中国农业科学 ›› 2014, Vol. 47 ›› Issue (2): 324-333.doi: 10.3864/j.issn.0578-1752.2014.02.012

• 土壤肥料·节水灌溉·农业生态环境 • 上一篇    下一篇

产芽孢木质素降解菌MN-8的筛选及其对木质素的降解

 李红亚, 李术娜, 王树香, 王全, 朱宝成   

  1. 河北农业大学生命科学学院,河北保定 071000
  • 收稿日期:2013-03-01 出版日期:2014-01-15 发布日期:2013-03-01
  • 通讯作者: 朱宝成,E-mail:zhu2222@126.com
  • 作者简介:李红亚,E-mail:lihy77@sina.com。李术娜,E-mail:bdlishuna@126.com。李红亚与李术娜为同等贡献作者
  • 基金资助:

    国家星火计划项目(2010GA620010)、河北省农业科技成果转化项目(12820911D)、河北省科技支撑计划项目(12226605)

Screening, Identification of Lignin-Degradating Bacillus MN-8 and Its Characteristics in Degradation of Maize Straw Lignin

 LI  Hong-Ya, LI  Shu-Na, WANG  Shu-Xiang, WANG  Quan, ZHU  Bao-Cheng   

  1. College of Life Sciences, Agricultural University of Hebei, Baoding 071000, Hebei
  • Received:2013-03-01 Online:2014-01-15 Published:2013-03-01

摘要: 【目的】分离、筛选产芽孢的高效木质素降解细菌,并进一步研究其对木质素的降解作用,为木质素微生物降解规模化应用提供理论依据。【方法】采用苯胺蓝(Azure-B)变色圈法,结合木质素降解酶活力测定从牛粪中分离筛选出产芽孢的木质素降解菌。通过形态特征观察、生理生化试验、16S rDNA以及gyrB序列分析对其中活性最强的菌株进行种属鉴定。利用菌株进行玉米秸秆堆积发酵,监测发酵过程中木质素过氧化物酶(LiP)酶活力、锰过氧化酶(MnP)酶活力以及玉米秸秆中木质素含量的变化,考察菌株对玉米秸秆木质素的降解作用。利用气相色谱-质谱联用(GC/MS)方法对菌株发酵后玉米秸秆中的木质素降解产物进行分析,推测菌株对木质素的降解机制。【结果】分离筛选到一株活性较高的产芽孢的木质素降解细菌MN-8,经形态特征观察、生理生化试验以及16S rDNA序列分析,鉴定菌株MN-8属于芽孢杆菌属(Bacillus)。利用16S rDNA序列分析发现MN-8菌株与地衣芽孢杆菌(Bacillus licheniformis)和解淀粉芽孢杆菌(B. amyloliquefaciens)同源性均高于99%。而基于gyrB序列构建的系统发育树显示,该菌株与解淀粉芽孢杆菌同源性最高,为99%。因此确定MN-8菌株为解淀粉芽孢杆菌。在玉米秸秆堆积发酵16 d后木质素降解率可达24%;发酵的6—8 d及10—12 d 两个阶段内,分别出现MnP酶活力及LiP的产酶高峰期,相对应两个阶段内秸秆木质素的降解最为显著;GC/MS分析显示菌株MN-8可将玉米秸秆中木质素降解成4-羟基-3,5二甲氧基苯乙酮等芳香族类化合物及短链脂肪酸类等小分子物质。【结论】高效木质素降解菌解淀粉芽孢杆菌MN-8可以通过断裂木质素单体之间的连接键β-O-4,高效降解秸秆木质素成为小分子芳香族化合物等物质,且其对秸秆木质素的降解依赖于LiP及MnP的产生。

关键词: 木质素降解 , 芽孢杆菌 , 筛选 , 鉴定 , 降解特性

Abstract: 【Objective】The objective of this study is to obtain the Bacillus with the high lignin-degradation ability and investigate its characteristics in degradation of maize straw lignin, and to provide a basis for microbiological degradation of lignin. 【Method】Bacillus with high ability for degrading lignin was isolated and screened from cattle manure by the decolorization of aniline blue in plate cultivation and the lignin degradation enzyme activity was determined. Among of the obtained Bacillus, the strain with higher ability for degrading lignin was identified through morphology, physiological and biochemical tests, 16S rDNA and gyrB sequence. Then the straw heap fermentation experiment was performed with the inoculation of the bacterial strain obtained above. The changes of lignin peroxidase (LiP), manganese peroxidase (MnP) and the content of lignin in maize straw in the fermentaiton process were monitored to study the lignin-degradation activity of the strain. Its mechanism for degrading lignin was clarified by the GC/MS analysis results of lignin-degradation products. 【Result】 A strain of spore bacterium MN-8 with high ability of lignin-degradation was isolated and screened. Based on the morphology, physiological and biochemical experiments and 16S rDNA sequence analysis, strain MN-8 was identified as Bacillus. The result of 16S rDNA sequence analysis showed that MN-8 was more than 99% similar with Bacillus licheniformis and B. amyloliquefaciens. But the phylogenetic tree based on gyrB sequences showed that strain MN-8 shared 99% identity with B. amyloliquefaciens. Combined with 16S rDNA and gyrB sequence analysis, the strain MN-8 was confirmed as B. amyloliquefaciens. After 16 d incubation in the process of straw heap fermentation, lignin degradation rate was up to 24%. In the two stages of 6-8 d and 10-12 d in the stacking fermentation, the maximum activities of MnP and LiP were appeared. Lignin was degradated apparently in the same stage. And the analysis results of degradation products by GC/MS showed that strain MN-8 could degradate lignin efficiently into some low-molecular-weight matter, such as aromatic compounds 4-hydroxy-3,5-dimethoxy-acetophenone and short chain fatty acids, etc. 【Conclusion】B. amyloliquefaciens MN-8 with high lignin-degradation ability was obtained. It could degradate straw lignin into small molecular substances such as aromatic compounds by the rupture of β-O-4 bond connected between lignin monomer. And its degradation capacity depends on the LiP and MnP.

Key words: lignin-degradation , bacillus , screening , identification , degradation characteristics