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1 Introduction

Lipids, natural oils, and fats are abundantly present in nature. Lipids are constituents of membranes of bacteria, archaea, and eukaryotes, while oils and fats are storage compounds for carbon and energy in all kinds of living organisms. Lipids are mostly esters of glycerol and long-chain fatty acids (LCFA) or, in the case of archaea, ethers of glycerol and long-chain alcohols. A substantial part of the dry weight of biomass is lipids, oils, and fats. These energy-rich compounds can be anaerobically degraded by methanogenic and sulfate-reducing communities. Methanogens and sulfate-reducing bacteria are not known to hydrolyze lipids. This suggests that other anaerobic microorganisms are responsible for the synthesis and excretion of lipases for the initial attack of lipids. A variety of sulfate-reducing bacteria is able to degrade LCFA coupled to the reduction of sulfate. However, methanogens do not degrade LCFA. In methanogenic environments, LCFA are degraded by proton-reducing acetogenic bacteria to acetate and hydrogen, which are substrates for the methanogens. For energetic reasons, the growth of proton-reducing acetogenic bacteria on LCFA is possible only if acetate and, in particular, hydrogen are efficiently removed by methanogens. This results in an obligately syntrophic growth driven by interspecies hydrogen transfer (Schink, 1997; Schink and Stams, 2006). In this chapter, we present information on the physiology of sulfate-reducing bacteria and syntrophic methanogenic communities that are able to grow on LCFA.

2 General Biochemical Pathway of LCFA Degradation

LCFA degradation pathways have not been studied extensively in methanogenic and sulfate-reducing communities at a biochemical or genetic level. Experiments with 14C-labeled LCFA indicated that degradation occurs by β-oxidation (Nuck and Federle, 1996; Weng and Jeris, 1976). Detailed studies on LCFA metabolism have been done in model organisms like Escherichia coli (DiRusso et al., 1999). LCFA biodegradation occurs through sequential steps: (1) LCFA adsorption to the cell surface, (2) LCFA uptake, and (3) LCFA conversion to lower molecular weight components via β-oxidation. A scheme of the β-oxidation cycle is shown in Fig. 1 . The end product in this cycle is acetyl-CoA.

Figure 1
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The β-oxidation pathway involved in LCFA degradation in sulfate-reducing bacteria, and acetogenic bacteria that grow in syntrophy with methanogens.

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E. coli possesses a metabolic pathway to degrade LCFA anaerobically in the presence of nitrate, fumarate, or trimethylamine-N-oxide that is distinct from the Fad enzymes used for aerobic LCFA metabolism (Campbell et al., 2003; Klein et al., 1971). FadIJ, like FadAB (Pramanik et al., 1979), appear to form a multienzyme complex that contains enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-oxoacyl-CoA thiolase activities (Campbell et al., 2003; Snell et al., 2002). In contrast to the gene organization in E. coli, genomic analyses of Syntrophus aciditrophicus (McInerney et al., 2007) and Syntrophomonas wolfei (http://www.jgi.doe.gov) indicate that separate genes encode for enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-oxoacyl-CoA thiolase activities. Both the S. aciditrophicus and S. wolfei genomes contain multiple homologs encoding not only for enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-oxoacyl-CoA thiolase activities but also for acetyl-CoA synthetase (AMP-forming) and acyl-CoA dehydrogenase activities (Table 1 ). Presumably, the homologs differ in chain-length or substrate (saturated vs. unsaturated fatty acids) specificity.

Table 1 Homologues of β-oxidation genes in the genomes of syntrophic acetogenic bacteria
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In sulfate-reducing bacteria that degrade LCFA completely to CO2, acetyl-CoA is further degraded via the acetyl-CoA cleavage pathway or a modified citric acid cycle (Schauder et al., 1986). Acetyl-CoA can also be converted to acetate as is the case for LCFA-degrading bacteria in methanogenic environments and in several sulfate-reducing bacteria. Acetate is then a substrate for acetoclastic methanogens (Methanosarcina and Methanosaeta) or acetate-utilizing sulfate-reducing bacteria (Desulfobacter, Desulfobacterium, Desulforhabdus and Desulfobacca).

3 Hydrogenation of Unsaturated Fatty Acids

Some fermentative microorganisms are capable of hydrogenating unsaturated fatty acids, without being able to grow by these conversions (Mackie et al., 1991). Several anaerobic ruminal and human gut bacteria with biohydrogenation capabilities, belonging to the genera Butyrivibrio, Pseudobutyrivibrio, “Fusocillus,” Borrelia, Roseburia, and Clostridium, have been isolated and characterized (Devillard et al., 2007; Fukuda et al., 2005; Hunter et al., 1976; Kemp et al., 1984; Kopecny et al., 2003; Maia et al., 2007; Moon et al., 2008; Sachan & Davis, 1969; van de Vossenberg and Joblin, 2003; Wallace et al., 2006).

Early studies suggested that the degradation of unsaturated LCFA, such as linoleic (C18:2) and oleic (C18:1) acids, proceeded via β-oxidation only after chain saturation (Novak and Carlson, 1970; Weng and Jeris, 1976). Canovas-Dias et al. (1991) detected palmitoleate (C16:1) as a transient product during oleate (C18:1) degradation, suggesting the occurrence of one β-oxidation step without prior chain saturation. Thus far, experimental data are lacking to define the exact pathway involved in the degradation of unsaturated LCFA. Nevertheless, an analysis of the free energy variation of different possible reactions involved in LCFA degradation can provide some insight into which pathways are most likely as illustrated for oleate in (Table 2 ).

Table 2 Gibbs free energy changes (at standard conditions) for hydrogenation and β-oxidation reactions (presumably) involved in conversion of unsaturated fatty acids
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The hydrogenation step is thermodynamically favorable as indicated by the Gibbs free energy change of oleate (C18:1) conversion to stearate (C18:0). The direct β-oxidation of oleate (C18:1) to palmitoleate (C16:1) is thermodynamically unfavorable under standard conditions (ΔG0′> 0). Oleate (C18:1) degradation by a combined hydrogenation and β-oxidation to form palmitate (C16:0) seems most likely, as the endergonic oxidation reaction could be driven by the exergonic hydrogenation reaction. In studies performed with anaerobic sludges, palmitate was indeed a main intermediate product in oleate degradation (Lalman and Bagley, 2001; Pereira et al., 2002).

4 LCFA Degradation by Sulfate-Reducing Communities

Baars (1930) described Vibrio rübentschickii, a sulfate-reducing bacterium that is able to grow on LCFA and short-chain fatty acids. However, it never became really clear whether or not this culture was a pure culture of a sulfate-reducing bacterium (Postgate and Campbell, 1966). Later, the ability of pure cultures of sulfate-reducing bacteria to grow on LCFA and short-chain fatty acids was shown by Widdel (1980). Currently, representatives of several genera of sulfate-reducing microorganisms are known to be able to grow on LCFA (Fig. 2 ). Table 3 lists some properties of sulfate-reducing bacteria that can grow on LCFA. Some of the sulfate-reducing bacteria that have been tested grow also on acetate, indicating that these sulfate-reducing bacteria degrade LCFA completely to CO2. However, many LCFA-degrading sulfate-reducing bacteria produce acetate as a side product.

Figure 2
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Phylogenetic tree of bacterial 16S rRNA gene sequences of the fatty-acid degrading sulfate-reducing and acetogenic bacteria described in the Tables 3 and 4 (fatty-acid degrading bacteria are shown in bold). The tree was based on 16S rRNA gene sequences and calculated using the ARB software package (Ludwig et al., 2004). Thermotoga lettingae (AF355615) was used as outgroup.

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Table 3 Selected characteristics of fatty-acid degrading sulfate-reducing bacteria
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Table 4 Selected characteristics of fatty-acid oxidizing acetogenic bacteria that grow in syntrophy with methanogens
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5 LCFA Degradation by Methanogenic Communities

Syntrophomonas sapovorans was the first-described LCFA-degrading bacterium that grows in syntrophy with methanogens (Roy et al., 1986). The first-defined butyrate-degrading culture, consisting of Syntrophomonas wolfei and Methanospirillum hungatei, was described earlier by McInerney et al. (1981). This bacterium is able to degrade fatty acids with a chain length of up to 8 carbon atoms. Syntrophomonas sapovorans grows on LCFA with more than 12 and up to 18 carbon atoms and is able to utilize unsaturated LCFA, such as oleate (C18:1) and linoleate (C18:2). To date, 14 syntrophic LCFA-degrading microorganisms have been obtained in pure culture or in coculture with hydrogen-consuming microorganisms (Table 4 ; Fig. 2 ). All these acetogenic bacteria are capable of anaerobically degrading fatty acids with more than 4 carbon atoms and up to 18 carbon atoms . They all belong to the families Syntrophomonadacea (McInerney, 1992; Zhao et al., 1993; Wu et al., 2006a) and Syntrophaceae (Jackson et al., 1999). LCFA higher than lauric acid (i.e., with more than 12 carbon atoms) are utilized only by Syntrophomonas sapovorans, S. saponavida, S. curvata, S. zehnderi, S. palmitatica, Thermosyntrophica lipolytica, and Syntrophus aciditrophicus. T. lipolytica also grows syntrophically with methanogens on lipids such as olive oil, utilizing only the liberated fatty acid moieties and releasing the glycerol. Two lipases are excreted by this bacterium (Salameh and Wiegel, 2007).

The LCFA degradation in methanogenic environments requires the syntrophic cooperation of LCFA-degrading bacteria and methanogens. Interspecies hydrogen transfer is a key process in methanogenesis. In 1967, Bryant and coworkers described for the first time an obligate syntrophic association between two microbial species (Bryant et al., 1967). They discovered that Methanobacillus omelianskii, originally believed to be a pure culture, (Stadtman and Barker, 1949; Barker, 1956) was not axenic. It was actually a coculture of a bacterium fermenting ethanol to acetate and H2 (the “S-organism”), and an archaeon (later named Methanobacterium bryantii; Balch et al., 1979) that uses H2 to reduce carbon dioxide to methane. The conversion of ethanol to acetate and H2 by the “S-organism” was thermodynamically possible only in the presence of M. bryantii, which keeps the H2 concentration low. The concept of interspecies hydrogen transfer originating from these observations was fundamental to understand how compounds such as propionate, butyrate, and LCFA are degraded in methanogenic environments. Table 5 summarizes the reactions involved in syntrophic LCFA degradation.

Table 5 Some reactions involved in LCFA degradation in methanogenic environments and the Gibbs free energy changes
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To sustain their metabolism, LCFA-degrading bacteria have to couple the oxidation of NADH and FADH2 to proton reduction, which is energetically difficult. The midpoint redox potential of the couple H+/H2 at pH 7 is −414 mV, whereas the midpoint redox potential of the couples of NAD+/NADH and FAD/FADH2 are −320 mV and −220 mV respectively (Thauer et al., 1977). The ΔG0′ values of the reactions NADH + H+ → NAD+ + H2 and FADH2 → FAD + H2 are about 18 and 38 kJ respectively. Methanogens are able to create a hydrogen partial pressure as low as 1 Pa. Under these conditions, the ΔG′ values of NADH and FADH2 oxidation are approximately −11 and + 9 kJ respectively. Thus, NADH oxidation, but not FADH2 oxidation, is feasible by the creation of a low hydrogen partial pressure by methanogens. It is not clear how LCFA-degrading bacteria solve the energetic problem of FADH2 oxidation coupled to hydrogen formation. Likely, a reversed electron transport mechanism is involved. The sequence analysis of the genes near fadK on the E. coli chromosome provides a clue about the nature of the reverse electron transport system involved in the syntrophic metabolism of LCFA. fadK mutants are impaired in anaerobic growth with fatty acids, and FadK is a putative acyl-CoA sythetase (Campbell et al., 2003). Genes ydiQRST are clustered with fadK on the E. coli chromosome, and ydiQRST have high sequence homology to fixABCX involved in the anaerobic carnitine metabolism in E. coli (Buchet et al., 1998; Walt and Kahn, 2002) and the reverse electron transport in N2-fixing bacteria (Earl et al., 1987; Edgren and Nordlund, 2004, 2006; Lindblad et al., 1996). The S. wolfei genome contains homologs to fixABCX, while S. aciditrophicus apparently uses another system for reverse electron transport (McInerney et al., 2007). Further research is needed to unravel the biochemistry of reverse electron transport.

6 Biogas Formation from LCFA Containing Waste Materials

LCFA are energy-rich compounds that are abundantly present in raw and waste materials (Table 6 ). Thus, biogas formation from LCFA-containing waste represents a sustainable technology. About 1 m3 methane can be produced from 1 kg of LCFA. Lipids and LCFA are present in domestic and industrial wastewaters. In domestic wastewater, generally, lipids represent 20–25% of the total organic matter with concentrations ranging from 40 to 100 mg/L (Quémeneur and Marty, 1994). Lipids/LCFA concentrations in industrial wastewaters are more variable and highly dependent of the industrial process. Concentrations of 11.2–22.4 g lipids/L were found in an industrial wastewater from a wool-scouring process (Becker et al., 1999). Also, a relatively high concentration of lipids, that is, 6.6 g/L, was measured in olive oil mill effluents (Beccari et al., 1998). Lower concentrations (0.4–1.7 g lipids/L) were detected in a sunflower oil mill wastewater with LCFA concentrations in the range 0.2–1.3 g/L (Saatci et al., 2003). Total lipids in dairy wastewaters range from 0.9 to 2.0 g/L (Kim et al., 2004).

Table 6 Saturated- and unsaturated-LCFA present in raw materials and wastewaters (shown as % of total LCFA) (adapted from Hwu, 1997)
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Wastewaters and waste streams that contain high concentrations of lipids and LCFA may yield high levels of methane in an anaerobic digestion process. A problem associated with anaerobic treatment of lipids and LCFA is their poor solubility. LCFA were found to be inhibitory for methanogens (Lalman and Bagley, 2001, 2002; Kim et al., 2004; Pereira et al., 2003, 2004). The inhibitory effects are reversible and are often associated with the interactions of lipids and LCFA with the cell wall, preventing the conversion of other compounds due to their physical interaction (Pereira et al., 2005). However, by applying a sequence of loading and digestion phases in anaerobic reactors, high rates of methanogenesis and complete methanogenesis could be obtained (Pereira et al., 2002, 2003, 2004). Recently, it was demonstrated that continuous high rate anaerobic treatment of LCFA was possible by applying a relatively short start-up period with three feeding and three feed less phases. Methane recovery of up to 72% was obtained when a bioreactor was fed with an organic loading rate of 21 kgCOD m−3 day−1 and retention time of 9h (Cavaleiro et al., 2009). These findings offer excellent prospects for a sustainable energy production from lipids and LCFA.

7 Research Needs

Numerous strains of LCFA-degrading sulfate-reducing bacteria and LCFA-degrading acetogenic bacteria that grow in syntrophy with methanogens have been isolated and characterized. Although these bacteria have been relatively well studied from the physiological point of view, biochemical and genetic studies of the pathways involved in LCFA degradation are scarce. The genomes of Desulfatibacillum alkenivorans and the acetogenic bacterium Syntrophomonas zehnderi are presently being sequenced (US Department of Energy, Joint Genome Institute). Comparison of the genomes of these bacteria and the genomes of related bacteria that are not well able to grow on LCFA may provide insight into LCFA-degrading pathways and the regulation of these pathways. In addition, it may allow an elucidation of the differences and similarities between degradation of saturated and unsaturated LCFA. Studies with methanogenic communities have shown that bacteria that were enriched with unsaturated fatty acids are able to degrade a wide range of saturated fatty acids, but the opposite is not the case (Sousa et al., 2007a, b).

As lipids and LCFA are highly energetic compounds, further research is needed to get insight into how such compounds can be efficiently and completely converted to biogas. The challenge is to implement the syntrophic nature of the microorganisms involved in the most appropriate reactor configuration and the most optimal process operation.