水稻秸秆低温复合菌系多样性及发酵动态

Microbial diversity of a community for ensiling rice straw at low temperature and fermentation dynamics

摘要:

摘要: 【目的】为解决中国寒冷地区水稻秸秆大面积废弃问题，加快低温地区水稻秸秆饲料转化，本文筛选了可以低温下加速秸秆发酵过程的微生物复合菌系，研究其微生物组成并跟踪其发酵动态。【方法】通过5℃下连续定向富集筛选，获得低温复合菌系。采用克隆文库方法分析复合菌系的组成。将复合菌系和商业接种剂(由Lactobacillus plantarum，Enterococcus faecium，L．salivarilus，Pediococcus acidilactici 组成) 分别接入稻秸进行10℃发酵。气质联机(GC－MS) 测定发酵产物的同时，通过变性梯度凝胶电泳检测微生物在发酵体系的定殖情况。采用定量PCR 方法追踪复合菌系组成菌在发酵过程中的动态。【结果】16S rDNA 克隆文库分析结果表明复合系主要由两种微生物组成，一种属乳酸杆菌(Lactobacillus)，一种属乳酸球菌(Leuconostoc)。10℃稻秸发酵结果表明，在发酵第6 天接种复合菌系处理的pH 已经下降到4.3，乳酸菌菌落 形成单位为2.9×109CFU/g 鲜样，而接种商业接种剂的处理pH 为5.3，乳酸菌菌落形成单位为3.6×108CFU/g鲜样; 在发酵30 d时，接种复合菌系处理的乳酸含量为8.1g/kg鲜样，接种商业接种剂处理的乳酸含量为2.0g/kg 鲜样。变性梯度凝胶电泳结果表明，在接种复合菌系的稻秸中，从发酵的第6天开始，检测到的微生物主要为L.sakei和Leuconostoc inhae，在整个发酵过程中，两菌一直存在; 在商业接种剂处理中，发酵第6 天检测到的微生物除其四种组成菌外，还包括Uncultured bacterium; 而在发酵第16 天和第30天，只检测到组成菌中的L．plantarum 和E．faecium。定量PCR 结果显示，接种复合菌系处理中，L．sakeiDNA 在发酵第6天达到41.0%，在发酵第16天已达到65%，Le inhae在发酵的第6天达到整个发酵过程中的最大值(5.5%)。【结论】接种复合菌系，可以有效促进水稻秸秆的低温发酵进程。复合菌系组成菌可以定殖在发酵体系中，并占据优势。复合菌系的关键菌为L.sakei。

Abstract:

Abstract: [Objective] To accelerate the conversion of rice straw into feeds in the low-temperature region，a microbial community was constructed by continuous enrichment cultivation． Microbial diversity and dynamics during the fermentation at 10℃ was analyzed．［Methods］The community was selected at 5℃ under static condition．To analyze the inoculating effects，the community and commercial inoculant (CI:composed of Lactobacillus plantarum，Enterococcus faecium，L.salivarilus，Pediococcus acidilactici) were respectively inoculated into the rice straw for 30 d fermentation at 10℃．Fermented products were detected by gas chromatography–mass spectrometry (GC-MS)．Composition microorganisms of the community were analyzed using cloning library．Microbial dynamics during the fermentation was detected by denatured gradient gel eletrophoresis (DGGE)．Quantitative PCR was used for tracking the composition microorganisms of the community during the fermentation．［Results］The results from 16S rDNA cloning library showed that the community was mainly composed of Lactobacillus spp. and Leuconostoc spp．At 6d fermentation，the pH and the lactic acid bacterial colony forming units (LAB CFUs) in the fermented rice straw with the community amounted to 4.3 and 2.9×109 CFU/g fresh matter (FM)，respectively．The pH and LAB CFUs with the CI were respectively 5.3 and 2.9×109 CFU/g FM．At 30 d fermentation，the lactic acid concentrations with the community and the CI were respectively 8. 1g / kg FM and 2.0g/kg FM． From DGGE patterns， both L． sakei and Leuconostoc inhae of the community were detected at 6d fermentation and existed during the fermentation． For the treatment with the CI，the uncultured bacterium was detected at 6d fermentation besides the composition microorganisms of the CI． At 16d and 30d fermentation，only L． plantarum and E． faecium were detected． Quantitative PCR showed DNA mass of L． sakei amounted to 41. 0% at 6d fermentation in the treatment with the community． At 16d，DNA mass of L． sakei was 65% ． The highest value (5.5% ) of DNA mass of Le inhae appeared at 6d of fermentation ［Conclusion］ The community could effectively colonize into the rice straw fermentation system and accelerate the fermentation process at low temperature． The dominating microorganism of the community was L． sakei at 10℃ ．