Autotrophic adaptive laboratory evolution of the acetogen Clostridium autoethanogenum delivers the gas-fermenting strain LAbrini with superior growth, products, and robustness.

Microbes able to convert gaseous one-carbon (C1) waste feedstocks are increasingly important to transition to the sustainable production of renewable chemicals and fuels. Acetogens are interesting biocatalysts since gas fermentation using Clostridium autoethanogenum has been commercialised. However, most acetogen strains need complex nutrients, display slow growth, and are not robust for bioreactor fermentations. In this work, we used three different and independent adaptive laboratory evolution (ALE) strategies to evolve the wild-type C. autoethanogenum to grow faster, without yeast extract and to be robust in operating continuous bioreactor cultures. Multiple evolved strains with improved phenotypes were isolated on minimal media with one strain, named "LAbrini", exhibiting superior performance regarding the maximum specific growth rate, product profile, and robustness in continuous cultures. Whole-genome sequencing of the evolved strains identified 25 mutations. Of particular interest are two genes that acquired seven different mutations across the three ALE strategies, potentially as a result of convergent evolution. Reverse genetic engineering of mutations in potentially sporulation-related genes CLAU_3129 (spo0A) and CLAU_1957 recovered all three superior features of our ALE strains through triggering significant proteomic rearrangements. This work provides a robust C. autoethanogenum strain "LAbrini" to accelerate phenotyping and genetic engineering and to better understand acetogen metabolism.

Analytical tools for unravelling the metabolism of gas-fermenting Clostridia.

Acetogens harness the Wood-Ljungdahl Pathway, a unique metabolic pathway for C1 capture close to the thermodynamic limit. Gas fermentation using acetogens is already used for CO-to-ethanol conversion at industrial-scale and has the potential to valorise a range of C1 and waste substrates to short-chain and medium-chain carboxylic acids and alcohols. Advances in analytical quantification and metabolic modelling have helped guide industrial gas fermentation designs. Further advances in the measurements of difficult to measure metabolites are required to improve kinetic modelling and understand the regulation of acetogen metabolism. This will help guide future synthetic biology designs needed to realise the full potential of gas fermentation in stimulating a circular bioeconomy.

Strategies to improve viability of a circular carbon bioeconomy-A techno-economic review of microbial electrosynthesis and gas fermentation.

A circular carbon bioeconomy has potential to halt atmospheric accumulation of greenhouse gases causing climate change and sustainably produce chemical, agricultural and fuel products. Here, we report application of a simplified technoeconomic assessment to critically review two approaches in this space - microbial electrosynthesis and gas fermentation. For microbial electrosynthesis, decoupling of surface-dependant abiotic process for electron delivery from volume-dependant biotic carbon fixation, is shown as the only economically viable strategy to scale-up due to comparatively low biofilm electron consumption rate. This is effectively an electrolyser-assisted gas fermentation system. Targeting high-value products, such as protein for human food consumption is one of the few pathways forward for electrolyser-assisted gas fermentation. Alternatively, gas fermentation of reformed biogas presents an interesting and potentially more sustainable implementation pathway to improve economic viability of chemicals. This critical review suggests linking water treatment resource recovery with gas fermentation is attractive for bioplastics and butanol in terms of competitiveness and market demand.

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

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.