Formate dehydrogenases and hydrogenases in syntrophic propionate-oxidizing communities : gene analysis and transcritional profiling

Many places on earth are without oxygen (anaerobic) such as rice paddy fields, swamps and sediments of freshwater lakes and oceans. When oxygen, nitrate or other electron acceptors are not present, organic material is degraded to carbon dioxide and methane by mixed microbial species that each have their own specific function in degradation. Anaerobic microbial communities are used in anaerobic digesters all over the world to treat organic waste and wastewater. Propionate is one of the most important intermediates in anaerobic digestion. It can only be degraded by propionate oxidizing bacteria when methanogenic archaea keep the concentration of the interspecies electron carriers, hydrogen and formate, low. However, little is known about the molecular mechanism of hydrogen and formate transfer. Hydrogenases are involved in hydrogen transfer and require Fe, Ni and/or Se for catalysis. Formate dehydrogenases that are involved in formate transfer require the trace metals W or Mo and in some cases Se for catalysis. However, the effect of W, Mo and Se limitation on the propionate degrading community of a UASB reactor and the transcription of formate dehydrogenase and hydrogenase encoding genes in this community was never examined. This would give more insight in formate transfer in the propionate degrading community of the UASB reactor and provide a method to study depletion of these metals in the reactor sludge. We used the genome sequences of the propionate degrading Syntrophobacter fumaroxidans and its syntrophic methanogenic partner, Methanospirillum hungatei to study molecular mechanisms of hydrogen and formate transfer in syntrophic cocultures and UASB reactor sludge, by gene analysis and molecular techniques. Gene analysis and microarray data determined formate dehydrogenase and hydrogenase encoding gene clusters in S. fumaroxidans and M. hungatei (Chapter 4). When S. fumaroxidans oxidizes propionate, reducing equivalents are generated by three intermediate reactions in the form of FADH2, NADH and reduced ferredoxin. We found by gene analysis (Chapter 2) and RT qPCR (Chapter 3) that the genes coding for four formate dehydrogenases, six hydrogenases and one formate hydrogen lyase of S. fumaroxidans and five formate dehydrogenases and three hydrogenases of M. hungatei were all transcribed during syntrophic and axenic growth. However, the transcription levels were dependent on the growth condition. Comparison of transcription levels also revealed that electrons from ferredoxin and NADH are simultaneously confurcated for hydrogen production by a cytoplasmic [FeFe]-hydrogenase. Moreover, results indicated that during syntrophic growth electrons from ferredoxin and NADH are confurcated to formate via a cytoplasmic formate dehydrogenase (FDH1). During syntrophic growth, the electrons generated at the level of FADH2, travel via a cytoplasmic oriented succinate dehydrogenase, menaquinones, cytochrome b and c to the periplasmic formate dehydrogenase (FDH2) (Chapter 5). When S. fumaroxidans is grown in pure culture with alternative electron acceptors such as sulfate and fumarate, electrons flow partly to FDH2, and partly to the periplasmic hydrogenase (Hyn). The energy gained from propionate conversion to methane, acetate, and carbon dioxide has to be shared by S. fumaroxidans and M. hungatei. When M. hungatei takes more energy, less energy remains for S. fumaroxidans. In this situation S. fumaroxidans up-regulates transcription of genes coding for an additional cytoplasmic confurcating hydrogenase (Hox) and the periplasmic hydrogenase (Hyn) that is coupled to succinate oxidation. In addition, S. fumaroxidans induces transcription of genes coding for the Rnf-complex and ferredoxin dependent hydrogenases and formate dehydrogenases. This provides the possibility to use the membrane potential for the energy dependent coupling of ferredoxin reduction to NADH oxidation. The designed RT qPCR primers were used in UASB reactor sludge from the alcohol distillery NEDALCO in Bergen op Zoom (Netherlands) to investigate the effect of trace elements depletion. A lab-scale UASB reactor was fed with propionate and synthetic medium without added W, Mo and Se. During the reactor run, Syntrophobacter spp. were the dominant propionate-oxidizers and M. hungatei the dominant hydrogen and formate using methanogen. However, when propionate degradation decreased, two other propionate-oxidizers; Pelotomaculum propionicicum and Smithella propionica became abundant (Chapter 6). RT qPCR showed that in this reactor run the transcription of genes coding for formate dehydrogenases and hydrogenases in S. fumaroxidans decreased while transcription of genes coding for formate dehydrogenases and hydrogenases in M. hungatei were more stable (Chapter 7). This research shows that RT qPCR is a fast technique that can give information on the active processes in a UASB reactor, and that trace element limitation and possible malfunctioning of UASB reactors can be predicted. With this PhD research we gained insight in the molecular mechanisms of hydrogen and formate transfer between S. fumaroxidans an M. hungatei in defined cocultures and in a propionate-fed UASB reactor. This contributes to the understanding of similar molecular mechanisms in other syntrophic microorganisms and may improve the performance of anaerobic digesters in the future.