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1.
Int J Mol Sci ; 23(2)2022 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-35055147

RESUMO

Strategies for depleting carbon dioxide (CO2) from flue gases are urgently needed and carbonic anhydrases (CAs) can contribute to solving this problem. They catalyze the hydration of CO2 in aqueous solutions and therefore capture the CO2. However, the harsh conditions due to varying process temperatures are limiting factors for the application of enzymes. The current study aims to examine four recombinantly produced CAs from different organisms, namely CAs from Acetobacterium woodii (AwCA or CynT), Persephonella marina (PmCA), Methanobacterium thermoautotrophicum (MtaCA or Cab) and Sulphurihydrogenibium yellowstonense (SspCA). The highest expression yields and activities were found for AwCA (1814 WAU mg-1 AwCA) and PmCA (1748 WAU mg-1 PmCA). AwCA was highly stable in a mesophilic temperature range, whereas PmCA proved to be exceptionally thermostable. Our results indicate the potential to utilize CAs from anaerobic microorganisms to develop CO2 sequestration applications.


Assuntos
Acetobacterium/enzimologia , Bactérias/enzimologia , Dióxido de Carbono/metabolismo , Anidrases Carbônicas/genética , Acetobacterium/genética , Anaerobiose , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Anidrases Carbônicas/química , Anidrases Carbônicas/metabolismo , Estabilidade Enzimática , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Temperatura
2.
Biotechnol J ; 17(5): e2100515, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35077002

RESUMO

The capability of four genetically modified Acetobacterium woodii strains for improved production of acetone from CO2 and hydrogen was tested. The acetone biosynthesis pathway was constructed by combining genes from Clostridium acetobutylicum and Clostridium aceticum. Expression of acetone production genes was demonstrated in all strains. In bioreactors with continuous gas supply, all produced acetic acid, acetone, and, surprisingly, isopropanol. The production of isopropanol was caused by an endogenous secondary alcohol dehydrogenase (SADH) activity at low gas-feeding rate. Although high amounts of the natural end product acetic acid of A. woodii were formed,14.5 mM isopropanol and 7.6 mM acetone were also detected, showing that this is a promising approach for the production of new solvents from C1 gases. The highest acetic acid, acetone, and isopropanol production was detected in the recombinant A. woodii [pJIR750_ac1t1] strain, with final concentrations of 438 mM acetic acid, 7.6 mM acetone, and 14.5 mM isopropanol. The engineered strain A. woodii [pJIR750_ac1t1] was found to be the most promising strain for acetone production from a gas mixture of CO2 and H2 and the formation of isopropanol in A. woodii was shown for the first time.


Assuntos
Dióxido de Carbono , Clostridium acetobutylicum , 2-Propanol , Ácido Acético , Acetobacterium , Acetona , Dióxido de Carbono/metabolismo , Clostridium acetobutylicum/metabolismo , Hidrogênio/metabolismo
3.
Appl Microbiol Biotechnol ; 106(4): 1447-1458, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35092454

RESUMO

Lactate has various uses as industrial platform chemical, poly-lactic acid precursor or feedstock for anaerobic co-cultivations. The aim of this study was to construct and characterise Acetobacterium woodii strains capable of autotrophic lactate production. Therefore, the lctBCD genes, encoding the native Lct dehydrogenase complex, responsible for lactate consumption, were knocked out. Subsequently, a gene encoding a D-lactate dehydrogenase (LDHD) originating from Leuconostoc mesenteroides was expressed in A. woodii, either under the control of the anhydrotetracycline-inducible promoter Ptet or under the lactose-inducible promoter PbgaL. Moreover, LDHD was N-terminally fused to the oxygen-independent fluorescence-activating and absorption-shifting tag (FAST) and expressed in respective A. woodii strains. Cells that produced the LDHD fusion protein were capable of lactate production of up to 18.8 mM in autotrophic batch experiments using H2 + CO2 as energy and carbon source. Furthermore, cells showed a clear and bright fluorescence during exponential growth, as well as in the stationary phase after induction, mediated by the N-terminal FAST. Flow cytometry at the single-cell level revealed phenotypic heterogeneities for cells expressing the FAST-tagged LDHD fusion protein. This study shows that FAST provides a new reporter tool to quickly analyze gene expression over the course of growth experiments of A. woodii. Consequently, fluorescence-based reporters allow for faster and more targeted optimization of production strains.Key points •Autotrophic lactate production was achieved with A. woodii. •FAST functions as fluorescent marker protein in A. woodii. •Fluorescence measurements on single-cell level revealed population heterogeneity.


Assuntos
Dióxido de Carbono , Ácido Láctico , Acetatos/metabolismo , Acetobacterium , Dióxido de Carbono/metabolismo , Fluorescência
4.
Appl Microbiol Biotechnol ; 105(23): 8989-9002, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34716461

RESUMO

One of the bottlenecks of the hydrogen production by dark fermentation is the low yields obtained because of the homoacetogenesis persistence, a metabolic pathway where H2 and CO2 are consumed to produce acetate. The central reactions of H2 production and homoacetogenesis are catalyzed by enzyme hydrogenase and the formyltetrahydrofolate synthetase, respectively. In this work, genes encoding for the formyltetrahydrofolate synthetase (fthfs) and hydrogenase (hydA) were used to investigate the diversity of homoacetogens as well as their phylogenetic relationships through quantitative PCR (qPCR) and next-generation amplicon sequencing. A total of 70 samples from 19 different H2-producing bioreactors with different configurations and operating conditions were analyzed. Quantification through qPCR showed that the abundance of fthfs and hydA was strongly associated with the type of substrate, organic loading rate, and H2 production performance. In particular, fthfs sequencing revealed that homoacetogens diversity was low with one or two dominant homoacetogens in each sample. Clostridium carboxivorans was detected in the reactors fed with agave hydrolisates; Acetobacterium woodii dominated in systems fed with glucose; Blautia coccoides and unclassified Sporoanaerobacter species were present in reactors fed with cheese whey; finally, Eubacterium limosum and Selenomonas sp. were co-dominant in reactors fed with glycerol. Altogether, quantification and sequencing analysis revealed that the occurrence of homoacetogenesis could take place due to (1) metabolic changes of H2-producing bacteria towards homoacetogenesis or (2) the displacement of H2-producing bacteria by homoacetogens. Overall, it was demonstrated that the fthfs gene was a suitable marker to investigate homoacetogens in H2-producing reactors. KEY POINTS: • qPCR and sequencing analysis revealed two homoacetogenesis phenomena. • fthfs gene was a suitable marker to investigate homoacetogens in H2 reactors.


Assuntos
Hidrogênio , Acetobacterium , Clostridiales , Eubacterium , Filogenia
5.
Metab Eng ; 68: 68-85, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34537366

RESUMO

Cheap and renewable feedstocks such as the one-carbon substrate formate are emerging for sustainable production in a growing chemical industry. We investigated the acetogen Acetobacterium woodii as a potential host for bioproduction from formate alone and together with autotrophic and heterotrophic co-substrates by quantitatively analyzing physiology, transcriptome, and proteome in chemostat cultivations in combination with computational analyses. Continuous cultivations with a specific growth rate of 0.05 h-1 on formate showed high specific substrate uptake rates (47 mmol g-1 h-1). Co-utilization of formate with H2, CO, CO2 or fructose was achieved without catabolite repression and with acetate as the sole metabolic product. A transcriptomic comparison of all growth conditions revealed a distinct adaptation of A. woodii to growth on formate as 570 genes were changed in their transcript level. Transcriptome and proteome showed higher expression of the Wood-Ljungdahl pathway during growth on formate and gaseous substrates, underlining its function during utilization of one-carbon substrates. Flux balance analysis showed varying flux levels for the WLP (0.7-16.4 mmol g-1 h-1) and major differences in redox and energy metabolism. Growth on formate, H2/CO2, and formate + H2/CO2 resulted in low energy availability (0.20-0.22 ATP/acetate) which was increased during co-utilization with CO or fructose (0.31 ATP/acetate for formate + H2/CO/CO2, 0.75 ATP/acetate for formate + fructose). Unitrophic and mixotrophic conversion of all substrates was further characterized by high energetic efficiencies. In silico analysis of bioproduction of ethanol and lactate from formate and autotrophic and heterotrophic co-substrates showed promising energetic efficiencies (70-92%). Collectively, our findings reveal A. woodii as a promising host for flexible and simultaneous bioconversion of multiple substrates, underline the potential of substrate co-utilization to improve the energy availability of acetogens and encourage metabolic engineering of acetogenic bacteria for the efficient synthesis of bulk chemicals and fuels from sustainable one carbon substrates.


Assuntos
Acetobacterium , Acetatos , Acetobacterium/genética , Fermentação , Formiatos
6.
Environ Microbiol ; 23(11): 6953-6964, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34448343

RESUMO

The acetogenic model bacterium Acetobacterium woodii is well-known to produce acetate by homoacetogenesis from sugars, but under certain conditions minor amounts of ethanol are produced in addition. Here, we have aimed to identify physiological conditions that increase electron and carbon flow towards ethanol production. Ethanol was only produced from fructose but not from H2  + CO2 , formate, pyruvate, lactate or alanine. In the absence of Na+ , the Wood-Ljungdahl pathway (WLP) of acetate formation is not functional. Therefore, the ethanol yield increased to 0.42 mol/mol (ethanol/fructose) with an ethanol/acetate ratio of 0.28 mol/mol. The presence of bicarbonate/CO2 stimulated electron and carbon flow through the WLP and led to less ethanol produced. Of the 11 potential alcohol dehydrogenase genes, the most upregulated during ethanologenesis was adh4. A deletion of adh4 led to an increase in ethanol production by 100% to a yield of 0.79 mol/mol (ethanol/fructose); this correlated with an increase in transcript abundance of adh6. In sum, our studies revealed low Na+ and bicarbonate/CO2 as factors that trigger ethanol formation and that a deletion of adh4 drastically increased ethanol formation in A. woodii.


Assuntos
Acetobacterium , Acetatos/metabolismo , Acetobacterium/genética , Acetobacterium/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Dióxido de Carbono/metabolismo , Etanol/metabolismo
7.
Appl Microbiol Biotechnol ; 105(14-15): 5861-5872, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34331557

RESUMO

In times of global climate change and the fear of dwindling resources, we are facing different considerable challenges such as the replacement of fossil fuel-based energy carriers with the coincident maintenance of the increasing energy supply of our growing world population. Therefore, CO2 capturing and H2 storing solutions are urgently needed. In this study, we demonstrate the production of a functional and biotechnological interesting enzyme complex from acetogenic bacteria, the hydrogen-dependent CO2 reductase (HDCR), in the well-known model organism Escherichia coli. We identified the metabolic bottlenecks of the host organisms for the production of the HDCR enzyme complex. Here we show that the recombinant expression of a heterologous enzyme complex transforms E. coli into a whole-cell biocatalyst for hydrogen-driven CO2 reduction to formate without the need of any external co-factors or endogenous enzymes in the reaction process. This shifts the industrial platform organism E. coli more and more into the focus as biocatalyst for CO2-capturing and H2-storage. KEY POINTS: • A functional HDCR enzyme complex was heterologously produced in E. coli. • The metabolic bottlenecks for HDCR production were identified. • HDCR enabled E. coli cell to capture and store H2 and CO2 in the form of formate.


Assuntos
Acetobacterium , Hidrogênio , Dióxido de Carbono , Deutério , Escherichia coli/genética , Formiatos
8.
Appl Environ Microbiol ; 87(14): e0283920, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33990298

RESUMO

Gas fermentation is a promising way to convert CO-rich gases to chemicals. We studied the use of synthetic cocultures composed of carboxydotrophic and propionigenic bacteria to convert CO to propionate. So far, isolated carboxydotrophs cannot directly ferment CO to propionate, and therefore, this cocultivation approach was investigated. Four distinct synthetic cocultures were constructed, consisting of Acetobacterium wieringae (DSM 1911T) and Pelobacter propionicus (DSM 2379T), Ac. wieringae (DSM 1911T) and Anaerotignum neopropionicum (DSM 3847T), Ac. wieringae strain JM and P. propionicus (DSM 2379T), and Ac. wieringae strain JM and An. neopropionicum (DSM 3847T). Propionate was produced by all the cocultures, with the highest titer (∼24 mM) being measured in the coculture composed of Ac. wieringae strain JM and An. neopropionicum, which also produced isovalerate (∼4 mM), butyrate (∼1 mM), and isobutyrate (0.3 mM). This coculture was further studied using proteogenomics. As expected, enzymes involved in the Wood-Ljungdahl pathway in Ac. wieringae strain JM, which are responsible for the conversion of CO to ethanol and acetate, were detected; the proteome of An. neopropionicum confirmed the conversion of ethanol to propionate via the acrylate pathway. In addition, proteins related to amino acid metabolism and stress response were highly abundant during cocultivation, which raises the hypothesis that amino acids are exchanged by the two microorganisms, accompanied by isovalerate and isobutyrate production. This highlights the importance of explicitly looking at fortuitous microbial interactions during cocultivation to fully understand coculture behavior. IMPORTANCE Syngas fermentation has great potential for the sustainable production of chemicals from wastes (via prior gasification) and flue gases containing CO/CO2. Research efforts need to be directed toward expanding the product portfolio of gas fermentation, which is currently limited to mainly acetate and ethanol. This study provides the basis for a microbial process to produce propionate from CO using synthetic cocultures composed of acetogenic and propionigenic bacteria and elucidates the metabolic pathways involved. Furthermore, based on proteomics results, we hypothesize that the two bacterial species engage in an interaction that results in amino acid exchange, which subsequently promotes isovalerate and isobutyrate production. These findings provide a new understanding of gas fermentation and a coculturing strategy for expanding the product spectrum of microbial conversion of CO/CO2.


Assuntos
Acetobacterium/metabolismo , Monóxido de Carbono/metabolismo , Deltaproteobacteria/metabolismo , Propionatos/metabolismo , Acetobacterium/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Cocultura , Deltaproteobacteria/efeitos dos fármacos , Fermentação , Proteoma/metabolismo , Acetato de Sódio/farmacologia
9.
Environ Microbiol ; 23(8): 4214-4227, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33989450

RESUMO

Acetogenic bacteria are already established as biocatalysts for production of high-value compounds from C1 substrates such as H2  + CO2 or CO. However, little is known about the physiology, biochemistry and bioenergetics of acetogenesis from formate, an interesting feedstock for biorefineries. Here, we analysed formate metabolism in the model acetogen Acetobacterium woodii. Cells grew optimally on 200 mM formate to an optical density of 0.6. Formate was exclusively converted to acetate (and CO2 ) with a ratio of 4.4:1. Transcriptome analyses revealed genes/enzymes involved in formate metabolism. Strikingly, A. woodii has two genes potentially encoding a formyl-THF synthetase, fhs1 and fhs2. fhs2 forms an operon with a gene encoding a potential formate transporter, fdhC. Deletion of fhs2/fdhC led to a reduced growth rate, formate consumption and optical densities. Acetogenesis from H2  + CO2 was accompanied by transient formate production; strikingly, formate reutilization was completely abolished in the Δfhs2/fdhC mutant. Take together, our studies gave the first detailed insights into the formatotrophic lifestyle of A. woodii.


Assuntos
Acetobacterium , Acetobacterium/genética , Metabolismo Energético , Formiatos , Óperon
10.
Metab Eng ; 66: 296-307, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33894339

RESUMO

Gas fermentation is a technology for producing platform chemicals as well as fuels and one of the most promising alternatives to petrochemicals. Medium-chained acids and alcohols such as hexanoate and hexanol are particularly interesting due to their versatile application. This study elucidated the pathway of chain elongation in native C6 compound-producing acetogens. Essential genes of Clostridium carboxidivorans for synthesis of medium-chained acids and alcohols were identified in order to demonstrate their catalytic activity in the acetogenic model organism Acetobacterium woodii. Two such gene clusters were identified, which are responsible for conversion of acetyl-CoA to butyryl-CoA by reverse ß-oxidation. Using RT-PCR it could be demonstrated that only genes of cluster 1 are expressed constitutively with simultaneous formation of C6 compounds. Based on genes from C. carboxidivorans, a modular hexanoyl-CoA synthesis (hcs) plasmid system was constructed and transferred into A. woodii. With the recombinant A. woodii strains AWO [pPta_hcs1], AWO [pPta_hcs2], AWO [pTet_hcs1], and AWO [pTet_hcs2] butyrate and hexanoate production under heterotrophic (1.22-4.15 mM hexanoate) and autotrophic conditions (0.48-1.56 mM hexanoate) with both hcs clusters could be detected. hcs Cluster 1 from C. carboxidivorans was transferred into the ABE-fermenting strain Clostridium saccharoperbutylacetonicum as well. For further analysis, genes were also cloned into the hcs plasmid system individually. The resulting recombinant C. saccharoperbutylacetonicum strains with just individual genes neither produced hexanoate nor hexanol, but the strains containing the entire gene cluster were capable of chain elongation. A production of 0.8 mM hexanoate and 5.2 mM hexanol in the fermentation with glucose could be observed.


Assuntos
Álcoois , Clostridium , Acetobacterium , Clostridium/genética
11.
Environ Microbiol ; 23(5): 2648-2658, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33817956

RESUMO

More than 2 million tons of glycerol are produced during industrial processes each year and, therefore, glycerol is an inexpensive feedstock to produce biocommodities by bacterial fermentation. Acetogenic bacteria are interesting production platforms and there have been few reports in the literature on glycerol utilization by this ecophysiologically important group of strictly anaerobic bacteria. Here, we show that the model acetogen Acetobacterium woodii DSM1030 is able to grow on glycerol, but contrary to expectations, only for 2-3 transfers. Transcriptome analysis revealed the expression of the pdu operon encoding a propanediol dehydratase along with genes encoding bacterial microcompartments. Deletion of pduAB led to a stable growth of A. woodii on glycerol, consistent with the hypothesis that the propanediol dehydratase also acts on glycerol leading to a toxic end-product. Glycerol is oxidized to acetate and the reducing equivalents are reoxidized by reducing CO2 in the Wood-Ljungdahl pathway, leading to an additional acetate. The possible oxidation product of glycerol, dihydroxyacetone (DHA), also served as carbon and energy source for A. woodii and growth was stably maintained on that compound. DHA oxidation was also coupled to CO2 reduction. Based on transcriptome data and enzymatic analysis we present the first metabolic and bioenergetic schemes for glycerol and DHA utilization in A. woodii.


Assuntos
Acetobacterium , Di-Hidroxiacetona , Acetobacterium/genética , Glicerol , Oxirredução
12.
Bioresour Technol ; 319: 124177, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33035863

RESUMO

Microbial electrosynthesis (MES) for CO2 valorization could be influenced by fluctuations in CO2 mass transfer and flow rates. In this study, we developed an efficient method for CO2 delivery to cathodic biofilm by directly sparging CO2 through the pores of ceramic hollow fiber wrapped with Ni-foam/carbon nanotube electrode, and obtained 45% and 77% higher acetate and methane production, respectively. This was followed by the MES stability test in response to fluctuations in CO2 flow rates varying from 0.3 ml/min to 10 ml/min. The biochemical production exhibited an increasing trend with CO2 flow rates, achieving higher acetate (47.0 ± 18.4 mmol/m2/day) and methane (240.0 ± 32.2 mmol/m2/day) generation at 10 ml/min with over 90% coulombic efficiency. The biofilm and suspended biomass, however, showed high resistance to CO2 flow fluctuations with Methanobacterium and Acetobacterium accounting for 80% of the total microbial community, which suggests the robustness of MES for onsite carbon conversion.


Assuntos
Acetobacterium , Dióxido de Carbono , Eletrodos , Metano , Methanobacterium
13.
Bioresour Technol ; 320(Pt A): 124289, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33129088

RESUMO

The present study aimed to demonstrate the utilization of unpurified industrial CO2 with low impurities for acetate production via microbial electrosynthesis (MES) for the first time. In MES experiments with CO2-rich brewery gas, the enriched mixed culture dominated by Acetobacterium produced 1.8 ± 0.2 g/L acetic acid at 0.26 ± 0.03 g/Lcatholyte/d rate and outperformed a pure culture of Clostridium ljungdahlii (1.1 ± 0.02 g/L; 0.138 ± 0.004 g/Lcatholyte/d). The electron recovery in acetic acid was also more for mixed culture (84 ± 13%) than C. ljungdahlii (42 ± 14%). Electrochemical analysis of biocathodes suggested the role of microbial biofilm in improved hydrogen electrocatalysis. In comparative gas fermentation tests, the mixed culture outperformed C. ljungdahlii and produced acetic acid at a similar level with both industrial and pure CO2 feedstocks. These results suggest the robustness and capability of the mixed microbial community for utilizing slightly impure industrial CO2 for bioproduction and presents a major advancement in MES technology.


Assuntos
Acetobacterium , Dióxido de Carbono , Clostridium , Eletrodos , Hidrogênio
14.
Environ Microbiol ; 23(1): 499-511, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33283462

RESUMO

Acetobacterium woodii utilizes the Wood-Ljungdahl pathway for reductive synthesis of acetate from carbon dioxide. However, A. woodii can also perform non-acetogenic growth on 1,2-propanediol (1,2-PD) where instead of acetate, equal amounts of propionate and propanol are produced as metabolic end products. Metabolism of 1,2-PD occurs via encapsulated metabolic enzymes within large proteinaceous bodies called bacterial microcompartments. While the genome of A. woodii harbours 11 genes encoding putative alcohol dehydrogenases, the BMC-encapsulated propanol-generating alcohol dehydrogenase remains unidentified. Here, we show that Adh4 of A. woodii is the alcohol dehydrogenase required for propanol/ethanol formation within these microcompartments. It catalyses the NADH-dependent reduction of propionaldehyde or acetaldehyde to propanol or ethanol and primarily functions to recycle NADH within the BMC. Removal of adh4 gene from the A. woodii genome resulted in slow growth on 1,2-PD and the mutant displayed reduced propanol and enhanced propionate formation as a metabolic end product. In sum, the data suggest that Adh4 is responsible for propanol formation within the BMC and is involved in redox balancing in the acetogen, A. woodii.


Assuntos
Acetatos/metabolismo , Acetobacterium/enzimologia , Álcool Desidrogenase/metabolismo , Proteínas de Bactérias/metabolismo , 1-Propanol/metabolismo , Acetaldeído/metabolismo , Acetobacterium/genética , Acetobacterium/crescimento & desenvolvimento , Álcool Desidrogenase/genética , Aldeídos/metabolismo , Proteínas de Bactérias/genética , Dióxido de Carbono/metabolismo , Etanol/metabolismo , Genoma Bacteriano , NAD/metabolismo , Oxirredução
15.
Bioresour Technol ; 323: 124573, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33360948

RESUMO

In this study, the impact of gas composition (i.e. CO, CO2 and H2 partial pressures) on CO2 utilization, growth, and acetate production was investigated in batch and continuous cultures of A. woodii. Based on an industrial blast furnace gas, H2 blending was used to study the impact of H2 availability on CO2 fixation alone and together with CO using idealized gas streams. With H2 available as an additional energy source, net CO2 fixation and CO, CO2 and H2 co-utilization was achieved in gas-limited fermentations. Using industrial blast furnace gas, up to 15.1 g l-1 acetate were produced in continuous cultures. Flux balance analysis showed that intracellular fluxes and total ATP production were dependent on the availability of H2 and CO. Overall, H2 blending was shown to be a suitable control strategy for gas fermentations and demonstrated that A. woodii is an interesting host for CO2 fixation from industrial gas streams.


Assuntos
Acetobacterium , Dióxido de Carbono , Fermentação , Hidrogênio
16.
NPJ Biofilms Microbiomes ; 6(1): 40, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-33056998

RESUMO

Cathode-driven applications of bio-electrochemical systems (BESs) have the potential to transform CO2 into value-added chemicals using microorganisms. However, their commercialisation is limited as biocathodes in BESs are characterised by slow start-up and low efficiency. Understanding biosynthesis pathways, electron transfer mechanisms and the effect of operational variables on microbial electrosynthesis (MES) is of fundamental importance to advance these applications of a system that has the capacity to convert CO2 to organics and is potentially sustainable. In this work, we demonstrate that cathodic potential and inorganic carbon source are keys for the development of a dense and conductive biofilm that ensures high efficiency in the overall system. Applying the cathodic potential of -1.0 V vs. Ag/AgCl and providing only gaseous CO2 in our system, a dense biofilm dominated by Acetobacterium (ca. 50% of biofilm) was formed. The superior biofilm density was significantly correlated with a higher production yield of organic chemicals, particularly acetate. Together, a significant decrease in the H2 evolution overpotential (by 200 mV) and abundant nifH genes within the biofilm were observed. This can only be mechanistically explained if intracellular hydrogen production with direct electron uptake from the cathode via nitrogenase within bacterial cells is occurring in addition to the commonly observed extracellular H2 production. Indeed, the enzymatic activity within the biofilm accelerated the electron transfer. This was evidenced by an increase in the coulombic efficiency (ca. 69%) and a 10-fold decrease in the charge transfer resistance. This is the first report of such a significant decrease in the charge resistance via the development of a highly conductive biofilm during MES. The results highlight the fundamental importance of maintaining a highly active autotrophic Acetobacterium population through feeding CO2 in gaseous form, which its dominance in the biocathode leads to a higher efficiency of the system.


Assuntos
Acetobacterium/fisiologia , Fontes de Energia Bioelétrica/microbiologia , Biofilmes/crescimento & desenvolvimento , Dióxido de Carbono/química , Acetatos/química , Acetobacterium/genética , Proteínas de Bactérias/genética , Condutividade Elétrica , Eletrodos , Oxirredutases/genética , Compostos de Prata/química
17.
Microb Biotechnol ; 13(6): 2044-2056, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32959527

RESUMO

Acetogenic bacteria have gained much attraction in recent years as they can produce different biofuels and biochemicals from H2 plus CO2 or even CO alone, therefore opening a promising alternative route for the production of biofuels from renewable sources compared to existing sugar-based routes. However, CO metabolism still raises questions concerning the biochemistry and bioenergetics in many acetogens. In this study, we focused on the two acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui which, so far, are the only identified acetogens harbouring a H2 -dependent CO2 reductase and furthermore belong to different classes of 'Rnf'- and 'Ech-acetogens'. Both strains catalysed the conversion of CO into the bulk chemical acetate and formate. Formate production was stimulated by uncoupling the energy metabolism from the Wood-Ljungdahl pathway, and specific rates of 1.44 and 1.34 mmol g-1  h-1 for A. woodii ∆rnf and T. kivui wild type were reached. The demonstrated CO-based formate production rates are, to the best of our knowledge, among the highest rates ever reported. Using mutants of ∆hdcr, ∆cooS, ∆hydBA, ∆rnf and ∆ech2 with deficiencies in key enzyme activities of the central metabolism enabled us to postulate two different CO utilization pathways in these two model organisms.


Assuntos
Acetobacterium , Monóxido de Carbono , Acetobacterium/genética , Formiatos , Thermoanaerobacter
18.
Sci Rep ; 10(1): 14872, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913242

RESUMO

Flavin-based electron bifurcation is a long hidden mechanism of energetic coupling present mainly in anaerobic bacteria and archaea that suffer from energy limitations in their environment. Electron bifurcation saves precious cellular ATP and enables lithotrophic life of acetate-forming (acetogenic) bacteria that grow on H2 + CO2 by the only pathway that combines CO2 fixation with ATP synthesis, the Wood-Ljungdahl pathway. The energy barrier for the endergonic reduction of NADP+, an electron carrier in the Wood-Ljungdahl pathway, with NADH as reductant is overcome by an electron-bifurcating, ferredoxin-dependent transhydrogenase (Nfn) but many acetogens lack nfn genes. We have purified a ferredoxin-dependent NADH:NADP+ oxidoreductase from Sporomusa ovata, characterized the enzyme biochemically and identified the encoding genes. These studies led to the identification of a novel, Sporomusa type Nfn (Stn), built from existing modules of enzymes such as the soluble [Fe-Fe] hydrogenase, that is widespread in acetogens and other anaerobic bacteria.


Assuntos
Acetobacterium/enzimologia , Proteínas de Bactérias/metabolismo , Ferredoxinas/metabolismo , Firmicutes/enzimologia , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Acetobacterium/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Anaerobiose , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Transporte de Elétrons , Elétrons , Firmicutes/genética , Hidrogenase/genética , Hidrogenase/isolamento & purificação , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/isolamento & purificação , Oxirredução , Homologia de Sequência de Aminoácidos
19.
Biochim Biophys Acta Bioenerg ; 1861(11): 148263, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32663477

RESUMO

rnf genes are widespread in anaerobic bacteria and hypothesized to encode a respiratory enzyme that couples exergonic reduction of NAD with reduced ferredoxin as a reductant to vectorial ion (Na+, H+) translocation across the cytoplasmic membrane. However, despite its importance for the physiology of these bacteria, little is known about the subunit composition and the function of subunits. Here, we have purified the entire Rnf complex from the acetogen Acetobacterium woodii or after its production in Escherichia coli. These studies revealed covalently bound flavin in RnfB and RnfD. Unfortunately, the complex did not catalyze electron transfer from reduced ferredoxin to NAD. We, therefore, concentrated on the two cytosolic subunits RnfC and RnfB. RnfC was produced in E. coli, purified and shown to have 8.3 mol iron and 8.6 mol sulfur per mol of the subunit, consistent with the presence of two [4Fe-4S] centers, which were verified by EPR analysis. Flavins could not be detected, but RnfC catalyzed NADH-dependent FMN reduction. These data confirm RnfC as NADH-binding subunit and FMN as an intermediate in the electron transport chain. RnfB could only be produced as a fusion to the maltose-binding protein. It contained 25 mol iron and 26 mol sulfur, consistent with the predicted six [4Fe4S] centers. The FeS centers in RnfB were reduced with reduced ferredoxin as reductant. These data are consistent with RnfB as the ferredoxin-binding subunit of the complex.


Assuntos
Acetobacterium/enzimologia , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Ferredoxinas/metabolismo , Mononucleotídeo de Flavina/metabolismo , NAD/metabolismo , Proteínas de Bactérias/genética , Transporte de Elétrons , Oxirredução
20.
Biochem Biophys Res Commun ; 527(2): 518-524, 2020 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-32423799

RESUMO

The Na+-translocating F1FO ATP synthase from Acetobacterium woodii (AwF-ATP synthase) with a subunit stoichiometry of α3:ß3:γ:δ:ε:a:b2:(c2/3)9:c1 represents an evolutionary path between ATP-synthases and vacuolar ATPases, by containing a heteromeric rotor c-ring, composed of subunits c1, c2 and c3, and an extra loop (γ195-211) within the rotary γ subunit. Here, the recombinant AwF-ATP synthase was subjected to negative stain electron microscopy and single particle analysis. The reference free 2D class averages revealed high flexibility of the enzyme, wherein the F1 and FO domains distinctively bended to adopt multiple conformations. Moreover, both the F1 and FO domains tilted relative to each other to a maximum extent of 28° and 30°, respectively. The first 3D reconstruction of the AwF-ATP synthase was determined which accommodates well the modelled structure of the AwF-ATP synthase as well as the γ195-211-loop. Molecular simulations of the enzyme underlined the bending features and flexibility observed in the electron micrographs, and enabled assessment of the dynamics of the extra γ195-211-loop.


Assuntos
Acetobacterium/enzimologia , Proteínas de Bactérias/ultraestrutura , ATPases Mitocondriais Próton-Translocadoras/ultraestrutura , Acetobacterium/química , Acetobacterium/ultraestrutura , Proteínas de Bactérias/análise , Imageamento Tridimensional , Microscopia Eletrônica , ATPases Mitocondriais Próton-Translocadoras/análise , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/análise , Proteínas Recombinantes/ultraestrutura
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