| 题名 | 生物还原法处理碱性含Cr(VI)废水的应用基础研究 |
| 作者 | 徐林 |
| 学位类别 | 博士 |
| 答辩日期 | 2012-06-02 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 刘会洲 |
| 关键词 | 生物还原法 碱性含Cr(VI)废水 Pannonibacter phragmitetus LSSE-09 细胞提取物 细菌固定化 海藻酸钠微囊 超顺磁性Fe3O4纳米颗粒 |
| 其他题名 | Basic and Applied Research on Bioreduction of Alkaline Cr(VI)-containing Wastewater |
| 学位专业 | 化学工程 |
| 中文摘要 | 从铬盐厂污泥中筛选出一株高效的碱性Cr(VI)还原菌Pannonibacter phragmitetus LSSE-09。该菌在有氧和无氧条件下均具备较强的Cr(VI)还原性能，最佳还原pH值为9.0。LSSE-09生长菌体具有很高的还原速率，37 °C时，有氧生长9 h和24 h能分别完全还原100和1000 mg/L Cr(VI)。无外加电子供体时，静息菌体无氧培养120 min即可将350 mg/L Cr(VI)全部还原，还原速率为1.46 mg Cr(VI)/g (干菌重)/min，而有氧还原速率只有0.21 mg/g/min。乳酸钠、醋酸钠、丙酮酸钠、甲酸钠、柠檬酸钠和葡萄糖作为外加电子供体, 均能明显提高LSSE-09在有氧、无氧时的Cr(VI)还原速率。醋酸钠对LSSE-09的还原性能强化作用最明显，3000 mg/L的醋酸钠能使无氧、有氧的还原速率分别提高至9.47 mg/g/min和4.42 mg/g/min，比不加电子供体时分别提高了5倍和20倍，还原速率明显高于现有报道。此外，LSSE-09还具备优良的重复使用性能，并且NO3-和SO42-对还原速率的抑制作用很小。实验探讨了P. phragmitetus LSSE-09的Cr(VI)还原机理。通过检测细胞各部分提取物的Cr(VI)还原性能，证明了LSSE-09的Cr(VI)还原酶是存在于细胞质内的可溶蛋白，与外层膜周质空间内的可溶蛋白及细胞膜蛋白无关。细胞提取物（S12）的最佳Cr(VI)还原pH值均为7.0，NADH、葡萄糖、醋酸盐、柠檬酸盐、丙酮酸盐和乳酸盐作为外加电子供体，能明显提高S12的酶活。其中，NADH对S12的酶活强化作用最明显，其有氧和无氧条件下的酶活分别是不加电子供体时的3.5倍和3.4倍。1.0 mmol/L的金属离子Mn2+，Cd2+，Fe3+和Hg2+对Cr(VI)还原酶具有抑制作用，而Cu2+能起到强化Cr(VI)还原酶活性的作用，使有氧和无氧条件下的相对酶活分别提高了29%和33%。在应用方面，为解决传统实心微球包埋固定化细菌活性低的问题，实验采用界面交联法制备出直径3 mm，壁厚0.1 mm的海藻酸钠空心微囊固定化LSSE-09。综合考虑还原速率、物理性能和对上清液中Cr(III)的脱除性能，确定最佳制备条件为：羧甲基纤维素钠（sodium carboxymethyl cellulose, CMC）20 g/L，海藻酸钠（sodium alginate, SA）5 g/L，CaCl2 0.1 mol/L，交联时间30 min。此时微囊的还原速率为4.20 mg/g/min，是相同条件下游离菌体的69.0%，高于传统实心微球包埋法。微囊能脱除63.7%的Cr(III)有机络合物，极大地降低了上清液中的Cr(III)含量。同时，该微囊还具备较好的循环使用和储存性能，使用8次后活性仅损失了22.7%。经过35天，4 °C储存，相对活性依然高达储存前的85.7%。此外，实验首次解决了碱性环境的细菌磁固定化问题。PEI修饰的Fe3O4超顺磁性纳米颗粒能够单分散在水中，长期静置不会发生沉降，颗粒平均直径约为15 nm，且具备超顺磁性，比饱和磁化强度和等电点分别为为62.3 emu/g和11.5。pH 9.0时，PEI修饰的磁颗粒能够通过静电吸附固定在细菌P. phragmitetus LSSE-09的表面，使磁固定化的细菌具有超顺磁性特征，比饱和磁化强度为16.3 emu/g，在外加磁场下分离迅速、方便。磁固定化细菌的还原活性与游离菌体相同，并且可以多次重复使用。此方法不仅首次解决了碱性Cr(VI)还原菌的磁固定化问题，而且对于所有在碱性环境发挥功能的细菌磁固定化具有借鉴意义。本论文工作为生物还原法处理碱性含Cr(VI)废水的工艺优化和机理研究提供了必要的基础数据、新颖的工艺思路和进一步的研究方向。 |
| 英文摘要 | A large quantity of alkaline Cr(VI)-containing wastewater exceeding the discharging limit have become a well-recognized biohazard in water pollution control due to the extremely toxic and carcinogenic effects of Cr(VI) on biological systems.`Bioreduction is an environmentally-friendly method that can reduce the Cr(VI) to Cr(III) which is much stable and less toxic, using a series of enzyme-catalyzed reactions. It provides easier operation process, lower cost and no-secondly pollution. Thus, bioreduction has large-scale potential application for Cr(VI)-containing wastewater treatment. To overcome the existing shortcomings of bioreduction including insufficient research on alkaline Cr(VI)-containing wastewater, lower reduction rates of reported bacterial strains and lack of research on reduction mechanisms, this thesis develops a applied basic research on bioreduction of alkaline Cr(VI)-containing wastewater from the point of isolating a high-efficient strain, investigating the Cr(VI)-reducing mechanism and improving the bioreduction activity of immobilized cells. A novel Cr(VI) resistant bacterial strain LSSE-09, identified as Pannonibacter phragmitetus, was isolated from industrial sludge of chromate factory. It has strong aerobic and anaerobic Cr(VI)-reduction potential under alkaline conditions, with optimal pH value of 9.0. At 37°C, growing cells of strain LSSE-09 could completely reduce 100 and 1000 mg/LCr(VI) to Cr(III) within 9 and 24 h respectively under aerobic condition. Resting cells showed higher anaerobic reduction potential with the rate of 1.46 mg/g (dry weight)/min, comparing with their aerobic reduction rate, 0.21 mg/g/min. External electron donors, such as lactate, acetate, formate, pyruvate, citrate and glucose could highly increase the reduction rate, especially for aerobic reduction. The presence of 3000 mg/Lacetate enhanced anaerobic and aerobic Cr(VI)-reduction rates up to 9.47 mg/g/minand 4.42 mg/g/minrespectivelyNO3- and SO42- had slightly negative effects on Cr(VI)-reduction rates. , which were 5 and 20 times faster than those without it. To the best of our knowledge, the reduction rates of strain LSSE-09 is obviously higher than other reports. Strain LSSE-09 could be repeatedly used and retained high specific Cr(VI)-reduction activity. To investigate the Cr(VI)-reducing mechanism, hexavalent chromate reductase was characterized by evaluating different cell-free extracts and was found to be localized in the cytoplasmic fractions of P. phragmitetus LSSE-09, not in the periplasmic and membrane fractions.The Cr(VI) reductase activity of cell-free extract (S12) was significantly improved by external electron donors, such as NADH, glucose, acetate, formate, citrate, pyruvate and lactate. The reductase activity was optimal at pH 7.0 with NADH as the electron donor. The aerobic and anaerobic Cr(VI)-reduction enhanced by 0.1 mmol/LNADH were respectively 3.5 and 3.4 times as high as that without adding NADH. The Cr(VI) reductase activity was inhibited by 1.0 mmol/L Mn2+, Cd2+, Fe3+, and Hg2+, whereas Cu2+ enhanced the chromate reductase activity by 29% aerobically and 33% anaerobically. To overcome the lower activity of bacteria entrapped in tranditional gel bead, P. phragmitetus LSSE-09 was encapsulated in liquid-core alginate-carboxymethyl cellulose capsules, with diameter of 3 mm, membrane thickness of 0.1 mm. Taking the physical properties of the capsules, the activity of encapsulated cells and total Cr (III) concentration in the supernatant into account, optimal conditions (sodium alginate 5 g/L; sodium carboxymethyl cellulose 20 g/L; CaCl2 0.1 mol/L; gelation time 30 min) for LSSE-09 encapsulation were determined. At optimal conditions, a relatively high reduction rate of 4.20 mg/g/min，which was 69.0% compared with that of free cells was obtained. Total Cr (III) concentration in the supernatant was significantly decreased after reduction, because 63.7% of the formed soluble organo-Cr(III) compounds compared with those of free cells were removed by the relatively smaller porous structure of alginate capsules. In addition, encapsulated LSSE-09 decreased 22.7% of activity after eight repeated cycles at 37°C, and 85.7% of its initial activity remained after 35-day storage at 4 °C. In addition, a novel bacteria separation and immobilization method under alkaline conditions was firstly developed by using superparamagnetic Fe3O4 nanoparticles (NPs). The polyethyleneimine (PEI)-modified NPs were monodispersed and the particle size was about 15 nm with a saturation magnetization of 62.3 emu/g and an isoelectric point (pI) of 11.5 at room temperature. At initial pH 9.0, P. phragmitetus LSSE-09 cells were immobilized by PEI-modified NPs via electrostatic attraction. The coated cells exhibited typical superparamagnetic behavior with a saturation magnetization of 16.3 emu/g, which provid quick and easy magnetic separation advantage with an external magnetic field. Compared to free cells, the coated cells not only had the same Cr(VI)-reduction activity but could also retain high specific Cr(VI)-reduction activity over six batch cycles. The results suggest that PEI-modified NPs could not only immobilize LSSE-09 but also has potential application for immobilizing bacteria which play roles under alkaline conditions. This thesis provides necessary basic data, novel process idea and further research direction for the process optimization and mechanisms of the research on bioreduction of alkaline Cr(VI)-containing wastewater. |
| 语种 | 中文 |
| 公开日期 | 2013-09-25 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1795]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 徐林. 生物还原法处理碱性含Cr(VI)废水的应用基础研究[D]. 中国科学院研究生院. 2012. |
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