沼液对枯草芽孢杆菌产孢的影响及菌剂初型的制备

发布时间：2018-05-31 15:43

本文选题：枯草芽孢杆菌 + 培养基　；参考：《东北农业大学》2017年硕士论文

【摘要】：枯草芽孢杆菌(Bacillus subtilis)是当今研究最为热门的生防菌,含有较为多样的生理习性,容易被分离培养,因能够产生多样抗性的内生芽孢,所以被认为是作为生防菌较好的一种选择。沼液是沼气在发酵后剩余的液体,不仅富含大量可快速被吸收的营养成分,而且还含有其它供微生物生长的物质,如氨基酸、多种微量元素、维生素和其他活性物质。随着我国农村沼气工程的规模不断扩大,沼液资源化安全处理与利用备受重视。利用的枯草芽孢杆菌D1是一株具有拮抗性的细菌,前期试验发现在牛粪沼液中可以生长,进一步优化该菌株利用沼液的能力,为沼液再利用提供了新的代谢途径。该菌株对水稻的立枯病具有较高的拮抗作用,为了能将其更好的保存下来,通过选取优良的载体和稳定的保护剂,在高效利用沼液的同时将其保存,这也为后期在生物农药上利用提供技术参数。但想要将其应用于菌剂的制备,菌体本身芽孢含量是影响实际应用的关键因素,采用分批培养法优化了枯草芽孢杆菌高效利用沼液的产孢培养基;利用比浊法和稀释平板涂布法来检测枯草芽孢杆菌的菌体数量及芽孢产率;利用火焰原子吸收光谱法分析了沼液中金属离的含量,优化产孢培养基成分后进行菌剂载体和保护剂的初步研究,旨在高附值利用沼液,降低生产成本的同时,枯草芽孢杆菌的芽孢产率得到提升,并得到稳定的保存,为后续的实际应用做出理论依据。得到的主要研究结论如下:(1)活化菌株并测定生长曲线。枯草芽孢杆菌D1是革兰氏阳性菌,对立枯病有显著的拮抗作用,在19h时为生长高峰期。(2)利用单因素试验确定枯草芽孢杆菌D1的最佳产孢培养基的最佳碳源是麸皮,最佳氮源是大豆粉;优化后得到一组最优解为培养基的成分为含量为2.45%的麸皮,含量为0.9%的大豆粉和含量为49.01%的沼液,芽孢产率为87.82%。(3)对沼液的性质进行测定。供试沼液中含N 1580 mg/Kg、P 379 mg/Kg、K 998 mg/Kg,该沼液的p H为7.52,总固体含量即TS%为5;沼液中含有能促进枯草芽孢杆菌D1生长的Ca~(2+)、Mn~(2+)、Mg~(2+)这三种离子,检测了沼液中的这三种离子的含量,分别为:Ca~(2+)14.8 mg/L、Mg~(2+)0.151 mg/L、Mn~(2+)0.08 mg/L。(4)对优化产孢培养基后获得的高产菌株剂型的载体及保护剂进行筛选试验,确定枯草芽孢杆菌D1固体剂型的最适载体为硅藻土,同时筛选出最佳保护剂是液体石蜡油,最终有效活菌数量可达到89.9%。
[Abstract]:Bacillus subtilisis is one of the most popular biocontrol bacteria, which contains various physiological habits and is easy to be isolated and cultured. Because of its ability to produce endophytic spores with various resistance, Bacillus subtilisis is considered as a better choice for biocontrol. Biogas liquid is the residual liquid of biogas after fermentation. It not only contains a large amount of nutrients which can be quickly absorbed, but also contains other substances for microbial growth, such as amino acids, trace elements, vitamins and other active substances. With the increasing scale of rural biogas project in China, more and more attention has been paid to the safe treatment and utilization of biogas liquid. Bacillus subtilis D1 was an antagonistic bacterium, which was found to be able to grow in cow manure biogas liquid in previous experiments, and further optimized the ability of the strain to utilize biogas liquid, which provided a new metabolic pathway for the reuse of biogas liquid. The strain has a high antagonistic effect on rice bacterial blight. In order to preserve it better, it can be preserved by selecting excellent carrier and stable protective agent, using biogas liquid efficiently at the same time. This also provides technical parameters for the later use of biological pesticides. In order to be used in the preparation of bacterial agents, the spores content of Bacillus subtilis was the key factor affecting the practical application. The spore production medium of Bacillus subtilis was optimized by batch culture. The bacterial number and spores yield of Bacillus subtilis were determined by turbidimetry and dilution plate coating, and the content of metal dissociation in biogas liquid was analyzed by flame atomic absorption spectrometry. After optimizing the composition of spore producing medium, the preliminary study of fungicide carrier and protective agent was carried out. The purpose of this study was to use biogas liquid with high yield value and reduce the production cost, at the same time, the spores yield of Bacillus subtilis was improved and kept stably. To provide the theoretical basis for the subsequent practical application. The main results are as follows: 1) activated strain and measured growth curve. Bacillus subtilis D1 is a Gram-positive bacterium, which has a significant antagonistic effect on bacterial blight. It is the peak period of growth at 19 h.) the best carbon source for spores production medium of Bacillus subtilis D1 is wheat bran by single factor test. The optimum nitrogen source was soybean powder, and the optimum composition of the medium was wheat bran (2.45%), soybean powder (0.9%) and biogas liquid (49.01%), and the spore yield was 87.82%. The biogas liquid contained N 1580 mg / kg P 379 mg / K g / K 998 mg / kg, pH = 7.52, and total solid content was 5%. The biogas liquid contained three kinds of ions, Ca~(2 Mn-MN 2 mg 2, which could promote the growth of Bacillus subtilis D1), and the contents of the three ions in the biogas liquid were detected. The optimal carrier and protectant of high yield strain obtained by optimizing the sporulation medium were determined to be diatomite as the best carrier of Bacillus subtilis D1 solid dosage form, which was 0.151 mg 路L ~ (-1) mg / L ~ (-1) mg 路L ~ (-1) 路L ~ (-1)% 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1). At the same time, the best protective agent is liquid paraffin oil, and the effective live bacteria quantity can reach 89. 9%.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S476.1;TQ450.6

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