本文選題：巨大芽孢桿菌　切入點(diǎn)：解磷能力　出處：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：溶磷細(xì)菌(簡稱磷細(xì)菌)是一類在土壤中具有很強(qiáng)溶磷能力的細(xì)菌的總稱。磷細(xì)菌可使土壤中不能被植物利用的磷化物轉(zhuǎn)變成可被利用的可溶性磷化物,故又稱溶磷細(xì)菌。溶磷細(xì)菌主要通過生產(chǎn)各種酶或酸來發(fā)揮自己的生物特性,溶磷細(xì)菌在磷資源循環(huán)的過程中起著不可或缺的作用,由于磷細(xì)菌可以將難溶性無機(jī)磷轉(zhuǎn)化為可溶性磷,提高作物對磷的利用率,因此在微生物肥料生產(chǎn)過程中應(yīng)用廣泛。實(shí)踐證明,磷細(xì)菌對小麥、甘薯、大豆、水稻等多種農(nóng)作物,以及蘋果、桃等果樹具有一定增產(chǎn)效果。常用在農(nóng)業(yè)生產(chǎn)中的解磷細(xì)菌有解磷巨大芽孢桿菌,俗稱為“大芽孢”磷細(xì)菌,此外,在農(nóng)業(yè)生產(chǎn)中常用到的還有其他芽孢桿菌和無色桿菌、假單胞菌等。本實(shí)驗(yàn)從作物根際土壤中分離高效解磷細(xì)菌。對于從土壤中分離到的菌株,通過平板法進(jìn)行初篩,根據(jù)解磷透明圈直徑和菌落直徑比值大小初步確定菌株解無機(jī)磷和有機(jī)磷的能力,然后通過液體搖瓶培養(yǎng),利用鉬銻抗比色法測定發(fā)酵上清液中有效磷含量,從而進(jìn)一步確定該分離菌株的解磷能力。通過實(shí)驗(yàn)篩選出1株具有高效解有機(jī)磷能力的細(xì)菌,在蛋黃培養(yǎng)基上可產(chǎn)生明顯的解磷圈,分解有機(jī)磷能力最強(qiáng),D/d比值為4,在卵磷脂液體培養(yǎng)基中培養(yǎng)5d后,發(fā)酵上清液中的有效磷達(dá)到最高值54.21mg/L,是對照組的45倍,可看出解磷效果顯著。通過形態(tài)特征、生理生化特性和16S rDNA序列分析等一系列試驗(yàn)對菌株進(jìn)行鑒定。通過篩選從作物根際土上分離得到的巨大芽孢桿菌BM2菌株,存于實(shí)驗(yàn)室保藏。采用單因素實(shí)驗(yàn)及響應(yīng)面份分析試驗(yàn)優(yōu)化了巨大芽孢桿菌菌株BM2的最佳發(fā)酵培養(yǎng)基以及最佳發(fā)酵條件。實(shí)驗(yàn)表明,巨大芽孢桿菌BM2最適生長條件優(yōu)化該菌株的最適生長溫度為32℃,最佳生長pH為7.2。該菌株發(fā)酵培養(yǎng)基的最佳碳源為可溶性淀粉,最佳氮源為豆粕粉,最佳無機(jī)鹽為磷酸二氫鉀。采用中心組合試驗(yàn)設(shè)計(jì),進(jìn)行響應(yīng)面分析結(jié)合實(shí)際值,確定最優(yōu)的液體發(fā)酵條件為可溶性淀粉48.85g/L,豆粕19.67g/L,pH為7.2,該設(shè)計(jì)的理論芽孢數(shù)為43.16×108 cfu/m L,實(shí)際測定的芽孢數(shù)為41.29×108 cfu/mL,與理論值差距很小。研究確定,菌株BM2的最佳發(fā)酵溫度為34℃,最佳接種量為3%,最佳發(fā)酵時(shí)間為32h,最佳裝液量為60m L/250m L。為了提高巨大芽孢桿菌BM2的生物穩(wěn)定性,通過對其進(jìn)行固定化優(yōu)化,確定巨大芽孢桿菌BM2固定化的最佳載體和交聯(lián)劑,最佳載體為濃度為4%的海藻酸鈉,最佳交聯(lián)劑為4%的氯化鈣,包菌量在10%,交聯(lián)時(shí)間在24h時(shí),菌株能保持其最佳活性,達(dá)到最佳效果。擬南芥生根實(shí)驗(yàn)中,培養(yǎng)基中添加了巨大芽孢桿菌BM2發(fā)酵液的擬南芥幼苗的生根情況較于對照組擬南芥幼苗的根長均有明顯的增加,且觀察到,添加了發(fā)酵液培養(yǎng)基中的擬南芥?zhèn)雀^于對照組發(fā)達(dá)。通過實(shí)驗(yàn)可知該菌株發(fā)酵液濃度為23cfu/m L,將BM2發(fā)酵液500倍時(shí),對擬南芥生根促進(jìn)效果最佳,根長相對于不加BM2菌劑的對照組增加了67.4%。稀釋倍數(shù)為50時(shí),對擬南芥生長促進(jìn)效果最不明顯。所有稀釋倍數(shù)處理下的擬南芥根長跟對照相比差異明顯。不同稀釋倍數(shù)處理的巨大芽孢桿菌BM2發(fā)酵液對黃瓜幼苗的生長都起到了促進(jìn)作用。隨著稀釋倍數(shù)的增加,對黃瓜幼苗生長的促進(jìn)作用越明顯,在500倍稀釋的發(fā)酵液處理下,與對照組相比差異顯著,黃瓜幼苗的株高提高了25%,黃瓜幼苗的鮮重比對照組提高37%,在1000倍稀釋的發(fā)酵處理下,與對照組相比差異顯著,黃瓜幼苗的株高提高了51%,黃瓜幼苗的根長比對照組提高了21%,黃瓜幼苗的鮮重比對照組提高39%。以上對比差異顯著(P0.05)。添加發(fā)酵液的處理能夠促進(jìn)黃瓜幼苗根長的增長。
[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，

本文編號：1643646