Gas fermentation offers both fossil carbon-free sustainable production of fuels and chemicals and recycling of gaseous and solid waste using gas-fermenting microbes. Bioprocess development, systems-level analysis of biocatalyst metabolism, and engineering of cell factories are advancing the widespread deployment of the commercialised technology. Acetogens are particularly attractive biocatalysts but effects of the key physiological parameter–specific growth rate (μ)—on acetogen metabolism and the gas fermentation bioprocess have not been established yet. Here, we investigate the μ-dependent bioprocess performance of the model-acetogen Clostridium autoethanogenum in CO and syngas (CO + CO2+H2) grown chemostat cultures and assess systems-level metabolic responses using gas analysis, metabolomics, transcriptomics, and metabolic modelling. We were able to obtain steady-states up to μ ∼2.8 day−1 (∼0.12 h−1) and show that faster growth supports both higher yields and productivities for reduced by-products ethanol and 2,3-butanediol. Transcriptomics data revealed differential expression of 1,337 genes with increasing μ and suggest that C. autoethanogenum uses transcriptional regulation to a large extent for facilitating faster growth. Metabolic modelling showed significantly increased fluxes for faster growing cells that were, however, not accompanied by gene expression changes in key catabolic pathways for CO and H2 metabolism. Cells thus seem to maintain sufficient “baseline” gene expression to rapidly respond to CO and H2 availability without delays to kick-start metabolism. Our work advances understanding of transcriptional regulation in acetogens and shows that faster growth of the biocatalyst improves the gas fermentation bioprocess.

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.

Gas fermentation offers both fossil carbon-free sustainable production of fuels and chemicals and recycling of gaseous and solid waste using gas-fermenting microbes. Bioprocess development, systems-level analysis of biocatalyst metabolism, and engineering of cell factories are advancing the widespread deployment of the commercialised technology. Acetogens are particularly attractive biocatalysts but effects of the key physiological parameter – specific growth rate (μ) – on acetogen metabolism and the gas fermentation bioprocess have not been established yet. Here, we investigate the (μ-dependent bioprocess performance of the model-acetogen Clostridium autoethanogenum in CO and syngas (CO+CO2+H2) grown chemostat cultures and assess systems-level metabolic responses using gas analysis, metabolomics, transcriptomics, and metabolic modelling. We were able to obtain steady-states up to μ ~2.8 day-1 (~0.12 h-1) and show that faster growth supports both higher yields and productivities for reduced by-products ethanol and 2,3-butanediol. Transcriptomics data revealed differential expression of 1,337 genes with increasing (μ) and suggest that C. autoethanogenum uses transcriptional regulation to a large extent for facilitating faster growth. Metabolic modelling showed significantly increased fluxes for faster growing cells that were, however, not accompanied by gene expression changes in key catabolic pathways for CO and H2 metabolism. Cells thus seem to maintain sufficient 'baseline' gene expression to rapidly respond to CO and H2 availability without delays to kick-start metabolism. Our work advances understanding of transcriptional regulation in acetogens and shows that faster growth of the biocatalyst improves the gas fermentation bioprocess.

Gas fermentation has emerged as a sustainable route to produce fuels and chemicals by recycling inexpensive one-carbon (C1) feedstocks from gaseous and solid waste using gas-fermenting microbes. Currently, acetogens that utilise the Wood-Ljungdahl pathway to convert carbon oxides (CO and CO2) into valuable products are the most advanced biocatalysts for gas fermentation. However, our understanding of the functionalities of the genes involved in theC1-fixing gene cluster and its closely-linked genes is incomplete. Here, we investigate the role of two genes with unclear functions - hypothetical protein (hp; LABRINI_07945) and CooT nickel binding protein (nbp; LABRINI_07950) - directly adjacent and expressed at similar levels to theC1-fixing gene cluster in the gas-fermenting model-acetogenClostridium autoethanogenum. Targeted deletion of either thehpornbpgene using CRISPR/nCas9, and phenotypic characterisation in heterotrophic and autotrophic batch and autotrophic bioreactor continuous cultures revealed significant growth defects and altered by-product profiles for both∆hpand∆nbpstrains. Variable effects of gene deletion on autotrophic batch growth on rich or minimal media suggest that both genes affect the utilisation of complex nutrients. Autotrophic chemostat cultures showed lower acetate and ethanol production rates and higher carbon flux to CO2and biomass for both deletion strains. Additionally, proteome analysis revealed that disruption of either gene affects the expression of proteins of theC1-fixing gene cluster and ethanol synthesis pathways. Our work contributes to a better understanding of genotype-phenotype relationships in acetogens and offers engineering targets to improve carbon fixation efficiency in gas fermentation.

The challenge of limiting global warming to below 1.5°C requires all industries to implement new technologies and change practices immediately. The aviation industry contributes 2% of human-induced CO2 emissions and 12% of all transport emissions. Decarbonising the aviation industry, which relies heavily on high-density liquid fuels, has been difficult to achieve. The problems are compounded by the continued reliance on so-called sustainable aviation fuels, which use first-generation agricultural feedstocks, creating a trade-off between biomass for food and feed and its use as a feedstock for energy generation. Decarbonising aviation is also challenging because of problems in developing electric aircraft. Alternative feedstocks already exist that provide a more feasible path towards decelerating climate change. One such alternative is to use gas fermentation to convert greenhouse gases (e.g. from food production and food waste) into fuels using microbial acetogens. Acetogens are anaerobic microorganisms capable of producing alcohols from gaseous CO, CO2 and H2. Australia offers feedstock resources for gas fermentation with abundant H2 and CO2 production in proximity to each other. In this review, we put forward the principles, approaches and opportunities offered by gas fermentation technologies to replace our dependency on fossil fuels for aviation fuel production in Australia.