土壤中高效解磷细菌的筛选及其发酵工艺的优化

发布时间：2018-03-21 11:27

  本文选题：巨大芽孢杆菌　切入点：解磷能力　出处：《山东农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：溶磷细菌(简称磷细菌)是一类在土壤中具有很强溶磷能力的细菌的总称。磷细菌可使土壤中不能被植物利用的磷化物转变成可被利用的可溶性磷化物,故又称溶磷细菌。溶磷细菌主要通过生产各种酶或酸来发挥自己的生物特性,溶磷细菌在磷资源循环的过程中起着不可或缺的作用,由于磷细菌可以将难溶性无机磷转化为可溶性磷,提高作物对磷的利用率,因此在微生物肥料生产过程中应用广泛。实践证明,磷细菌对小麦、甘薯、大豆、水稻等多种农作物,以及苹果、桃等果树具有一定增产效果。常用在农业生产中的解磷细菌有解磷巨大芽孢杆菌,俗称为“大芽孢”磷细菌,此外,在农业生产中常用到的还有其他芽孢杆菌和无色杆菌、假单胞菌等。本实验从作物根际土壤中分离高效解磷细菌。对于从土壤中分离到的菌株,通过平板法进行初筛,根据解磷透明圈直径和菌落直径比值大小初步确定菌株解无机磷和有机磷的能力,然后通过液体摇瓶培养,利用钼锑抗比色法测定发酵上清液中有效磷含量,从而进一步确定该分离菌株的解磷能力。通过实验筛选出1株具有高效解有机磷能力的细菌,在蛋黄培养基上可产生明显的解磷圈,分解有机磷能力最强,D/d比值为4,在卵磷脂液体培养基中培养5d后,发酵上清液中的有效磷达到最高值54.21mg/L,是对照组的45倍,可看出解磷效果显著。通过形态特征、生理生化特性和16S rDNA序列分析等一系列试验对菌株进行鉴定。通过筛选从作物根际土上分离得到的巨大芽孢杆菌BM2菌株,存于实验室保藏。采用单因素实验及响应面份分析试验优化了巨大芽孢杆菌菌株BM2的最佳发酵培养基以及最佳发酵条件。实验表明,巨大芽孢杆菌BM2最适生长条件优化该菌株的最适生长温度为32℃,最佳生长pH为7.2。该菌株发酵培养基的最佳碳源为可溶性淀粉,最佳氮源为豆粕粉,最佳无机盐为磷酸二氢钾。采用中心组合试验设计,进行响应面分析结合实际值,确定最优的液体发酵条件为可溶性淀粉48.85g/L,豆粕19.67g/L,pH为7.2,该设计的理论芽孢数为43.16×108 cfu/m L,实际测定的芽孢数为41.29×108 cfu/mL,与理论值差距很小。研究确定,菌株BM2的最佳发酵温度为34℃,最佳接种量为3%,最佳发酵时间为32h,最佳装液量为60m L/250m L。为了提高巨大芽孢杆菌BM2的生物稳定性,通过对其进行固定化优化,确定巨大芽孢杆菌BM2固定化的最佳载体和交联剂,最佳载体为浓度为4%的海藻酸钠,最佳交联剂为4%的氯化钙,包菌量在10%,交联时间在24h时,菌株能保持其最佳活性,达到最佳效果。拟南芥生根实验中,培养基中添加了巨大芽孢杆菌BM2发酵液的拟南芥幼苗的生根情况较于对照组拟南芥幼苗的根长均有明显的增加,且观察到,添加了发酵液培养基中的拟南芥侧根较于对照组发达。通过实验可知该菌株发酵液浓度为23cfu/m L,将BM2发酵液500倍时,对拟南芥生根促进效果最佳,根长相对于不加BM2菌剂的对照组增加了67.4%。稀释倍数为50时,对拟南芥生长促进效果最不明显。所有稀释倍数处理下的拟南芥根长跟对照相比差异明显。不同稀释倍数处理的巨大芽孢杆菌BM2发酵液对黄瓜幼苗的生长都起到了促进作用。随着稀释倍数的增加,对黄瓜幼苗生长的促进作用越明显,在500倍稀释的发酵液处理下,与对照组相比差异显著,黄瓜幼苗的株高提高了25%,黄瓜幼苗的鲜重比对照组提高37%,在1000倍稀释的发酵处理下,与对照组相比差异显著,黄瓜幼苗的株高提高了51%,黄瓜幼苗的根长比对照组提高了21%,黄瓜幼苗的鲜重比对照组提高39%。以上对比差异显著(P0.05)。添加发酵液的处理能够促进黄瓜幼苗根长的增长。
[Abstract]:Phosphate solubilizing bacteria (the phosphorus bacteria) is a kind of general strong ability of phosphorus solubilizing bacteria in soil. Phosphorus bacteria can make changes in the soil can not be phosphide by plants into soluble phosphide can be used, it is also called the solubilizing bacteria. Phosphorus bacteria mainly through the production of enzymes or acids. To play their own biology, phosphorus bacteria play an indispensable role in the process of phosphorus resource circulation, because the phosphorus bacteria can be insoluble inorganic phosphate into soluble phosphorus, improve the utilization rate of crop phosphorus, so the application of fertilizer production in the process of microbial widely. Practice has proved that the phosphate solubilizing bacteria on wheat. Sweet potato, soybean, rice and other crops, and apple, peach and other fruit trees has a certain yield increasing effect. The commonly used phosphate solubilizing bacteria in agricultural production in a solubilizing Bacillus megaterium, commonly known as "spore" phosphorus bacteria, in addition, in agricultural production. In common to the other Bacillus and Pseudomonas, Achromobacter, etc. the experimental separation of efficient phosphate solubilizing bacteria from the rhizosphere soil. The strains isolated from the soil, were screened by plate method, according to the P transparent circle diameter and colony diameter ratio of small initially determined the ability of strains the solution of inorganic phosphorus and organic phosphorus, and then through the liquid shake flask, the ratio of molybdenum antimony determination of available phosphorus in the fermentation supernatant in color method, so as to further determine the phosphate solubilizing ability of isolated strains. The experiment screened 1 strains with high degradation ability of organic phosphorus bacteria in the egg yolk medium can produce phosphorus loop solutions obviously, decomposition of organic phosphorus was the strongest, the ratio of D/d is 4, the lecithin liquid culture medium 5D, effective phosphorus in fermentation supernatant reached a maximum value of 54.21mg/L is 45 times higher than that of control group, the phosphate solubilizing effect significantly . according to the morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis of a series of tests of the strains were identified by screening. The isolated Bacillus megaterium BM2 from rhizosphere soil, stored in the laboratory preservation. By single factor experiment and response surface analysis test the optimum fermentation optimization of Bacillus strain BM2 the culture medium and the optimal fermentation conditions. Experiments showed that Bacillus megaterium BM2 the optimum growth conditions to optimize the strain optimum temperature is 32 DEG C, the optimal growth of pH was the best carbon source of the strain 7.2. fermentation medium was soluble starch, the best nitrogen source was soybean powder, the best inorganic salt as using potassium dihydrogen phosphate. The central composite experimental design and response surface analysis was carried out according to the actual value, to determine the optimal fermentation conditions for 48.85g/L of soluble starch, soybean meal 19.67g/L, pH is 7.2, the number of the design theory of Bacillus 涓，

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