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

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 family Syntrophomonadaceae. The other two enrichment cultures contained different bacterial populations in which the majority of members belonged to the phylum Firmicutes and the class Deltaproteobacteria. After several attempts to isolate these dominant bacteria, strain MPA, belonging to the family Syntrophomonadaceae, and strain TOL, affiliated with the phylum Firmicutes, were successfully isolated. Strain MPA converts palmitate to acetate and methane in syntrophic association with Methanospirillum hungatei. Even though strain TOL assimilated [13 C]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 family Syntrophomonadaceae, as most anaerobic LCFA-degrading microbes do, but may also be found in phylogenetically diverse bacterial groups.To date, anaerobic (methanogenic) treatment processes have been widely applied to the treatment of municipal and industrial waste and wastewater because of demonstrable performance and cost-saving advantages (30,34,45). To expand the applications of these processes, many engineers and researchers are now being challenged to treat more-complex waste and wastewater containing anthropogenic compounds and/or compounds that are recalcitrant to biodegradation (17). This type of treatment has been applied to lipid-rich wastes and wastewater since the early stages of development of anaerobic treatment technologies for the following reasons: (i) lipid-rich waste and wastewater are widely found in certain food processing industries such as dairy, edible oil, and slaughterhouses (22, 33); and (ii) lipids have a high theoretical methane yield in comparison with that of other organic substrates (22). However, most of the previous studies of methanogenic processes with lipid-rich wastewater found them to be less stable and able to accommodate lower organic-loading rates (see, for example, references 37 and 47) than other types of waste and wastewater. This may be due in part to the acute toxicity of long-chain fatty acids (LCFA), which are the main constituent and hydrolysate of lipids in the anaerobic consortium. LCFA give rise to substrate toxicity in anaerobic microbes (10,18,20) and tend to adsorb onto the bi...

Stimulation of Methane Generation from Nonproductive Coal by Addition of Nutrients or a Microbial Consortium

“…The degradation of LCFA (19,20), aromatics (12,47), and alkanes (1, 51) has been observed in methanogenic consortia. The degradation of these compounds in the absence of a terminal electron acceptor (TEA) for respiration involves the syntrophic cooperation of microorganisms; i.e., the reactions are thermodynamically driven by the removal of H 2 (32) and result in accumulation of acetate and propionate (Fig.…”

“…Methanogen growth and activity may have been inhibited by the acute toxicity of LCFA (20) or aromatics (37,43). Hydrogenotrophic cell numbers declined between day 8 and day 39 as organics in solution increased.…”

“…6. Proposed model of biogenic methane generation from coal based on the current study and previous work in other laboratories (9,11,12,15,19,20,37,51). H 2 may be removed by methanogenesis, acetogenesis, or by a bacterial partner with a TEA.…”

“…The nutrient solution, defined as the medium, consisted of NaHCO 3 (2.5 g/liter), NH 4 Cl (0.5 g/liter), NaH 2 PO 4 (0.5 g/liter), and KCl (0.1 g/liter), sparged with N 2 /CO 2 (80: 20), and a complete suite of trace minerals (including 200 M SO 4 ) and vitamins (24) formulated to sustain methanogenic growth (but with coal providing the source of organic carbon and energy). In the nutrient-free solution, NH 4 Cl and NaH 2 PO 4 were replaced by an equivalent concentration of NaCl.…”

Identification of Methanoculleus spp. as Active Methanogens during Anoxic Incubations of Swine Manure Storage Tank Samples

“…The DNA stable isotope probing (DNA-SIP) method (10,11) is appropriate for identifying microorganisms that are actively involved in specific assimilatory processes under conditions that approximate those found in an environment of interest. This cultivation-independent approach relies on the incorporation of an isotopically heavy substrate into the nucleic acids of metabolically active organisms in complex microbial communities, providing information on microbial networks participating in a wide range of metabolic functions, including methanogenesis in soils (12), sediments (13), and sludges (14) and from different substrates (15,16).…”