Sekiguchi Y, Kamagata Y, Nakamura K, Ohashi A, Harada H
Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan.
Appl Environ Microbiol. 1999 Mar;65(3):1280-8. doi: 10.1128/AEM.65.3.1280-1288.1999.
16S rRNA-targeted in situ hybridization combined with confocal laser scanning microscopy was used to elucidate the spatial distribution of microbes within two types of methanogenic granular sludge, mesophilic (35 degrees C) and thermophilic (55 degrees C), in upflow anaerobic sludge blanket reactors fed with sucrose-, acetate-, and propionate-based artificial wastewater. The spatial organization of the microbes was visualized in thin sections of the granules by using fluorescent oligonucleotide probes specific to several phylogenetic groups of microbes. In situ hybridization with archaeal- and bacterial-domain probes within granule sections clearly showed that both mesophilic and thermophilic granules had layered structures and that the outer layer harbored mainly bacterial cells while the inner layer consisted mainly of archaeal cells. Methanosaeta-, Methanobacterium-, Methanospirillum-, and Methanosarcina-like cells were detected with oligonucleotide probes specific for the different groups of methanogens, and they were found to be localized inside the granules, in both types of which dominant methanogens were members of the genus Methanosaeta. For specific detection of bacteria which were previously detected by whole-microbial-community 16S ribosomal DNA (rDNA)-cloning analysis (Y. Sekiguchi, Y. Kamagata, K. Syutsubo, A. Ohashi, H. Harada, and K. Nakamura, Microbiology 144:2655-2665, 1998) we designed probes specific for clonal 16S rDNAs related to unidentified green nonsulfur bacteria and clones related to Syntrophobacter species. The probe designed for the cluster closely related to Syntrophobacter species hybridized with coccoid cells in the inner layer of the mesophilic granule sections. The probe for the unidentified bacteria which were clustered with the green nonsulfur bacteria detected filamentous cells in the outermost layer of the thermophilic sludge granule sections. These results revealed the spatial organizations of methanogens and uncultivated bacteria and their in situ morphologies and metabolic functions in both mesophilic and thermophilic granular sludges.
采用16S rRNA靶向原位杂交结合共聚焦激光扫描显微镜，以阐明在以蔗糖、乙酸盐和丙酸盐为基础的人工废水进料的上流式厌氧污泥床反应器中，中温（35℃）和高温（55℃）两种产甲烷颗粒污泥内微生物的空间分布。通过使用针对几个微生物系统发育组的荧光寡核苷酸探针，在颗粒的薄片中可视化微生物的空间组织。在颗粒切片中与古菌域和细菌域探针进行原位杂交，清楚地表明中温颗粒和高温颗粒均具有分层结构，外层主要含有细菌细胞，而内层主要由古菌细胞组成。用针对不同产甲烷菌群的寡核苷酸探针检测到了类甲烷八叠球菌、类甲烷杆菌、类甲烷螺菌和类甲烷囊菌细胞，发现它们位于颗粒内部，在这两种颗粒中，主要的产甲烷菌都是甲烷八叠球菌属的成员。为了特异性检测先前通过全微生物群落16S核糖体DNA（rDNA）克隆分析检测到的细菌（Y. Sekiguchi、Y. Kamagata、K. Syutsubo、A. Ohashi、H. Harada和K. Nakamura，《微生物学》144:2655 - 2665，1998），我们设计了针对与未鉴定的绿色非硫细菌相关的克隆16S rDNA以及与互营杆菌属物种相关的克隆的特异性探针。针对与互营杆菌属物种密切相关的簇设计的探针，与中温颗粒切片内层的球状细胞杂交。针对与绿色非硫细菌聚类的未鉴定细菌设计的探针，在高温污泥颗粒切片的最外层检测到丝状细胞。这些结果揭示了中温和高温颗粒污泥中产甲烷菌和未培养细菌的空间组织及其原位形态和代谢功能。
