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Formate Dehydrogenases and Hydrogenases in Syntrophic Propionate-Oxidizing Communities : = Gene Analysis and Transcritional Profiling.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Formate Dehydrogenases and Hydrogenases in Syntrophic Propionate-Oxidizing Communities :/
Reminder of title:	Gene Analysis and Transcritional Profiling.
Author:	Worm, Petra.
Description:	1 online resource (139 pages)
Notes:	Source: Dissertations Abstracts International, Volume: 83-03, Section: B.
Contained By:	Dissertations Abstracts International83-03B.
Subject:	Physiology. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=28237461click for full text (PQDT)
ISBN:	9798516038402

Formate Dehydrogenases and Hydrogenases in Syntrophic Propionate-Oxidizing Communities : = Gene Analysis and Transcritional Profiling.
Worm, Petra.

Formate Dehydrogenases and Hydrogenases in Syntrophic Propionate-Oxidizing Communities :Gene Analysis and Transcritional Profiling. - 1 online resource (139 pages)

Source: Dissertations Abstracts International, Volume: 83-03, Section: B.

Thesis (Ph.D.)--Wageningen University and Research, 2010.

Includes bibliographical references

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.

Electronic reproduction.
Ann Arbor, Mich. :
ProQuest,
2023

Mode of access: World Wide Web

ISBN: 9798516038402Subjects--Topical Terms:

518431
Physiology.
Index Terms--Genre/Form:

542853
Electronic books.

Formate Dehydrogenases and Hydrogenases in Syntrophic Propionate-Oxidizing Communities : = Gene Analysis and Transcritional Profiling.
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245 1 0 $a Formate Dehydrogenases and Hydrogenases in Syntrophic Propionate-Oxidizing Communities : $b Gene Analysis and Transcritional Profiling.
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500 $a Source: Dissertations Abstracts International, Volume: 83-03, Section: B.
500 $a Advisor: Stams, A. J. M.; Plugge, C. M.
502 $a Thesis (Ph.D.)--Wageningen University and Research, 2010.
504 $a Includes bibliographical references
520 $a 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.
520 $a Veel plekken op aarde zijn zuurstofloos (anaeroob), zoals moerassen, rijstvelden of de sedimenten in diepe meren en oceanen. Als geen zuurstof, nitraat of andere electronenacceptoren aanwezig zijn, wordt organisch materiaal afgebroken tot kooldioxide en methaan door een mengsel van microbiele soorten die elk een eigen specifieke functie hebben in de afbraak. Wereldwijd worden anaerobe bioreactors gebruikt voor de reiniging van organisch afval en afvalwater. Propionaat is een van de meest belangrijkste tussenproducten in anaerobe afbraak. Het kan alleen worden afgebroken door propionaat-oxiderende bacterien als methanogene archaea de concentratie van de electronenmediatoren, waterstof en formiaat, laag houden. Er is nog weinig bekend over de moleculaire mechanismen van waterstof en formiaat overdracht. Hydrogenases zijn nodig voor waterstof overdracht en hebben de spore metalen Fe, Ni en/of Se nodig voor katalyse. Formiaat dehydrogenases die betrokken zijn bij formiaat overdracht hebben de spore metalen W of Mo en in sommige gevallen Se nodig voor katalyse. Echter, het effect van W, Mo en Se limitatie op de propionaat afbrekende consortia in een UASB reactor en de transcriptie van genen die coderen voor formiaat dehydrogenases en hydrogenases in deze consortia, is nog niet eerder bestudeerd. Dit zou meer inzicht kunnen geven in formiaat overdracht in de propionaat afbrekende consortia van de UASB reactor en zou een methode kunnen opleveren om limitatie van deze metalen in de reactor materiaal te analyseren. We hebben gebruik gemaakt van de genoomsequenties van de propionaatafbrekende Syntrophobacter fumaroxidans en de syntrofe methanogene partner, Methanospirillum hungatei om de moleculaire mechanismen van waterstof- en formiaatoverdracht in reincultures, syntrofe cultures en UASB reactor materiaal middels genanalyse en moleculaire technieken te bestuderen. Genclusters die coderen voor formiaat dehydrogenases en hydrogenases in S. fumaroxidans en M. hungatei zijn bepaald met behulp van genanalyse en microarray gegevens (Hoofdstuk 4). Als S. fumaroxidans propionaat oxideert worden reductie-equivalenten geproduceerd in de vorm van FADH2, NADH en gereduceerd ferredoxine. Wij vonden met genanalyse (Hoofdstuk 2) en RT qPCR (Hoofdstuk 3) dat de genen die coderen voor vier formiaat dehydrogenases, zes hydrogenases en een formiaat waterstof lyase van S. fumaroxidans en vijf formiaat dehydrogenases en drie hydrogenases van M. hungatei allemaal afgeschreven worden in syntroof gegroeide cultures en reincultures. Echter, de hoeveelheid transcripten is afhankelijk van de groeiconditie. Het vergelijken van transcriptie niveaus toonde aan dat elektronen van ferredoxine en NADH tegelijk worden samengebracht (geconfurceerd) om waterstof te vormen met een cytoplasmatisch [FeFe]-hydrogenase. Verder wijzen onze resultaten erop dat tijdens sytrofe groei elektronen van ferredoxine en NADH geconfurceerd worden om formiaat te maken via een cytoplasmatisch formiaat dehydrogenase (FDH1). Tijdens syntrofe groei gaan elektronen in de vorm van FADH2 via een cytoplasmatisch georienteerd succinaat dehydrogenase, menaquinonen, cytochroom b en c naar het periplasmatisch formiaat dehydrogenase (FDH2) (Hoofdstuk 5). Als S. fumaroxidans wordt gegroeid in reincultures met externe elektronacceptoren, zoals sulfaat en fumaraat, gaan de elektronen gedeeltelijk naar FDH2 en gedeeltelijk naar het periplasmatisch hydrogenase (Hyn). De energie die verkregen wordt uit de omzetting van propionaat tot methaan, acetaat en kooldioxide, moet gedeeld worden door S. fumaroxidans en M. hungatei. Als M. hungatei meer energie wegneemt, blijft er minder energie over voor S. fumaroxidans. Dit verhoogt in S. fumaroxidans transcriptie van genen die coderen voor een van de andere cytoplasmatische confurcerende hydrogenases (Hox) en het periplasmatisch hydrogenase (Hyn) dat is gekoppeld aan succinaat oxidatie. Daarnaast induceert S. fumaroxidans genen die coderen voor het Rnf-complex en ferredoxine-afhankelijke hydrogenases en formiaat dehydrogenases. Dit zorgt voor de mogelijkheid om gebruik te maken de membraanpotentiaal die nodig is voor de energieafhankelijke koppeling van ferredoxin reductie en NADH oxidatie. De ontworpen RT qPCR primers zijn gebruikt om te onderzoeken wat het effect was van het niet toevoegen van spore elementen op UASB reactor materiaal van de alcohol distilleerderij NEDALCO in Bergen op Zoom. Een lab-schaal UASB reactor werd gevoed met propionaat en met synthetisch medium zonder toegevoegd W, Mo en Se. Tijdens de reactor run waren Syntrophobacter spp. de dominante propionaatoxideerders en was M.hungatei de dominante waterstof- en formiaat- gebruikende methanogeen. Echter, in de loop van de reactorrun verminderde de propionaatafbraak en begonnen twee andere propionaatoxideerders Pelotomaculum propionicicum en Smithella propionica te groeien. RT qPCR toonde aan dat in deze reactorrun de transcriptie van genen die coderen voor formiaat dehydrogenases en hydrogenases in S. fumaroxidans verminderde terwijl transcriptie van formiaat dehydrogenases en hydrogenases in M.hungatei stabiel bleef (Hoofdstuk 7). Dit onderzoek laat verder zien dat RT qPCR een snelle techniek is die informatie kan geven over de actieve processen in een UASB reactor en dat limitatie van spore elementen en het mogelijk slecht functioneren van UASB reactor kan worden voorspeld. Met dit promotieonderzoek hebben we inzicht gekregen in de moleculaire mechanismen van waterstof- en formiaatoverdracht tussen S. fumaroxidans en M.hungatei in gedefinieerde cocultures en in een UASB reactor. Hierdoor kunnen we vergelijkbare moleculaire mechanismen in andere syntrofe cultures beter begrijpen wat bijvoorbeeld kan bijdragen tot betere anaerobe bioreactors in de toekomst.
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