Environmental Microbiology Reports (2010) 2(4), 489–499 doi:10.1111/j.1758-2229.2010.00147.x Minireview Syntrophic butyrate and propionate oxidation processes: from genomes to reaction mechanismsemi4_147 489..499 Nicolai Müller,1† Petra Worm,2† Bernhard Schink,1* a cytoplasmic fumarate reductase to drive energy- Alfons J. M. Stams2 and Caroline M. Plugge2 dependent succinate oxidation. Furthermore, we 1Faculty for Biology, University of Konstanz, D-78457 propose that homologues of the Thermotoga mar- Konstanz, Germany. itima bifurcating [FeFe]-hydrogenase are involved 2Laboratory of Microbiology, Wageningen University, in NADH oxidation by S. wolfei and S. fumaroxidans Dreijenplein 10, 6703 HB Wageningen, the Netherlands. to form hydrogen. Summary Introduction In anoxic environments such as swamps, rice fields In anoxic environments such as swamps, rice paddy fields and sludge digestors, syntrophic microbial communi- and intestines of higher animals, methanogenic commu- ties are important for decomposition of organic nities are important for decomposition of organic matter to matter to CO2 and CH4. The most difficult step is the CO2 and CH4 (Schink and Stams, 2006; Mcinerney et al., fermentative degradation of short-chain fatty acids 2008; Stams and Plugge, 2009). Moreover, they are the such as propionate and butyrate. Conversion of these key biocatalysts in anaerobic bioreactors that are used metabolites to acetate, CO2, formate and hydrogen is worldwide to treat industrial wastewaters and solid endergonic under standard conditions and occurs wastes. Different types of anaerobes have specified only if methanogens keep the concentrations of these metabolic functions in the degradation pathway and intermediate products low. Butyrate and propionate depend on metabolite transfer which is called syntrophy degradation pathways include oxidation steps of (Schink and Stams, 2006). The study of syntrophic coop- comparably high redox potential, i.e. oxidation of eration is essential to understand methanogenic conver- butyryl-CoA to crotonyl-CoA and of succinate to sions in different environments (Mcinerney et al., 2008). fumarate, respectively, that require investment of The most difficult step in this degradation is the conver- energy to release the electrons as hydrogen or sion of short-chain fatty acids such as propionate and formate. Although investigated for several decades, butyrate. Under standard conditions (PH2 of 1 atm, sub- the biochemistry of these reactions is still not com- strate and product concentrations of 1 M, temperature pletely understood. Genome analysis of the butyrate- 298°K), propionate and butyrate oxidation to H2, formate oxidizing Syntrophomonas wolfei and Syntrophus and acetate are endergonic reactions (Table 1). In anoxic aciditrophicus and of the propionate-oxidizing Syn- environments, methanogenic Archaea maintain low H2, trophobacter fumaroxidans and Pelotomaculum ther- formate and acetate concentrations which make propi- mopropionicum reveals the presence of energy- onate and butyrate degradation feasible (Stams and transforming protein complexes. Recent studies Plugge, 2009). Syntrophic propionate and butyrate oxida- indicated that S. wolfei uses electron-transferring fla- tions involve energy-dependent reactions that are bio- voproteins coupled to a menaquinone loop to drive chemically not fully understood. However, recently butyryl-CoA oxidation, and that S. fumaroxidans uses several novel reactions were discussed to perform energy a periplasmic formate dehydrogenase, cytochrome transformation in other bacteria. These reactions will be b:quinone oxidoreductases, a menaquinone loop and summarized in this report with respect to their possible implications in syntrophic fatty acid oxidation. Moreover, the genomes of two propionate degraders (Syntropho- bacter fumaroxidans and Pelotomaculum thermopropio- Received 3 November, 2009; accepted 9 January, 2010. *For nicum) and two butyrate degraders (Syntrophomonas correspondence. E-mail [email protected]; Tel. (+49) 7531 882140; Fax (+49) 7531 884047. †Both authors contrib- wolfei and Syntrophus aciditrophicus) have been uted equally. sequenced (Mcinerney et al., 2007; Kosaka et al., 2008). © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd 490 N. Müller et al. Table 1. Standard free reaction enthalpies of fatty acid oxidation and methane production. Reaction DG0′ (kJ per reaction) - - Propionate + 2H2O → Acetate + CO2 + 3H2 Eq. 1 +76.0 - - - + Propionate + 2H2O + 2CO2 → Acetate + 3 HCOO + 3H Eq. 1a +65.3 - - + Butyrate + 2H2O → 2 Acetate + H + 2H2 Eq. 2 +48.3 - - - + Butyrate + 2H2O + 2CO2 → 2 Acetate + 2 HCOO + 2H Eq. 2a +38.5 4H2 + CO2 → CH4 + 2H2O Eq. 3 -131.7 - + 4 HCOO + 4H → CH4 + 3CO2 + 2H2O Eq. 4 -144.5 - + CH3COO + H → CH4 + CO2 Eq. 5 -36 Values calculated from the standard free formation enthalpies of the reactants at a concentration of 1 M, pH 7.0 and T = 25°C according to Thauer and colleagues (1977). Based on genome analysis we propose that novel energy- (Wofford et al., 1986). Electrons are released in the oxi- transforming reactions are involved in syntrophic butyrate dation of butyryl-CoA to crotonyl-CoA and in the oxidation and propionate degradation. of 3-hydroxybutyryl-CoA to acetoacetyl-CoA at -250 mV (Gustafson et al., 1986). As the standard midpoint redox potentials of these reducing equivalents are too high for Topic 1. Known energy-conserving mechanisms in reduction of protons to form H [-414 mV (Thauer et al., syntrophic butyrate and propionate degradation 2 1977; Schink, 1997)], it was postulated that an energy- Butyrate degradation dependent reversed electron transport is required to over- come the redox potential difference (Thauer and Morris, Butyrate oxidizers known to date belong to two groups of 1984). The partner organism keeps the hydrogen partial bacteria within the family Syntrophomonadaceae and the pressure low, thus raising the redox potential of proton order Syntrophobacterales. Formerly classified as reduction to a level around -300 mV (Schink, 1997). The Clostridia, the members of the family Syntrophomona- butyrate-oxidizing organism has to sacrifice part of the daceae have been reassigned to a new family within the gained ATP to shift electrons to this redox potential, and order Clostridiales, based on their 16S rRNA sequence the remaining ATP can be used for biosynthesis and (Zhao et al., 1993). Members of this family are S. wolfei, growth. As such fractional amounts of ATP cannot be Syntrophomonas bryantii [formerly Syntrophospora bry- provided by substrate level phosphorylation such energy antii (Wu et al., 2006)], Syntrophomonas erecta, Syntro- transformations have to be coupled to the cytoplasmic phomonas curvata, Syntrophomonas zehnderi and membrane (Thauer and Morris, 1984). Indeed, hydrogen Thermosyntropha lipolytica. production from butyrate has been shown to be sensitive The second group of syntrophic butyrate degraders to the protonophore CCCP and the ATPase inhibitor belongs to the Syntrophobacterales, an order of the DCCD, thus providing evidence for participation of a deltaproteobacteria subdivision. Organisms of this group transmembrane proton potential (Wallrabenstein and are S. aciditrophicus and Syntrophus buswellii. Several Schink, 1994). However, the underlying biochemical other Syntrophus strains such as Syntrophus gentianae mechanisms remained enigmatic until the completion of are able to oxidize benzoate or other aromatic com- the genome sequence of S. aciditrophicus (Mcinerney pounds syntrophically but these processes are not con- et al., 2007). It was stated that electrons released in sidered in this article. Remarkably, all these organisms butyryl-CoA oxidation are transferred to components of are restricted to the use of saturated or unsaturated fatty the membrane where they reduce NAD+ to NADH in an acids. Alternative substrates or alternative electron accep- endergonic manner, e.g. through an rnf-coded oxi- tors to grow these bacteria in pure culture have not been doreductase, and the necessary energy would be sup- found yet for these two groups of butyrate-oxidizing plied by a sodium ion gradient which in turn is provided by bacteria. ATP-dependent proton efflux and a sodium/proton anti- In all known butyrate-oxidizing bacteria, the beta- porter. In S. wolfei, however, a different reaction mecha- oxidation pathway is used (Wofford et al., 1986; Schink, nism has to be active since the genome of this bacterium 1997; Mcinerney et al., 2007). First, butyrate is activated does not contain rnf genes which will be discussed later in to butyryl-CoA with acetyl-CoA by a CoA transferase. this review (Müller et al., 2009). Further oxidation proceeds via crotonyl-CoA and 3-hydroxybutyryl-CoA to acetoacetyl-CoA which is Propionate degradation cleaved to two acetyl-CoA moieties. One of these is invested in butyrate activation, and the other one forms Several bacterial strains are known to degrade pro- ATP via phosphotransacetylase and acetate kinase pionate in syntrophic association with methanogens: © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 2, 489–499 Syntrophic butyrate and propionate oxidation processes 491 S. fumaroxidans, S. wolinii, S. pfennigii, S. sulfatire- Topic 2. Mechanisms for energy conservation in ducens, P. thermopropionicum, P. schinkii, P. propioni- anaerobic microorganisms cicum, Smithella propionica and Desulfotomaculum Electron transport phosphorylation thermobenzoicum ssp. thermosyntrophicum.