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Applied and Environmental Microbiology, August 2000, p. 3608-3615, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Cultivation and In Situ Detection of a Thermophilic Bacterium Capable of Oxidizing Propionate in Syntrophic Association with Hydrogenotrophic Methanogens in a Thermophilic Methanogenic Granular Sludge

Hiroyuki Imachi,1 Yuji Sekiguchi,1,* Yoichi Kamagata,2 Akiyoshi Ohashi,1 and Hideki Harada1

Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188,1 and National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Tsukuba, Ibaraki 305-8566,2 Japan

Received 3 February 2000/Accepted 29 May 2000

The thermophilic, anaerobic, propionate-oxidizing bacterial populations present in the methanogenic granular sludge in a thermophilic (55°C) upflow anaerobic sludge blanket reactor were studied by cultivation and in situ hybridization analysis. For isolation of propionate-degrading microbes, primary enrichment was made with propionate as the sole energy source at 55°C. After several attempts to purify the microbes, a thermophilic, syntrophic, propionate-oxidizing bacterium, designated strain SI, was isolated in both pure culture and coculture with Methanobacterium thermoautotrophicum. Under thermophilic (55°C) conditions, strain SI oxidized propionate, ethanol, and lactate in coculture with M. thermoautotrophicum. In pure culture, the isolate was found to ferment pyruvate. 16S ribosomal DNA sequence analysis revealed that the strain was relatively close to members of the genus Desulfotomaculum, but it was only distantly related to any known species. To elucidate the abundance and spatial distribution of organisms of the strain SI type within the sludge granules, a 16S rRNA-targeted oligonucleotide probe specific for strain SI was developed and applied to thin sections of the granules. Fluorescence in situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells were present in the middle and inner layers of the thermophilic granule sections and that they formed close associations with hydrogenotrophic methanogens. They accounted for approximately 1.1% of the total cells in the sludge. These results demonstrated that strain SI was one of the significant populations in the granular sludge and that it was responsible for propionate oxidation in the methanogenic granular sludge in the reactor.

* Corresponding author. Mailing address: Department of Environmental Systems Engineering, Nagaoka University of Technology, Kamitomioka 1603-1, Nagaoka, Niigata 940-2188, Japan. Phone: 81-258-47-9623. Fax: 81-258-47-9623. E-mail: skgc{at}vos.nagaokaut.ac.jp.

Applied and Environmental Microbiology, August 2000, p. 3608-3615, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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