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

Abstract

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 13 C-labeled palmitate (1 mM) as a substrate. After 8 to 19 days of incubation, we could detect 13 C-labeled bacterial rRNA. A density-resolved terminal restriction fragment length polymorphism fingerprinting analysis showed distinct bacterial populations in 13 C-labeled and unlabeled rRNA fractions. The bacterial populations in the 13 C-labeled rRNA fractions were identified by cloning and sequencing of reversetranscribed 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.Lipid is a one of the major organic fractions of wastes/ wastewaters, and lipid-rich wastes/wastewaters are widely found in certain food processing industries, such as dairy, edible oil, and slaughterhouses (20). Because lipids have a high theoretical methane yield in comparison with other organic substances, methanogenic treatment has been applied to lipidrich wastes/wastewaters but resulted in low organic loading rates (see, for example, references 16 and 50) compared to that seen for other types of wastes/wastewaters. This is at least partly due to the acute toxicity of long-chain fatty acids (LCFA), which are the main constituent and hydrolysate of lipids in the anaerobic consortium. LCFA can cause substrate toxicity in anaerobic microorganisms (see, for example, references 18 and 44) and tend to adsorb onto the biomass and flow out of the reactor.Under methanogenic conditions, LCFA degradation requires a syntrophic association of LCFA-degrading anaerobes and hydrogenotrophic methanogens, because the oxidation of LCFA is thermodynamically unfavorable in such environments unless the consumption of reducing equivalents (hydrogen and/or formate) is coupled with oxidation (37). Due to the syntrophic metabolism and toxicity of LCFA, isolation of LCFA-degrading syntrophs is difficult. Thus, information on LCFA-degrading bacteria in pure culture is based on Syntrophomonas species (10, 21, 36, 47, 54) and on Thermosyntropha lipolytica...

Detection of Active Butyrate-Degrading Microorganisms in Methanogenic Sludges by RNA-Based Stable Isotope Probing

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