Microbial conversion of CO₂ into value-added products

The escalation of atmospheric CO₂ levels has stimulated the advancement of carbon capture and utilization technologies, aimed at sequestering CO₂ and transforming it into valuable chemicals. Within this context, biological conversion of CO₂ by microorganisms has gathered significant interest, leveraging the inherent ability of autotrophic microbes to utilize CO₂ as a feedstock for growth and the production of industrially relevant metabolites. In particular, acetogenic bacteria carry out the conversion of CO₂ to acetyl-CoA and subsequently to acetate, utilizing H₂ as an energy source through the Wood Ljungdahl pathway (WLP). Despite the efficiency of this pathway, which stands as the sole known natural carbon-fixing mechanism not reliant on ATP consumption, limitations such as low biomass production and growth rates hinder the industrial exploitation of these microbes. Mixotrophic growth, a metabolic mode consisting of simultaneous conversion of CO₂ and an organic substrate, typically a hexose sugar, offers a potential way to address these challenges by supplying additional ATP produced via glycolysis. This thesis aimed at identifying a promising strategy to enable CO₂ fixation by microorganisms and assess its potential for development of a CO₂- consuming bioprocess. After that, the work focused on clarifying the metabolism of acetogens during mixotrophic growth and evaluating the potential for industrial application.

Initially, an analysis of the literature on microbial CO₂ conversion revealed the principal themes and challenges investigated over the past decade, during which this field has witnessed an exponential increase in interest, as indicated by the number of publications over years. Subsequently, the potential of mixotrophic growth for industrial biotechnological applications is explored.
Screening of seven acetogenic strains afterwards allowed to identify potential candidates for further development, considering their capability to simultaneously convert H₂, CO₂, and fructose to produce acetate. Further investigation has unveiled differences among mixotrophic strains, particularly concerning the contribution of carbons and electrons metabolized by the WLP to overall metabolism, and the allocation of carbon to different products. Nevertheless, across all mixotrophic strains, the WLP has been observed to contribute more than 50% of carbon and electrons during the exponential growth phase.
The potential of mixotrophic fermentation, particularly by the strain Clostridium ljungdahlii, for industrial application was then evaluated through techno-economic analysis. For this analysis, various process scenarios were considered, all of them focusing on the minimum selling price of the desired product, acetic acid. Results indicated that the CO₂-based technology presently lacks competitiveness compared to the existing acetic acid production processes. However, advancements in metabolic and process engineering, alongside evolving
carbon mitigation policies, may reshape the landscape in the coming years.
Moreover, alternative approaches should also be considered, such as integrating mixotrophic fermentation within a two-stage fermentation framework, in which acetate produced by acetogens is further converted into higher-value products, thereby circumventing costly downstream acetic acid processing. Additionally, exploring bacterial engineering to produce diverse compounds may represent a viable solution. This approach was investigated by enhancing phenotype predictions through a genome-scale metabolic model, incorporating enzyme pool constraints, and employing the OptKnock algorithm to forecast potential genetic engineering interventions for enhancing production fluxes of acetate, ethanol, lactate, and 2,3-butanediol during syngas and mixotrophic fermentation.
The present work increased the current knowledge on acetogenic mixotrophy with particular interest for their potential in development of CO₂-based bioprocesses for production of valuable chemicals. The relevance of the Wood Ljungdahl pathway on the overall mixotrophic metabolism was highlighted as mixotrophic strains showed high rates of CO₂ fixation also in presence of high fructose concentrations. Moreover, it was presented the first TEA study on the potential application of a mixotrophic acetogen in industrial setup; the results showed potential bottlenecks to tackle for future development of this technology. Moreover, an enhanced metabolic model was generated by inclusion of enzymatic constrains to a genome scale model, which can be used for accurate predictions of genetic engineering strategies to broaden the spectrum of products by C. ljungdhalii.