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1、 重庆大学博士学位论文RL-2生物絮凝剂的研制及絮凝机理研究姓名：罗平申请学位级别：博士专业：环境工程指导教师：罗固源20051001 中文摘要摘要目前，国内外在微生物絮凝剂的作用规律及絮凝机理等方面的研究还不够详细、系统，只提出了一些假说，如粘质假说、酯合学说、菌体外纤维素纤丝学说、架桥学说等。由于产絮凝剂的微生物种类繁多，絮凝剂的组成、结构复杂，对于同一应用体系，不同的微生物絮凝剂有不同的作用机制。本文对高效絮凝剂絮凝性能、絮凝作用规律、絮凝机理以及实际应用等方面进行了重点研究，其主要工作如下：（1）本研究从活性污泥及土壤中筛选得到 4株(RL-2，NH-4，RT-4及 NL-3)具有较高
2、絮凝活性(絮凝率90%)的微生物。进一步优化筛选得出 RL-2菌株产生的絮凝剂性能最为优异，根据其个体形态特征、菌落特征及生理生化实验，初步鉴定为短芽孢杆菌（Bacillus brevis）。在菌体培养过程中发现，絮凝活性与菌生长量呈正相关；且絮凝剂主要分布在发酵液中，仅有少量附着在细胞表面，说明絮凝剂是由 RL-2菌在细胞内合成并分泌到胞外的胞外絮凝剂。（2）RL-2产生的絮凝剂絮凝性能优异，具有实际应用价值。为了最大限度的提高絮凝活性，对 RL-2产絮凝剂的培养进行了条件优化试验研究，得到了最佳培养基配方为：以葡萄糖为碳源，酵母膏+（NH4）2SO4为氮源，在初始 pH值7.0，培养温度
3、30，通气量 120150r/min，培养 3d后，经乙醇及氯化十六烷基吡啶铵沉淀，可获得胞外生物絮凝剂产品，其产率高达 5.1320g/L。本研究发现了短芽孢杆菌能够产生微生物絮凝剂，且絮凝活性高，性能稳定。（3）用食品加工行业的高浓度有机废水，如味精废水、豆制品废水、罐头废水、啤酒废水、中药废水及乳品废水为替代培养基，对 RL-2菌进行培养，优化筛选。结果表明，RL-2菌在不外加碳、氮源的情况下，可直接利用一定浓度的味精废水作为养料产生胞外生物絮凝剂，从而为 RL-2菌找到了一种较为廉价的替代培养基，大大降低了 RL-2絮凝剂的生产成本。因此，味精废水的利用既降低了生物絮凝剂 RL-2的生
4、产成本，又为该类废水处理提供了新的方法。该菌可利用工业废弃物生长，生长条件粗放，且所产絮凝剂使用范围广，因此对其进行了深入的研究。（4）采用化学分析、仪器分析等多种方法对微生物絮凝剂 RL-2的成分及絮凝特性的分析研究表明，RL-2絮凝剂的有效成分是一种无毒的高聚物多糖。薄层色谱分析表明，该多糖是由葡萄糖、甘露糖及葡萄糖醛酸等单糖构成的杂多糖，红外光谱分析结果表明该絮凝剂分子链上分布着羟基、羧基等多种极性基团，故絮凝性能优异。絮凝特性的研究表明该絮凝剂具有热稳定性；能溶于酸、碱溶液I 重庆大学博士学位论文中但不溶于有机溶剂中；用粘度法测得该絮凝剂的平均分子量为 3.64106Da（道尔顿）。与
5、无机及有机高分子絮凝剂对高岭土悬液的絮凝活性相比，性能优，用量少。（5）絮凝剂结构分析、Zeta电位测定及氢键和离子键检测结果表明，絮凝剂和高岭土之间的结合力为氢键，絮凝过程存在架桥作用，其絮凝机理为：絮凝剂和高岭土颗粒在絮凝剂的活性部位以氢键结合，然后再经过“架桥”作用絮凝沉淀。Ca2+在絮凝过程中主要起压缩颗粒双电层，吸附电中和，降低动电势的作用。该絮凝剂性能优异的原因在于分子链上分布着羟基、羧基等较多的极性基团，且极性基团在分子链上分布均匀，分子量大，絮凝剂分子容易与胶体颗粒结合，更重要的是 RL-2具有糖链结构，分子链伸展良好，增加了絮凝剂分子的有效长度。（6）RL-2菌产生的生物絮凝
6、剂能够有效的絮凝处理各种实际废水，尤其适用于食品和发酵行业的絮凝提纯过程。试验结果表明，对含有有机悬浮物的味精废水的絮凝效果好，既可以获得副产品：饲料添加剂，又可减少企业排污量。关键词：微生物絮凝剂，菌种鉴定，絮凝作用，物质结构，絮凝机理，实际应用II 英文摘要ABSTRACTAt present, the flocculating mechanisms of microbial flocculants have not beenstudied in detail, only put forward some hypotheses, for example, adhesion hypothes
7、is,esterification theory, extracellular fiber theory and bridging theory. So there are manyflocculant-producing strains, and complicate componant and construction of theflocculant, with the same object of study, different bioflocculants have differentflocculating mechanism. The key research of this
8、paper are the flocculatingperformance research, flocculation, and the flocculating mechanism as well as actualapplication, the following works have been mainly carried out in this research.（1）4 strains produced bioflocculants ( RL-2, NH-4, RT-4 and NL-3) werescreened from soil and activated sludge s
9、amples. Further optimization selection, aflocculant-producing strain Bacillus sp.RL-2 with high flocculating activity was isolatedfrom the activated sludge of the wastewater treatment plant in Chongqing. indicated thatRL-2 strain produces flocculant performance most excellent, it is identified as Ba
10、cillusbrives based on its colony character，morphological and physiological characteristics,using the partial 16S rRNA sequencing method. The bioflocculant was released into theculture broth in parallel with cell growth , and the flocculant main distributed in thebroth, only a little adhered in the i
11、nterface of cell. This suggests that the bioflocculantwas produced by biosynthesis and secrete extracellular biopolymeric flocculant.（2）For the raising flocculating activity, The optimum conditions for producingflocculant were set up. The favourable substrate and culture condition for flocculantform
12、ation are：glucose as carbon source, yeast extract and（NH4）2SO4 as nitrogensource, medium initial pH, culture temperature and the speed of shake are7.0, 30 and120150r/min, respectively. RL-2 produced the bioflocculant during the latelogarithmic growth in the batch culture, and it could be recovered f
13、rom supernatant byethanol and cetylpridinium chloride precipitation. The productivity of the flocculantreached as high as 5.1320g/L. In this paper, we found Bacillus brives can producebioflocculant, has better flocculating effects, and stable property.（3）In order to reduce cost of media, the strain
14、Bacillus brives RL-2 wascultivated in various organic wastewater from food production such as monosodiumglutamate wastewater(MSG) wastewater, the wastewater of bean products, cannedwastewater, brewing effluent, the wastewater of traditional Chinese medicine andIII 重庆大学博士学位论文milkings wastewater, and
15、selected favourable wastewater as medium for flocculantproduction. The result showed that the strain RL-2 could rapidly grow and reproducewell in under the condition of not extra carbon source, at the sametime it alsosynthesized intracellularly and excreted extracellularly flocculant. So, we have found akind of relatively low-priced nutriment for RL-2 bacterium, and the production cost offlocculant RL-2 have greatly r
