蚕沙解磷菌株SEM-5的分离鉴定及其溶磷作用

Isolation and identification of phosphate-solubilizing strain SEM-5 from silkworm excrement and its phosphorus dissolution function

【目的】蚕沙中不仅富含营养物质,还含有大量微生物资源,筛选出蚕沙中的高效解磷菌并对其解磷机理进行探讨,为微生物肥料的开发利用提供资源。【方法】以高温发酵期的蚕沙为材料,通过无机磷筛选培养基定向筛选获得耐高温的解磷菌1株;通过生理生化及分子生物学方法进行分类鉴定;利用钼锑抗比色法和高效液相色谱法进行有效磷和有机酸含量的定量分析;通过SPSS 11.0对相关数据进行统计分析。【结果】从高温堆肥发酵过程中的蚕沙中筛选获得1株白色圆形菌落的菌株,菌体长杆状、革兰氏染色阳性;通过基因组测序获得其全长基因组大小为5324477 bp,GC含量为37.73%,37个Scaffolds,73个Contigs,注释基因5628个、tRNA 48个及rRNA 1个;基因组序列比对分析鉴定为巨大芽孢杆菌,命名为SEM-5。该菌株具有较强的耐受盐(NaCl浓度≤10%)碱(pH 5~10)及高温(60℃)能力;对不同无机磷的解磷效果不同,其中对钙盐来源的无机磷溶解效率显著高于磷酸铝和磷酸铁(P<0.05,下同)。对不同发酵时间SEM-5菌株发酵液的有效磷浓度、有机酸含量和发酵液pH等进行检测及相关分析,结果表明不同发酵时间的SEM-5菌株发酵液中有效磷浓度与不同有机酸的相关性不同;在0~14 d的发酵过程中,有效磷浓度与乙酸和酒石酸含量呈极显著正相关(P<0.01,下同),与乳酸、琥珀酸和丙酸含量呈显著正相关;与发酵液pH和草酸含量分别呈极显著和显著负相关,与苹果酸和柠檬酸含量则无显著相关性(P>0.05)。【结论】分离自蚕沙的巨大芽孢杆菌(SEM-5菌株)具有很好的环境适应性,耐盐碱和高温,且能高效地溶解释放钙盐中的磷元素,具有良好的微生物肥料开发应用潜力。

【Objective】Silkworm excrement is not only rich in nutrients,but also a variety of functional microorganisms.In this study,in order to provide resources for the development and utilization of microbial fertilizer,some high efficient phosphate dissolving bacteriawere screened and its phosphate dissolving mechanism was discussed preliminarily.【Method】A high temperature resistant strain with high efficiency of inorganic phosphorus hydrolysis was screened from the silkworm excrement through the inorganic phosphorus screening medium,and classified using the methods of physiology,biochemistry and molecular biology;the soluble phosphorus and the organic acid contents were quantitatively determined by the methods of the molybdenum antimony anti colorimetry and the high performance liquid chromatography(HPLC),respectively.The relevant data was statistical analyzed by the SPSS statistics software.【Result】A strain was screened from silkworm excrement during high temperature composting fermentation.The strain was white round colony,rod-shaped and gram positive.Full-length genome sequence was obtained by genome sequencing.The genome size was 5324477 bp,GC content was 37.73%,37 Scaffolds,73 Contigs,a total of 5628 genes were predicted along with 48 tRNAs and one rRNA.It was classified as Bacillus megaterium,named SEM-5,which had a marked function of dissolving inorganic phosphorus and strong tolerance of salt(NaCl concentration≤10%),alkali(pH 5-10)and high temperature(60).The result showed that the dissolving inorganic phosphorus ability of the strain on different sources was different.The dissolution efficiency of inorganic phosphorus from calcium salt was significantly higher than that of aluminum phosphate and iron phosphate(P<0.05,the same below).The data of available phosphorus concentration,organic acid and pH of SEM-5 strain fermentation broth in different fermentation times were detected and analyzed,and correlation analysis showed that the correlation between the concentration of soluble phosphorus and different organic acids was different in different fermentation times.The concentration of soluble phosphorus in fermentation broth had an extremely significant positive correlation with the content of acetic acid and tartaric acid(P<0.01,the same below),and had a significant positive correlation with the content of lactic acid,succinic acid,propionic acid in fermentation broth,and had a extremely significant negative,significant negative correlation with pH of the fermentation broth and the content of oxalic acid,respectively,but had no significant difference with malic acid and citric acid contents(P>0.05),from the beginning to day14 of the fermentation.【Conclusion】B.megaterium isolated from silkworm excrement(strain SEM-5)has good environmental adaptability,including tolerance to salt,alkali and high temperature,and exhibits a good dissolving and releasing ability for inorganic phosphorus from insoluble calcium source.It has a good application potential and development value of microbial fertilizer.