Identification and Cultivation of Anaerobic, Syntrophic Long-Chain Fatty Acid-Degrading Microbes from Mesophilic and Thermophilic Methanogenic Sludges

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Abstract
We investigated long-chain fatty acid (LCFA)-degrading anaerobic microbes by enrichment, isolation, and RNA-based stable isotope probing (SIP). Primary enrichment cultures were made with each of four LCFA substrates (palmitate, stearate, oleate, or linoleate, as the sole energy source) at 55°C or 37°C with two sources of anaerobic granular sludge as the inoculum. After several transfers, we obtained seven stable enrichment cultures in which LCFAs were converted to methane. The bacterial populations in these cultures were then subjected to 16S rRNA gene-based cloning, in situ hybridization, and RNA-SIP. In five of seven enrichment cultures, the predominant bacteria were affiliated with the familySyntrophomonadaceae. The other two enrichment cultures contained different bacterial populations in which the majority of members belonged to the phylumFirmicutesand the classDeltaproteobacteria. After several attempts to isolate these dominant bacteria, strain MPA, belonging to the familySyntrophomonadaceae, and strain TOL, affiliated with the phylumFirmicutes, were successfully isolated. Strain MPA converts palmitate to acetate and methane in syntrophic association withMethanospirillum hungatei. Even though strain TOL assimilated [13C]palmitate in the original enrichment culture, strain TOL has not shown the ability to degrade LCFAs after isolation. These results suggest that microbes involved in the degradation of LCFAs under methanogenic conditions might not belong only to the familySyntrophomonadaceae, as most anaerobic LCFA-degrading microbes do, but may also be found in phylogenetically diverse bacterial groups.

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