在形态的E．coli JM109（pGEX-AZR）投加到厌氧序批式生物反应器（AnSBR）中，考察其对生物脱色过程的强化作用，投加游离态和大孔泡沫固定菌体的系统表现出类似的强化性能，脱色能力及抗浓度负荷冲击的能力都高于投加海藻酸钠包埋菌体的强化系统和对照系统。在运行过程中取污泥总DNA进行指纹分析的结果表明，E．coli JM109（pGEX-AZR）能够在强化系统维持较高的丰度，不同形式E．coli JM109（pGEX-AZR）的投加丰富了污泥群落的多样性。更多还原

【Abstract】 It has been aimed to investigate growth characteristics of a genetically engineered microorganism （GEM） Escherichia coli JM 109 （pGEX-AZR） and its ability to decolorize azo dyes. For the ecological safety, E. coli JM109 （pGEX-AZR） was fixed in the MBR to prevent the release of the GEM into the environment. Meanwhile, bio-augmentation of Escherichia coli JM109 （pGEX-AZR） to azo dyes decolorization was studied, and the kinetics and structure changes of microbial commnunity in augmented system were revealed by modern molecular techniques.The optimal growth conditions of E. coli JM109 （pGEX-AZR） are as follows: glucose 5 g·L^-1, NH₄C10.3 g·L^-1, inorganic salt medium (KH₂PO₄ 2.0 g·L^-1, Na₂HPO₄ 1.3 g·L^-1, MgSO₄ 2.0 g·L^-1, CaCl₂ 0.0364 g·L^-1, FeCl₃ 0.00025 g·L^-1), pH=7.5, inoculation amount 10 mL·L^-1, temperature 35℃. When the optical density at 660 nm (OD₆₆₀) of E. coli JM109 （pGEX-AZR） was up to 1.7～1.8, IPTG was added the culture concentration of 1 mmol L^-1, and induction was conducted for another 8 h. Attempt to use lactose instead of IPTG inducing expression of azoductase by the recombinant strain E. coli JM109. Lactose was added so as to the final concentration to 40 mmol·L^-1, recombinant protein expression concentration and azoreductase activity are 93.6% and 85.7% as much as that given by the strain induced by IPTG.The co-metabolism kinetics of acid red GR by the E. coli JM109 （pGEX-AZR） was studied using glucose as primary substrate. The process of glucose utilization by E. coli JM109 （pGEX-AZR） can be described by the Monod equation, where the maximum specific biodegradation rate coefficientμ_{max g} is 0.07657 g·g^-1·h^-1, half-velocity constant K_g is 0.3324 g·L^-1. Acid red GR exerted non-competitive inhibition on the glucose utilization, an inhibition coefficient K_ig of 0.213 g·L^-1. The kinetics of the acid red GR decolorization by the E. coli JM109 （pGEX-AZR） agrees with Andrews model, the kinetic parameters, r_dye,max, K_s and K_i, have been found to be 42.45 mg L^-1 h^-1, 584.93 mg L1 and 556.89 mg L^-1 respectively. The biomass yield coefficient of E. coli JM109 （pGEX-AZR） is 0.1 g·g^-1, the biomass transformation capacity is 121.2mg·g^-1, the endogenous decay constant is 0.0378 d^-1.The decolorization of several kinds of azo dyes can be executed by the E. coli JM 109 （pGEX-AZR） . The optimal conditions of decolorization are anaerobic conditions and pH 7-8. The decolorization rate increases with the increase of temperature between 25～40℃. The process of decolorization of acid red GR is non-inhibit in the presence ot NaCl （1～5%）, NaSO₄ （1～5%）. In contrast, the addition of NaNO₃ （1～5%） had an adverse effect on the decolorization rate, this may be the competition in reduction reaction between the nitrate and the azo bonds, and the nitrate is obviously a better electron acceptor than the azo bond.Similar to acid red GR decolorization by suspended E. coli JM109 （pGEX-AZR）, acid red GR decolorization by the immobilized cells on macro-porous foam and the sodium-alginate immobilized cells can be also described by the same Andrews model. The kinetic parameters r_dey,max and K_s,. by the sodium-alginate immobilized cells decreased and K_i increased compared with suspended cell, which might be due to the low biological activity and slow mass transfer. The kinetic parameters r_dye,max, K_s. and K_i by the immobilized cells on macro-porous foam carriers increased compared with suspended cell, which might be due to the improvement of stability.E. coli JM109 （pGEX-AZR） was used for the decolorization of acid red GR in the submerged anaerobic membrane bioreactor. The results from the study show that, the bioreactor with E. coli JM109 （pGEX-AZR） has a high capacity for decolorization, decolorization rate of the supernatant effluent is of 88%～93%, decolorization rate and COD removal rates of the membrane effluent are about 96% and 56%～72%, respectively. It is obvious that the membrane separation process can enhance the wastewater treatment effect. The concentration ofE. coli JM109 （pGEX-AZR） had grown from 1.97 g·L^-1 to 2.12 g·L^-1 at the beginning, and finally had kept at the concentration between 1.60 g·L^-1 and 1.80 g·L^-1.The E. coli JM109 （pGEX-AZR） was tested in anaerobic sequencing batch reactors （AnSBR） in order to enhance the acid red GR decolorization. The continuous operations of the four bioreactors with different E. coli JM109 （pGEX-AZR） immobilization supports showed that the E. coli JM109 （pGEX-AZR） showed bio-augmentation in AnSBRs with suspended and immobilized on macro-porous foam carriers. The tolerance to acid red GR concentration shock and the decolorization rate in these two bio-augmented AnSBRs were higher than those of the other two systems, control system and bio-augmented AnSBRs system with the sodium-alginate immobilized cells. Changes in microbial community were detected with ribosomal intergenic spacer analysis （RISA） and amplified ribosomal DNA restriction analysis （ARDRA）, which revealed that the introduced E. coli JM109 （pGEX-AZR） is persistent in the augmented systems and maintained higher metabolic activity.更多还原