Challenges to explore uncultivable but functional bacteria in the rumen

本研究旨在通过分子生物学技术探讨非培养瘤胃细菌的生态学和功能。通过对纤维相关细菌（紧密附着在稻草茎上的细菌）的16 SrDNA序列比较分析，得到以下结果：1.发现了未鉴定的瘤胃细菌，主要分为3个类群（U1 ~ U3）。U1属于PFB门，由与拟杆菌属密切相关的细菌组成，而U2是与木纤维消化细菌阿氏梭菌密切相关的细菌。U3与瘤胃半纤维素降解菌反刍真杆菌（Eubacterium ruminantium）亲缘关系最近。所开发的检测方法灵敏度高，重现性可靠，并允许快速监测每个细菌群。结果表明，在瘤胃培养的秸秆中，共分离出14个细菌种或类群。3.在紧密粘附的细菌中，产琥珀酸纤维杆菌（Fibrobacter succinogenes）的生物量最大，其次是U2。此外，U2显示的时间过程中的茎就像F.琥珀酸。这些结果表明U2对瘤胃纤维的消化有显著的促进作用。4.扫描电镜下在茎上观察到许多具有突起结构的梭状芽孢杆菌，这些细菌可能是U2细菌。因此，在培养前采用热处理接种的方法，可以使U2在秸秆上富集并分离。

The present study is to explore ecology and function of uncultivated rumen bacteria through molecular biological techniques. By comparative 16SrDNA sequencing analysis of fiber-associated bacteria (bacteria tightly adherent to rice straw stems), the following results were obtained.1.So far uncharacterized rumen bacteria were found and mainly grouped into 3 clusters (U1 through U3). U1 was in PFB phylum comprising bacteria closely related to Bacteroides, while U2 was the group close to the wood fiber digesting bacterium Clostridium aldrichii. U3 had the hemicellulolytic rumen bacterium Eubacterium ruminantium as a closest relative.2.PCR primers specific to U2 and U3 were designed for developing real-time PCR assays to quantitate these groups. The developed assays were highly sensitive, reliable in reproducibility, and allowed rapid monitoring of each bacterial group. Such reliable assays were established for total 14 bacterial species or groups that were associated with ruminally incubated straw stems.3.Of tightly adherent bacteria, Fibrobacter succinogenes, a potent fibrolytic species, was major in its biomass followed by U2. Also, U2 showed time-course on stems just as F.succinogenes. These results strongly suggest significant contribution of U2 to rumen fiber digestion.4.Since many Clostridium-like bacteria with protuberance structure were observed on stems under scanning electronic microscopy, these might be U2 bacteria. Therefore, U2 could be enriched on straw sterns and isolated by using heat treatment to inoculum before cultivation.