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Enhancing CO2-Valorization Using Clostridium autoethanogenum for Sustainable Fuel and Chemicals Production.

ABSTRACT: Acetogenic bacteria can convert waste gases into fuels and chemicals. Design of bioprocesses for waste carbon valorization requires quantification of steady-state carbon flows. Here, steady-state quantification of autotrophic chemostats containing Clostridium autoethanogenum grown on CO2 and H2 revealed that captured carbon (460 ± 80 mmol/gDCW/day) had a significant distribution to ethanol (54 ± 3 C-mol% with a 2.4 ± 0.3 g/L titer). We were impressed with this initial result, but also observed limitations to biomass concentration and growth rate. Metabolic modeling predicted culture performance and indicated significant metabolic adjustments when compared to fermentation with CO as the carbon source. Moreover, modeling highlighted flux to pyruvate, and subsequently reduced ferredoxin, as a target for improving CO2 and H2 fermentation. Supplementation with a small amount of CO enabled co-utilization with CO2, and enhanced CO2 fermentation performance significantly, while maintaining an industrially relevant product profile. Additionally, the highest specific flux through the Wood-Ljungdahl pathway was observed during co-utilization of CO2 and CO. Furthermore, the addition of CO led to superior CO2-valorizing characteristics (9.7 ± 0.4 g/L ethanol with a 66 ± 2 C-mol% distribution, and 540 ± 20 mmol CO2/gDCW/day). Similar industrial processes are commercial or currently being scaled up, indicating CO-supplemented CO2 and H2 fermentation has high potential for sustainable fuel and chemical production. This work also provides a reference dataset to advance our understanding of CO2 gas fermentation, which can contribute to mitigating climate change.

SUBMITTER: Heffernan JK

PROVIDER: S-EPMC7135887 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Enhancing CO2-Valorization Using Clostridium autoethanogenum for Sustainable Fuel and Chemicals Production.

Heffernan James K JK Valgepea Kaspar K de Souza Pinto Lemgruber Renato R Casini Isabella I Plan Manuel M Tappel Ryan R Simpson Sean D SD Köpke Michael M Nielsen Lars K LK Marcellin Esteban E

Frontiers in bioengineering and biotechnology 20200327

Acetogenic bacteria can convert waste gases into fuels and chemicals. Design of bioprocesses for waste carbon valorization requires quantification of steady-state carbon flows. Here, steady-state quantification of autotrophic chemostats containing Clostridium autoethanogenum grown on CO2 and H2 revealed that captured carbon (460 ± 80 mmol/gDCW/day) had a significant distribution to ethanol (54 ± 3 C-mol% with a 2.4 ± 0.3 g/L titer). We were impressed with this initial resul ...[more]

PMID: 32292775

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Project description:UNLABELLED:The future sustainable production of chemicals and fuels from nonpetrochemical resources and reduction of greenhouse gas emissions are two of the greatest societal challenges. Gas fermentation, which utilizes the ability of acetogenic bacteria such as Clostridium autoethanogenum to grow and convert CO2 and CO into low-carbon fuels and chemicals, could potentially provide solutions to both. Acetogens fix these single-carbon gases via the Wood-Ljungdahl pathway. Two enzyme activities are predicted to be essential to the pathway: carbon monoxide dehydrogenase (CODH), which catalyzes the reversible oxidation of CO to CO2, and acetyl coenzyme A (acetyl-CoA) synthase (ACS), which combines with CODH to form a CODH/ACS complex for acetyl-CoA fixation. Despite their pivotal role in carbon fixation, their functions have not been confirmed in vivo By genetically manipulating all three CODH isogenes (acsA, cooS1, and cooS2) of C. autoethanogenum, we highlighted the functional redundancies of CODH by demonstrating that cooS1 and cooS2 are dispensable for autotrophy. Unexpectedly, the cooS1 inactivation strain showed a significantly reduced lag phase and a higher growth rate than the wild type on H2 and CO2 During heterotrophic growth on fructose, the acsA inactivation strain exhibited 61% reduced biomass and the abolishment of acetate production (a hallmark of acetogens), in favor of ethanol, lactate, and 2,3-butanediol production. A translational readthrough event was discovered in the uniquely truncated (compared to those of other acetogens) C. autoethanogenum acsA gene. Insights gained from studying the function of CODH enhance the overall understanding of autotrophy and can be used for optimization of biotechnological production of ethanol and other commodities via gas fermentation. IMPORTANCE:Gas fermentation is an emerging technology that converts the greenhouse gases CO2 and CO in industrial waste gases and gasified biomass into fuels and chemical commodities. Acetogenic bacteria such as Clostridium autoethanogenum are central to this bioprocess, but the molecular and genetic characterization of this microorganism is currently lacking. By targeting all three of the isogenes encoding carbon monoxide dehydrogenase (CODH) in C. autoethanogenum, we identified the most important CODH isogene for carbon fixation and demonstrated that genetic inactivation of CODH could improve autotrophic growth. This study shows that disabling of the Wood-Ljungdahl pathway via the inactivation of acsA (encodes CODH) significantly impairs heterotrophic growth and alters the product profile by abolishing acetate production. Moreover, we discovered a previously undescribed mechanism for controlling the production of this enzyme. This study provides valuable insights into the acetogenic pathway and can be used for the development of more efficient and productive strains for gas fermentation.

Project description:UnlabelledMost acetogens can reduce CO2 with H2 to acetic acid via the Wood-Ljungdahl pathway, in which the ATP required for formate activation is regenerated in the acetate kinase reaction. However, a few acetogens, such as Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei, also form large amounts of ethanol from CO2 and H2. How these anaerobes with a growth pH optimum near 5 conserve energy has remained elusive. We investigated this question by determining the specific activities and cofactor specificities of all relevant oxidoreductases in cell extracts of H2/CO2-grown C. autoethanogenum. The activity studies were backed up by transcriptional and mutational analyses. Most notably, despite the presence of six hydrogenase systems of various types encoded in the genome, the cells appear to contain only one active hydrogenase. The active [FeFe]-hydrogenase is electron bifurcating, with ferredoxin and NADP as the two electron acceptors. Consistently, most of the other active oxidoreductases rely on either reduced ferredoxin and/or NADPH as the electron donor. An exception is ethanol dehydrogenase, which was found to be NAD specific. Methylenetetrahydrofolate reductase activity could only be demonstrated with artificial electron donors. Key to the understanding of this energy metabolism is the presence of membrane-associated reduced ferredoxin:NAD(+) oxidoreductase (Rnf), of electron-bifurcating and ferredoxin-dependent transhydrogenase (Nfn), and of acetaldehyde:ferredoxin oxidoreductase, which is present with very high specific activities in H2/CO2-grown cells. Based on these findings and on thermodynamic considerations, we propose metabolic schemes that allow, depending on the H2 partial pressure, the chemiosmotic synthesis of 0.14 to 1.5 mol ATP per mol ethanol synthesized from CO2 and H2.ImportanceEthanol formation from syngas (H2, CO, and CO2) and from H2 and CO2 that is catalyzed by bacteria is presently a much-discussed process for sustainable production of biofuels. Although the process is already in use, its biochemistry is only incompletely understood. The most pertinent question is how the bacteria conserve energy for growth during ethanol formation from H2 and CO2, considering that acetyl coenzyme A (acetyl-CoA), is an intermediate. Can reduction of the activated acetic acid to ethanol with H2 be coupled with the phosphorylation of ADP? Evidence is presented that this is indeed possible, via both substrate-level phosphorylation and electron transport phosphorylation. In the case of substrate-level phosphorylation, acetyl-CoA reduction to ethanol proceeds via free acetic acid involving acetaldehyde:ferredoxin oxidoreductase (carboxylate reductase).

Dataset Information

Enhancing CO2-Valorization Using Clostridium autoethanogenum for Sustainable Fuel and Chemicals Production.

Publications

Enhancing CO<sub>2</sub>-Valorization Using <i>Clostridium autoethanogenum</i> for Sustainable Fuel and Chemicals Production.

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