一株产絮凝剂菌株的特性及其培养条件优化

发布时间：2018-06-26 19:03

  本文选题：甲基营养型芽孢杆菌 + 微生物絮凝剂　； 参考：《广西大学》2015年硕士论文

【摘要】：采用常规的平板划线分离方法从土壤样品中分离得到130多种具有絮凝活性的菌株,通过菌株发酵液对高岭土悬浊液的絮凝率考察产絮凝剂菌株的絮凝活性,得到了一株具有较高絮凝活性的菌株C412。通过平板菌落形态,生理生化试验和16S rDNA及GyrB同源性序列分析结果,确定菌株C412为甲基营养型芽孢杆菌。从絮凝活性分布发现菌株C412的胞外分泌物起到了主要的絮凝作用,然后将发酵液离心后,从上清液中提取到微生物絮凝剂。利用紫外和红外等光谱分析,发现微生物絮凝剂MBF412的化学组分主要由多糖构成。通过化学方法检测菌株C412分泌的胞外微生物絮凝剂MBF412,其含有57.3%的总糖和1.7%的总蛋白质含量。利用凝胶渗透色谱GPC发现微生物絮凝剂MBF412的分子量为3.47×105 g/mol,属于天然高分子物质。采用响应面方法优化了菌株生长的培养基组分(g/L)：可溶性淀粉(19.73),牛肉膏(1.35),硫酸铵(3.05),氯化钠(1),磷酸二氢钾(2),磷酸氢二钾(5)和硫酸镁(0.05),pH为7.63。在此培养基中进行验证实验,产絮凝剂菌株C412对高岭土悬浊液的最佳絮凝活性可达98.27%,微生物絮凝剂的产率约为4.25 g/L。并且,菌株C412在高浓度下的间歇式发酵表明：在摇瓶中的常规接种发酵时,菌株出现最佳絮凝活性的时间为13～17小时；在1.5 L发酵罐中发酵时,菌株出现最佳絮凝活性的时间缩短到2-6小时,这为低成本规模化生产微生物絮凝剂提供了有效的思路。同时微生物絮凝剂的相关特性研究表明：该微生物絮凝剂具有较佳的耐热性和耐酸碱性,从pH(4～13)和温度(4～60℃)均具有较佳的絮凝率,具有较广的应用范围,这归功于微生物絮凝剂的主要组分多糖和极性官能团。通过荧光定量PCR方法建立了甲基营养型芽孢杆菌中的絮凝相关基因katE,pepT,sacA及scrR和菌株絮凝活性之间的关系。此外,利用Zeta电位,初步探索了微生物絮凝过程中涉及到的两步沉淀机理(电中性机理和架桥机理)。本论文也证明了微生物絮凝剂MBF412的潜在工业应用价值。
[Abstract]:More than 130 strains with flocculating activity were isolated from soil samples by conventional plate line separation method. The flocculating activity of flocculant producing strains was investigated by the flocculation rate of the fermentation broth on kaolin suspensions. A strain C412 with high flocculation activity was obtained. Based on the results of colony morphology, physiological and biochemical tests, 16s rDNA and GyrB homology analysis, the strain C412 was identified as methyl-nutritious Bacillus. The extracellular secretion of strain C412 was found to play a major role in flocculation from the distribution of flocculating activity. Then the fermentation broth was centrifuged and the microbial flocculant was extracted from the supernatant. The chemical composition of microbial flocculant MBF412 was mainly composed of polysaccharides by UV and IR spectra. The extracellular microbial flocculant MBF412 secreted by strain C412 contained 57.3% of total sugar and 1.7% of total protein. The molecular weight of microbial flocculant MBF412 was found to be 3.47 脳 10 ~ 5 g / mol by gel permeation chromatography (GPC). The medium components (g / L) of strain growth were optimized by response surface method: soluble starch (19.73), beef paste (1.35), ammonium sulfate (3.05), sodium chloride (1), potassium dihydrogen phosphate (2), potassium hydrogen phosphate (5) and magnesium sulfate (0.05) at pH 7.63. The optimum flocculation activity of flocculant producing strain C412 on kaolin suspension was 98.27 and the yield of microbial flocculant was about 4.25 g / L. In addition, the batch fermentation of strain C412 at high concentration showed that the optimum flocculation activity of the strain was 1317 hours when the strain was inoculated in shaking flask, and when fermented in 1.5 L fermentor. The optimum flocculation time of the strain was shortened to 2-6 hours, which provided an effective way for the production of microbial flocculant on a low cost scale. At the same time, the relative characteristics of the microbial flocculant showed that the microbial flocculant had better heat resistance and acid and alkali resistance, and had a better flocculation rate from pH (4 ~ 13) and temperature (40 ~ 60 鈩，

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