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.