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Syntrophic methanogenic associations between acetogenic bacteria and methanogenic archaea are essential for the complete mineralization of organic compounds to methane and CO2. Propionate and butyrate are important intermediates in anaerobic digestion. In the absence of inorganic electron acceptors these short chain fatty acids can only be degraded if the products acetate, hydrogen and formate, are kept low by methanogens. However, when sulfate is available the conditions change, and propionate and butyrate can be oxidized coupled to sulfate reduction. Several sulfate-reducing bacteria are able to grow in syntrophic associations with methanogens, but others not.
In this thesis, a functional analysis of protein domains was performed on a selected group of bacteria with the ability to grow on short chain fatty acids alone, or in syntrophy with methanogens. Genome analysis revealed that the presence of periplasmic formate dehydrogenases, most probably involved in interspecies electron transfer, differentiated syntrophic from non-syntrophic butyrate and propionate degraders.
Moreover, the metabolic flexibility of the propionate-degrading bacterium Syntrophobacter fumaroxidans was investigated. S. fumaroxidans can couple propionate oxidation to sulfate reduction or can degrade propionate in syntrophic lifestyle with H2 and formate scavenging microorganisms. Propionate-grown cultures of S. fumaroxidans with sulfate as electron acceptor, or in syntrophy with Methanospirillum hungatei or Desulfovibrio desulfuricans were studied. We found that S. fumaroxidans is prone to oxidize propionate in syntrophy despite the availability of sulfate to grow on its own.
A comparative proteomic analysis of propionate degradation by S. fumaroxidans in five growth conditions, including axenic and cocultures, was performed. This analysis gave a thorough overview of the propionate metabolism of S. fumaroxidans. Details on the energy conservation mechanisms and electron transfer to syntrophic partners were obtained. The results indicate that confurcating hydrogenases and formate dehydrogenases are important energy converting enzymes in propionate degradation by S. fumaroxidans. Moreover, three formate dehydrogenases fulfil an important role in the syntrophic lifestyle. Furthermore, the proteomic profile of S. fumaroxidans grown with sulfate revealed in detail the sulfate respiratory pathway of this model bacterium. The abundance of a putatively confurcating protein complex detected only in sulfate-grown cells, is an important finding. This confurcating complex has similarities to heterodisulfide reductases, proteins known to bifurcate electrons in methanogenic archaea. The detection of membrane-associated proteins usually involved in sulfate reduction in all growth conditions leaves room for research on the role of these complexes in electron transfer during syntrophic lifestyle.
Understanding the interactions between propionate-oxidizing syntrophic consortia also involved the investigation of the syntrophic partners of S. fumaroxidans. We analysed the proteome of M. hungatei, Methanobacterium formicicum and D. desulfuricans grown in syntrophy and in pure culture with H2/CO2 or formate. Although both methanogens can grow on hydrogen and formate, the molecular mechanisms studied in this thesis, points to the use of hydrogen in M. formicicum, and of formate in M. hungatei, as electron carriers in their metabolism.
Lastly, the microbial community involved in pot ale digestion in an anaerobic membrane bioreactor was analysed using 16S rRNA next-generation sequencing. The robustness of the reactor to high loading tests and the effect on the microbial composition was discussed. Moreover, on-line monitoring of hydrogen in the biogas showed a rapid response to disturbances in the proper performance of the reactor. Thus, our study supports the use of on-line H2 measurements as an early warning indicator of process instability.
The detailed study and analysis of the molecular mechanisms for energy conservation and interspecies electron transfer discussed in this thesis increases our understanding of electron fluxes occurring in methanogenic syntrophic consortia. These types of analyses are necessary to unravel the black-box ecology of anaerobic biotechnology and the global carbon flux.
|Qualification||Doctor of Philosophy|
|Award date||3 Apr 2018|
|Place of Publication||Wageningen|
|Publication status||Published - 2018|