174 research outputs found
Formate dehydrogenases and hydrogenases in syntrophic propionate-oxidizing communities : gene analysis and transcritional profiling
No full textMany 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. <br/
Growth- and substrate-dependent transcription of formate dehydrogenase and hydrogenase coding genes in Syntrophobacter fumaroxidans and Methanospirillum hungatei
No full textTranscription of genes coding for formate dehydrogenases (fdh genes) and hydrogenases (hyd genes) in Syntrophobacter fumaroxidans and Methanospirillum hungatei was studied following growth under different conditions. Under all conditions tested, all fdh and hyd genes were transcribed. However, transcription levels of the individual genes varied depending on the substrate and growth conditions. Our results strongly suggest that in syntrophically grown S. fumaroxidans cells, the [FeFe]-hydrogenase (encoded by Sfum_844-46), FDH1 (Sfum_2703-06) and Hox (Sfum_2713-16) may confurcate electrons from NADH and ferredoxin to protons and carbon dioxide to produce hydrogen and formate, respectively. Based on bioinformatic analysis, a membrane-integrated energy-converting [NiFe]-hydrogenase (Mhun_1741-46) of M. hungatei might be involved in the energy-dependent reduction of CO2 to formylmethanofuran. The best candidates for F420-dependent N5,N10-methyl-H4 MPT and N5,N10,-methylene-H4MPT reduction are the cytoplasmic [NiFe]-hydrogenase and FDH1. 16S rRNA ratios indicate that in one of the triplicate co-cultures of S. fumaroxidans and M. hungatei, less energy was available for S. fumaroxidans. This led to enhanced transcription of genes coding for the Rnf-complex (Sfum_2694-99) and of several fdh and hyd genes. The Rnf-complex probably reoxidized NADH with ferredoxin reduction, followed by ferredoxin oxidation by the induced formate dehydrogenases and hydrogenase
Energy conservation mechanisms and electron transfer in syntrophic propionate-oxidizing microbial consortia
No full textSyntrophic 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.</p
Ecophysiology of sulfate-reducing bacteria and syntrophic communities in marine anoxic sediments
No full textPropionate, butyrate, acetate, hydrogen and formate are the major intermediates of organic matter degradation. Sulfate-reducing bacteria (SRB) contribute significantly to the consumption of these substrates in sulfate-rich marine sediments. In sulfate-depleted sediments, however, complete degradation of propionate or butyrate is only possible via syntrophic cooperation of acetogenic bacteria and methanogenic archaea. Despite that the predominance of SRB in sulfate-rich and methanogens in sulfate-depleted sediments was reported, recent studies showed that both types of microorganism could be present in upper and lower parts of marine sediments. In this thesis, propionate and butyrate conversions and the involved microbial community in sulfate, sulfate-methane transition and methane zone sediment of Aarhus Bay, Denmark were studied using sediment slurry incubations. Interspecies hydrogen transfer and coexistence during acetate degradation were investigated in mixed pure cultures. In Chapter 2, interspecies hydrogen transfer between aceticlastic Methanosaeta concilii and hydrogenotrophic microorganisms, Desulfovibrio vulgaris or Methanococcus maripaludis, was investigated. Additionally, coexistence of M. concilii and Desulfobacter latus growing on acetate under sulfidogenic conditions was studied. The results of Chapter 2 showed that D. vulgaris could reduce sulfate and grow on leaked hydrogen from M. concilii. Hydrogen leakage from M. concilii provides an explanation for biogeochemical zonation both for competitive (e.g. acetate) and non-competitive substrates (methyl compounds), and this indicates the possible coexistence of SRB and methanogens in sulfate-rich environments. In chapter 3 and 4, long term incubations were examined focusing on butyrate and propionate conversion and the microbial community dynamics in sediment slurry enrichments at different sulfate (o, 3 and 20 mM) concentrations and incubation temperatures (10°C and 25°C). Sulfate reduction is the dominant process for butyrate and propionate conversion in Aarhus Bay sediments. In the absence of sulfate, both substrates can be converted efficiently, indicating the presence of syntrophic communities throughout the sediment. The fluctuating methane concentrations and the enrichment of anaerobic methanotrophic archaea (ANME) during butyrate and propionate conversion at 10°C suggest the occurrence of anaerobic oxidation of methane (AOM) in sulfate-methane transition zone (SMTZ) of Aarhus Bay. The microbial community involved in butyrate and propionate conversions were investigated using next generation sequencing (NGS) of the 16S rRNA amplicon sequencing. The enriched sulfate-reducing bacteria at high sulfate concentration (20 mM) were different when butyrate and propionate were used as substrate. Desulfosarcina and Desulfobacterium dominate the butyrate-converting slurries (Chapter 3), whereas Desulfosarcina, Desulfobulbus and Desulforhopalus are the main SRB in propionate-converting slurries (Chapter 4). The increase in the relative abundance of Desulfobacteraceae and Desulfobulbaceae in SZ, SMTZ and MZ sediment slurries suggests the presence of sulfate reducers throughout the anoxic sediment column. In the absence of sulfate, Syntrophomonas and Cyrptanaerobacter become dominant which suggests their role in syntrophic butyrate and propionate conversion, respectively. These results were further supported in Chapter 6. The increase in the relative abundance of Syntrophomonas in the presence of sulfate (Chapter 3) and some members of Desulfobacteraceae (Chapter 4) in the absence of sulfate shows the metabolic flexibility of the microorganisms at different sulfate concentrations. Temperature has an impact on the microbial community (Chapter 4) and IPL composition (Chapter 5) in enrichment slurries. Cryptanaerobacter is dominant at 25°C, and, Desulfobacteraceae (Desulfofaba), especially Desulfobulbaceae members (Desulfobulbus, Desulforhopalus) become dominant at 10°C at 0 and 3 mM sulfate concentrations in propionate-amended enrichment slurries. In butyrate-amended slurries, Clostridiales have higher relative abundance at 10°C regardless of the sulfate concentration and the sediment depth which supports important role of Clostridiales in butyrate conversion in marine sediments. Archaeal community analyses revealed the dominance of hydrogenotrophic methanogens belonging to Methanomicrobiales in both butyrate- and propionate-converting slurries (Chapter 3 and 4) and enrichment cultures (Chapter 6) regardless of the sediment depth, the incubation temperature and the presence of sulfate, which indicate that they are the main syntrophic partners of butyrate and propionate degraders. The other syntrophic partner organisms are the aceticlastic methanogenic families: Methanosarcinaceae and Methanosaetaeceae. The presence of methane-oxidizing archaea (ANME-1b) in low temperature SMTZ slurries together with Desulfobacteraceae (Chapter 3 and 4) suggests the occurrence of anaerobic oxidation of methane (AOM) in SMTZ of Aarhus Bay. In conclusion, this thesis confirms the presence and activity of methanogens in sulfate-rich, and SRB in sulfate-depleted marine sediments; and their involvement in butyrate, propionate and acetate conversion. Novel bacterial and archaeal members enriched in the sediment slurries are likely involved in propionate, butyrate and acetate conversions at different depths of marine sediments in addition to known the cultured species
Microbiology of thermophilic anaerobic syngas conversion
No full textTese de doutoramento em Engenharia Química e BiológicaBiological syngas fermentation can be used for novel biotechnological applications. Syngas can be produced
from biomass and recalcitrant wastes; fermentation of this gas is therefore an environmentally
friendly process with foreseen applications in the production of biofuels and chemicals. This thesis reports
the use of thermophilic anaerobic mixed cultures for syngas and carbon monoxide (CO) conversion,
as well as the isolation and characterization of two new thermophilic bacterial strains - one CO-utilizing
bacterium and one CO-tolerant bacterium.
Stable thermophilic enrichments converting syngas or CO at 55 oC were obtained by long term exposure
of a thermophilic anaerobic suspended sludge to these substrates. Enrichments were successively
transferred (for over a year) with syngas and pure CO, as sole carbon and energy source. CO partial
pressure was increased from 0:09 to 0:88 bar during the enrichment procedure. Enrichment cultures
initiated with syngas produced mainly acetate, while hydrogen was the main product detected in enrichment
cultures initially incubated with CO. Bacteria branching within the families Peptococcaceae
and Thermoanaerobacteraceae were present in syngas and CO enrichment cultures. Syngas enrichment
cultures were composed of two predominant species related to Desulfotomaculum and Caloribacterium
genera, while bacteria assigned to Thermincola and Thermoanaerobacter genera were abundant in CO
enrichment cultures.
The e ect of sulfate on CO conversion by the obtained syngas-degrading enriched culture was also
investigated. Although the enrichment culture could convert CO into mainly acetate both in the presence
and in the absence of sulfate, CO conversion was faster when sulfate was present. Identi cation of the
predominant microorganisms revealed the presence of Desulfotomaculum-like bacteria.
A novel CO-tolerating bacterium, Thermoanaerobacter carboxyditolerans, was isolated from the syngasdegrading
enriched culture. This bacterium is closest related to Thermoanaerobacter thermohydrosulfu-
ricus (97% identity based on 16S rRNA gene sequence). Although T. carboxyditolerans does not utilize
CO, it is able to grow in the presence of high CO concentrations (pCO = 1:7 bar). Other Thermoanaer-
obacter species showed tolerance to CO, namely T. thermohydrosulfuricus, T. brockii subsp. nnii, T.
pseudethanolicus and T. wiegelii ; growth of these bacteria was not substantially a ected by CO concentrations
ranging from 25% to 100% (pCO from 0:425 to 1:7 bar). Nevertheless, hydrogen production by
those species decreased with increasing CO partial pressure.
A new thermophilic hydrogenogenic carboxydotrophic bacterium, Moorella stamsii, could be isolated
from the CO-degrading enrichment. This bacterium is able to utilize CO coupled to the production of
hydrogen. M. stamsii is phylogenetically related to Moorella glycerini (97% identity based on 16S rRNA
gene sequences).
With this PhD research we gained insight in the ecophysiology of thermophilic anaerobic conversion of
syngas and CO. Results presented in this thesis are important for prospecting biotechnological processes
for the production of added-values compounds, such as hydrogen and/or acetate.A fermentação biológica de gás de síntese pode ser usada como um novo processo biotecnológico para produção de biocombustíveis. O gás de síntese pode ser produzido a partir de biomassa florestal ou de resíduos recalcitrantes; a fermentação biológica deste gás é um processo ambiental e economicamente sustentável, com potenciais aplicações na produção de biocombustíveis e outros compostos químicos de elevado interesse. Nesta tese são descritos estudos que utilizam culturas mistas, em condições termofílicas, com o objetivo de estudar a conversão de gás de síntese e de monóxido de carbono (CO) por essas culturas. Descreve-se também o isolamento e caracterização fisiológica de duas novas bactérias; uma das bactérias isoladas è capaz de utilizar o CO e a outra é tolerante a elevadas concentrações de CO.
Culturas estáveis e enriquecidas em gás de síntese ou CO, em condições termofílicas 55º C foram conseguidas
após longa exposição da biomassa suspensa anaeróbia (usada como inóculo) a estes substratos. As culturas
foram sucessivamente transferidas para meio novo (durante mais de um ano), com gás de síntese ou CO como
única fonte de carbono e energia. Durante o processo de enriquecimento das culturas, a pressão parcial de
CO foi aumentando, começou por 0:09 bar até atingir 0:88 bar. As culturas iniciadas com gás de síntese como
substrato, produziram maioritariamente acetato, enquanto que as culturas que foram inicialmente incubadas com
CO produziram maioritariamente hidrogénio. Verificou-se que a comunidade bacteriana dominante presente nas
culturas enriquecidas em gás de síntese e CO pertence às famílias Peptococcaceae and Thermoanaerobacteraceae.
Os enriquecimentos em gás de síntese apresentaram duas espécies dominantes, filogeneticamente relacionadas com os genéros Desulfotomaculum e Caloribacterium, enquanto que bactérias pertencentes aos géneros Thermincola e Thermoanaerobacter estavam abundantemente presentes nos enriquecimentos em CO.
O efeito do sulfato na conversão de CO pela cultura enriquecida em gás de síntese anteriormente obtida foi
também objeto de estudo. Embora a cultura enriquecida consiga converter CO em acetato nas duas condições
testadas (presença/ausência de sulfato), a utilização de CO foi mais rápida na presença de sulfato. A identificação
dos microrganismos predominantes revelou a presença de organismos filogeneticamente próximos do género Desulfotomaculum.
A partir da cultura enriquecida em gás de síntese foi isolada uma nova bactéria: Thermoanaerobacter carboxy-
ditolerans. Esta bactéria está filogeneticamente relacionada com Thermoanaerobacter thermohydrosulfuricus (97% similaridade, com base na sequência do gene 16S). T. carboxyditolerans não utiliza CO, mas tem a capacidade de crescer na presença de altas concentrações de CO (pCO = 1:7 bar). Outras espécies do género Thermoanaerobacter (T. thermohydrosulfuricus, T. brockii subsp. nnii, T. pseudethanolicus e T. wiegelii) apresentaram também tolerância ao CO. O crescimento destas bactérias não foi significativamente afetado pela presença de CO em concentrações que variavam entre 25% a 100% de CO (pCO entre 0:425 e 1:7 bar). No entanto, verificou-se a diminuição da produção de hidrogénio, com o aumento da pressão parcial de CO no meio.
Uma nova bactéria termofílica, hidrogenogénica, carboxidotrófica, Moorella stamsii, foi isolada a partir da
cultura enriquecida em CO. M. stamsii consegue converter o CO em hidrogénio. E filogenticamente próxima de
Moorella glycerini (97% similaridade, com base na sequência do gene 16S).
Com os trabalhos de investigação decorrentes deste doutoramento, aumentamos os nossos conhecimentos
relativamente à ecologia, fisiologia e microbiologia da conversão anaeróbia do gás de síntese e do monóxido de
carbono, em condições termofílicas. Os resultados obtidos neste trabalho foram importantes, numa perspetiva
futura de aplicar processos biotecnológicos para a produção de compostos com interesse, como o hidrogénio ou o
acetato
Anoxic media design, preparation and considerations
No full textExclusion of oxygen from growth media is essential for the growth of anoxic prokaryotes. In general, anaerobic techniques focus on the use of deaerated boiled growth media. Successful enrichment, isolation, and cultivation of anoxic prokaryotes critically depend on the choice of appropriate growth media and incubation conditions. This chapter discusses the requirements of anoxic prokaryotes for growth in the laboratory and different existing methods for their cultivation
Formate Formation and Formate Conversion in Biological Fuels Production
No full textBiomethanation is a mature technology for fuel production. Fourth generation biofuels research will focus on sequestering CO2 and providing carbon-neutral or carbon-negative strategies to cope with dwindling fossil fuel supplies and environmental impact. Formate is an important intermediate in the methanogenic breakdown of complex organic material and serves as an important precursor for biological fuels production in the form of methane, hydrogen, and potentially methanol. Formate is produced by either CoA-dependent cleavage of pyruvate or enzymatic reduction of CO2 in an NADH- or ferredoxin-dependent manner. Formate is consumed through oxidation to CO2 and H2 or can be further reduced via the Wood-Ljungdahl pathway for carbon fixation or industrially for the production of methanol. Here, we review the enzymes involved in the interconversion of formate and discuss potential applications for biofuels productio
Biogas
No full textBiogas production represents a fascinating process for the recovery of nutrients and renewable energy from various organic waste streams. The process is of interest for the production of value-added chemicals by mixed cultures and can also be applied in combined bioenergy production systems. Strategies and opportunities for optimization of biogas quality and quantity are presented
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