Acetate augmentation boosts the ethanol production rate and specificity by Clostridium ljungdahlii during gas fermentation with pure carbon monoxide

Schulz, Sarah; Molitor, Bastian; Angenent, Largus T.

doi:10.1016/j.biortech.2022.128387

Cited by 11 publications

(8 citation statements)

References 29 publications

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“…Thus, the superior performance of LAbrini in continuous bioreactor cultures and its ability to grow in the absence of YE is due to its genotype and the selection strategy employed in its development. While growth without YE has been reported for acetogens before (Martin et al, 2015;Schulz et al, 2023), it is unclear if these phenotypes were the result of adaptation or evolution. Further studies are required to link mutations to its phenotype, key structural/functional attributes of the ACS/CODH enzyme complex and its regulation.…”

Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

“…ALE also afforded an Eubacterium limosum strain with higher CO tolerance and faster growth (Jin et al, 2022; Kang et al, 2020). Additionally, C. ljungdahlii and C. autoethanogenum have been adapted by continuous bioreactor cultivation to grow without YE (Martin et al, 2015; Schulz et al, 2023), but it is not clear if the phenotype was the result of adaptation (i.e. reversible) or evolution (i.e.…”

Section: Introductionmentioning

confidence: 99%

“…ALE was also recently used in Clostridium carboxidivorans to expand its product spectrum and improve growth on CO 2 +H 2 (22) and in Eubacterium limosum for higher CO tolerance and faster growth (23, 24). Additionally, C. ljungdahlii and C. autoethanogenum have been adapted by continuous bioreactor cultivation to grow without YE (25, 26), but it is not clear if the phenotype was the result of physiological adaptation (i.e. reversible) or evolution (i.e.…”

Section: Introductionmentioning

confidence: 99%

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Autotrophic adaptive laboratory evolution of the acetogenClostridium autoethanogenumdelivers the gas-fermenting strain LAbrini with superior growth, products, and robustness

Ingelman¹,

Heffernan

Harris

et al. 2023

Preprint

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Microbes able to convert gaseous one-carbon (C1) waste feedstocks are of growing importance in transitioning to the biosustainable production of renewable chemicals and fuels. Acetogens are particularly interesting biocatalysts since gas fermentation usingClostridium autoethanogenumhas already been commercialised. Most non-commercial acetogen strains, however, need complex nutrients, display slow growth, and are not sufficiently robust for routine bioreactor fermentations. In this work, we used three adaptive laboratory evolution (ALE) strategies to evolve the wild-type model-acetogenC. autoethanogenumto grow faster, without complex nutrients and to be robust in operation of continuous bioreactor cultures. Seven evolved strains with improved phenotypes were isolated on minimal medium with one strain, named "LAbrini" (LT1), exhibiting superior performance in terms of the maximum specific growth rate, product profile, and robustness in continuous cultures. Differing performance of the strains between bottle batch and continuous cultures shows the importance of testing novel strains in industrially relevant continuous fermentation conditions. Interestingly, a very distinct transcriptome profile linked to a potential CO toxicity phenotype was observed in bioreactor cultures for one evolved strain. Whole-genome sequencing of the seven evolved strains identified 25 mutations with two genomic regions under stronger evolutionary pressure. Our analysis also suggests that the genotypic changes that are potentially responsible for the improved phenotypes may serve as useful candidates for metabolic engineering of cell factories. This work provides the robustC. autoethanogenumstrain LAbrini to the academic community to speed up phenotyping and genetic engineering, improve quantitative characterisation of acetogen metabolism, and facilitate the generation of high-quality steady-state datasets.

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Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Autotrophic adaptive laboratory evolution of the acetogenClostridium autoethanogenumdelivers the gas-fermenting strain LAbrini with superior growth, products, and robustness

Ingelman¹,

Heffernan

Harris

et al. 2023

Preprint

View full text Add to dashboard Cite

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“…However, the increased CO uptake rate was compensated with higher rates of acetate reduction to ethanol. This is likely to be related to the redox state of the electron carriers and the fact that acetate was available in the cultivation broth (Diender et al, 2019; Mahamkali et al, 2020; Richter et al, 2016; Schulz et al, 2023; Xu et al, 2020). Effectively, the product spectrum changed as a function of the mass transfer rate.…”

Section: Discussionmentioning

confidence: 99%

A novel experimental method to determine substrate uptake kinetics of gaseous substrates applied to the carbon monoxide‐fermenting Clostridium autoethanogenum

Allaart,

Korkontzelos,

Sousa

et al. 2024

Biotech & Bioengineering

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Syngas fermentation has gained momentum over the last decades. The cost‐efficient design of industrial‐scale bioprocesses is highly dependent on quantitative microbial growth data. Kinetic and stoichiometric models for syngas‐converting microbes exist, but accurate experimental validation of the derived parameters is lacking. Here, we describe a novel experimental approach for measuring substrate uptake kinetics of gas‐fermenting microbes using the model microorganism Clostridium autoethanogenum. One‐hour disturbances of a steady‐state chemostat bioreactor with increased CO partial pressures (up to 1.2 bar) allowed for measurement of biomass‐specific CO uptake‐ and CO2 production rates (, ) using off‐gas analysis. At a pCO of 1.2 bar, a of −119 ± 1 mmol g−1X h−1 was measured. This value is 1.8–3.5‐fold higher than previously reported experimental and kinetic modeling results for syngas fermenters. Analysis of the catabolic flux distribution reveals a metabolic shift towards ethanol production at the expense of acetate at pCO 0.6 atm, likely to be mediated by acetate availability and cellular redox state. We characterized this metabolic shift as acetogenic overflow metabolism. These results provide key mechanistic understanding of the factors steering the product spectrum of CO fermentation in C. autoethanogenum and emphasize the importance of dedicated experimental validation of kinetic parameters.

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Acetic acid, growth rate, and mass transfer govern shifts in CO metabolism of Clostridium autoethanogenum

Elisiário

Hecke

Wever

et al. 2023

Appl Microbiol Biotechnol

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Syngas fermentation is a leading microbial process for the conversion of carbon monoxide, carbon dioxide, and hydrogen to valuable biochemicals. Clostridium autoethanogenum stands as a model organism for this process, showcasing its ability to convert syngas into ethanol industrially with simultaneous fixation of carbon and reduction of greenhouse gas emissions. A deep understanding on the metabolism of this microorganism and the influence of operational conditions on fermentation performance is key to advance the technology and enhancement of production yields. In this work, we studied the individual impact of acetic acid concentration, growth rate, and mass transfer rate on metabolic shifts, product titres, and rates in CO fermentation by C. autoethanogenum. Through continuous fermentations performed at a low mass transfer rate, we measured the production of formate in addition to acetate and ethanol. We hypothesise that low mass transfer results in low CO concentrations, leading to reduced activity of the Wood–Ljungdahl pathway and a bottleneck in formate conversion, thereby resulting in the accumulation of formate. The supplementation of the medium with exogenous acetate revealed that undissociated acetic acid concentration increases and governs ethanol yield and production rates, assumedly to counteract the inhibition by undissociated acetic acid. Since acetic acid concentration is determined by growth rate (via dilution rate), mass transfer rate, and working pH, these variables jointly determine ethanol production rates. These findings have significant implications for process optimisation as targeting an optimal undissociated acetic acid concentration can shift metabolism towards ethanol production. Key points • Very low CO mass transfer rate leads to leaking of intermediate metabolite formate. • Undissociated acetic acid concentration governs ethanol yield on CO and productivity. • Impact of growth rate, mass transfer rate, and pH were considered jointly. Graphical abstract

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Acetate augmentation boosts the ethanol production rate and specificity by Clostridium ljungdahlii during gas fermentation with pure carbon monoxide

Cited by 11 publications

References 29 publications

Autotrophic adaptive laboratory evolution of the acetogenClostridium autoethanogenumdelivers the gas-fermenting strain LAbrini with superior growth, products, and robustness

Autotrophic adaptive laboratory evolution of the acetogenClostridium autoethanogenumdelivers the gas-fermenting strain LAbrini with superior growth, products, and robustness

A novel experimental method to determine substrate uptake kinetics of gaseous substrates applied to the carbon monoxide‐fermenting Clostridium autoethanogenum

Acetic acid, growth rate, and mass transfer govern shifts in CO metabolism of Clostridium autoethanogenum

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