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Applied and Environmental Microbiology, July 2007, p. 4119-4127, Vol. 73, No. 13
0099-2240/07/$08.00+0     doi:10.1128/AEM.00362-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Diversity of Anaerobic Microorganisms Involved in Long-Chain Fatty Acid Degradation in Methanogenic Sludges as Revealed by RNA-Based Stable Isotope Probing

Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan,¹ Subground Animalcule Retrieval (SUGAR) Program, Extremobiosphere Research Center, Japan Agency for Marine-Earth Science & Technology (JAMSTEC), Yokosuka, Kanagawa 237-0061, Japan,² Department of Civil Engineering, Tohoku University, Sendai, Miyagi 980-8579, Japan³

Received 14 February 2007/ Accepted 27 April 2007

Long-chain fatty acid (LCFA) degradation is a key step in methanogenic treatment of wastes/wastewaters containing high concentrations of lipids. However, despite the importance of LCFA-degrading bacteria, their natural diversity is little explored due to the limited availability of isolate information and the lack of appropriate molecular markers. We therefore investigated these microbes by using RNA-based stable isotope probing. We incubated four methanogenic sludges (mesophilic sludges MP and MBF and thermophilic sludges TP and JET) with ¹³C-labeled palmitate (1 mM) as a substrate. After 8 to 19 days of incubation, we could detect ¹³C-labeled bacterial rRNA. A density-resolved terminal restriction fragment length polymorphism fingerprinting analysis showed distinct bacterial populations in ¹³C-labeled and unlabeled rRNA fractions. The bacterial populations in the ¹³C-labeled rRNA fractions were identified by cloning and sequencing of reverse-transcribed 16S rRNA. Diverse phylogenetic bacterial sequences were retrieved, including those of members of the family Syntrophaceae, clone cluster MST belonging to the class Deltaproteobacteria, Clostridium clusters III and IV, phylum Bacteroidetes, phylum Spirochaetes, and family Syntrophomonadaceae. Although Syntrophomonadaceae species are considered to be the major fatty acid-degrading syntrophic microorganisms under methanogenic conditions, they were detected in only two of the clone libraries. These results suggest that phylogenetically diverse bacterial groups were active in situ in the degradation of LCFA under methanogenic conditions.

Published ahead of print on 4 May 2007.

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