恶臭假单胞菌的培养优化及冷风干燥工艺的研究

发布时间：2018-02-11 19:45

  本文关键词： 恶臭假单胞菌 培养基 冷风干燥 石油降解菌 协同降解　出处：《华中农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：石油污染的生物修复是当前的研究热点,石油降解菌大部分属于革兰氏阴性菌。然而,革兰氏阴性菌普遍存在不耐干燥,不耐保藏的问题,这限制了革兰氏阴性菌菌剂的产品化及其应用。假单胞菌属于革兰氏阴性菌,在生物修复、生物转化、生物防治等领域有广阔的应用前景。本文以一株能降解石油的恶臭假单胞菌(Pseudomonas putida)J7为研究对象,分别从培养基优化、干燥方法的选择、冷风干燥制备活菌粉工艺的优化、协同菌株的选择及其对模拟含油湖水修复等方面展开研究。具体研究结果如下:(1)研究完成了恶臭假单胞菌J7培养基成分的优化。分别对碳源、氮源、无机盐采用单因素及正交试验,优化得到恶臭假单胞菌的培养基配方为:葡萄糖6g/L、酵母粉12 g/L、Mg SO4×7H20 3.5 g/L、KH2PO4 2.5 g/L、Ca Cl2 1.5 g/L,培养后OD600为10.71,活菌数达到2.15×1010 CFU/m L,达到国内最高培养水平。(2)研究了冷风干燥制备恶臭假单胞菌菌粉的工艺。分别研究了不同载体与浓缩菌液混合比下的冷风干燥曲线,以及成品菌粉含水率、载体、载体预处理、保护剂对冷风干燥制备恶臭假单胞菌菌粉的干燥存活率及保藏稳定性的影响。优化后的冷风干燥制备工艺为:复合载体为硅藻土和碱处理玉米芯粉,质量比为1:2,载体与浓缩菌液混合比为1:2,保护因子(W/W)为甘露醇7%、谷氨酸钠5%、甘油1%,成品含水率控制在7%以下。干燥菌粉活菌数达1.03×1011CFU/g,干燥存活率74.63%,显著高于喷雾干燥(23.95%),说明冷风干燥是一种适合恶臭假单胞菌干燥的技术。优化工艺制备的冷风干燥菌粉,保藏稳定性显著提高。室温保藏150d活菌数为2.13×109 CFU/g,而未优化的对照组菌粉,室温保藏90 d活菌数仅为3.26×108 CFU/g;4℃保藏条件下,对照组菌粉活菌数为1.84×1010 CFU/g,优化组活菌数为3.36×1010CFU/g,提高将近1倍。(3)初步研究石油降解菌剂的应用。筛选出与恶臭假单胞菌J7协同降解的菌株为短小芽孢杆菌Y11(Bacillus pumilus Y11)、地衣芽孢杆菌B36(Baclicus lincheniformis B36)。在含油湖水摇瓶体系中,对比单一菌株,混合菌株石油降解率提高13.0%。添加有机营养剂比无机营养剂更利于石油微生物降解,降解率提高15.2%,且降解体系C/N为20:1时较适。静态模拟的含油湖水体系中,投加混合菌株及有机营养剂能使石油降解效率从20.1%提高到56.6%。且投加制备的石油降解菌菌粉的处理组,依然保持相当的石油去除率(54.7%)。
[Abstract]:Bioremediation of petroleum pollution is a hot research topic at present. Most of the petroleum degrading bacteria belong to Gram-negative bacteria. However, Gram-negative bacteria are generally intolerant to drying and preservation. This limits the production of Gram-negative bacteria and their application. Pseudomonas belong to Gram-negative bacteria, bioremediation, biotransformation, In this paper, a strain of Pseudomonas putida)J7, which can degrade petroleum, was used as the research object. The optimization of culture medium, the selection of drying methods and the optimization of the technology of preparing live bacteria powder by cold air drying were studied in this paper. The selection of synergistic strains and their remediation of simulated oil-bearing lake water were studied. The results are as follows: 1) the optimization of the composition of the culture medium of Pseudomonas oxysporum J7 was carried out. The carbon sources and nitrogen sources were obtained, respectively. Inorganic salts were tested by single factor and orthogonal test. The optimized medium formula of Pseudomonas odour was: glucose 6g / L, yeast powder 12g / L mg SO4 脳 7H203.5 g / L KH2PO4 2.5 g / L KH2PO4 2.5 g / L Ca Cl2 1.5 g / L, OD600 10.71, viable bacteria 2.15 脳 1010 CFU/m / L, which reached the highest culture level in China. The technology of Pseudomonas odoratum powder. The drying curves of cold air under the mixing ratio of different carrier and concentrated bacteria solution were studied. And the moisture content of finished product powder, carrier, carrier pretreatment, The effect of protective agent on the survival rate and preservation stability of the preparation of Pseudomonas odour powder by cold air drying. The optimized preparation technology of cold air drying is as follows: the composite carrier is diatomite and the corn cob powder is treated with alkali. The mass ratio is 1: 2, the mixture ratio of carrier to concentrated bacteria is 1: 2, the protection factor W / W is mannitol 7, sodium glutamate 5, glycerol 1, the moisture content of finished product is controlled below 7%. The number of viable bacteria in dried powder is 1.03 脳 10 11 CFU / g, and the drying survival rate is 74.63, which is significantly higher than that in spray drying. It shows that cold air drying is a suitable drying technology for Pseudomonas odour. The number of live bacteria stored at room temperature for 150 days was 2.13 脳 109 CFU / g, while that of control group was only 3.26 脳 10 ~ 8 CFU / g at 4 鈩，

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