The global reliance on fossil fuels necessitates the development of sustainable alternatives. Microbial conversion of renewable biomass into everyday products presents a promising approach. Succinic acid (SA) is a key bio-based building block with diverse applications across various industries. Its use as a precursor for high-value chemicals like 1,4-butanediol and tetrahydrofuran, and as a monomer for biodegradable polymers such as polybutylene succinate, highlights its industrial importance. While metabolically engineered bacteria like *Escherichia coli*, *Corynebacterium glutamicum*, and *Mannheimia succiniciproducens* have shown impressive SA production, their sensitivity to low pH necessitates neutralizing agents (like lime or NaOH) during fermentation. Subsequent reacidification with strong acids (e.g., H2SO4) leads to gypsum (CaSO4) byproduct, posing environmental challenges and adding to downstream processing (DSP) costs. This study aims to overcome these limitations by employing an acid-tolerant yeast, *Issatchenkia orientalis*, to achieve high-titer SA production at low pH, thus minimizing DSP costs and the environmental impact. The research focuses on metabolic engineering strategies to enhance SA production in *I. orientalis*, scaling up the process, and performing techno-economic and life cycle assessments to establish its industrial viability and sustainability.

Extensive research has focused on engineering microorganisms for SA production. Bacteria have demonstrated high SA yields but require neutral pH fermentation, resulting in high DSP costs and environmental concerns due to gypsum waste. Yeasts, being more tolerant to low pH conditions, offer potential cost and environmental advantages. Previous work demonstrated the potential of *Issatchenkia orientalis* for SA production, but further improvement was needed to achieve industrial-scale yields. Other yeast species, particularly *Yarrowia lipolytica*, have shown promising results, reaching high titers and yields, but often under specific conditions (such as neutral pH or complex media) limiting scalability and economic feasibility. This study leverages previous insights while focusing on improving *I. orientalis* performance at low pH, targeting high titers and yields in industrially relevant media like sugarcane juice.

The study employed a multi-stage approach involving metabolic engineering of *I. orientalis*, fermentation optimization, scale-up, and techno-economic analysis. Initially, *I. orientalis* strain SA was further engineered by introducing a dicarboxylic acid transporter (*SpMAE1*) to improve SA export, resulting in strain SA/MAEI. Ethanol and glycerol byproduct pathways were then targeted for deletion to improve NADH availability, leading to strain SA/MAEI/pdcA/gpdA. The study then explored the co-fermentation of glucose and glycerol to enhance NADH supply and increase SA production. Additional gene deletions (g3473, encoding a dicarboxylic acid importer, and NDE, encoding an external NADH dehydrogenase) aimed at further optimizing cytosolic NADH availability for SA synthesis. Glycerol consumption was further improved via overexpression of *PaGDH* and endogenous DAK. Shake flask fermentations, bench-top bioreactor experiments (0.3 L and 0.1 L working volume), and pilot-scale fermentations (30 L working volume in a 75 L bioreactor) were conducted to evaluate the engineered strains. Fed-batch fermentations at pH 3 were performed to exploit the acid tolerance of *I. orientalis*. Sugarcane juice was used as a real-world substrate for fermentation. Succinic acid crystallization, recovery, and purity analysis were conducted using a two-stage vacuum distillation and crystallization method. Finally, techno-economic analysis (TEA) and life cycle assessment (LCA) were performed using BioSTEAM, a Python-based platform, to evaluate the economic and environmental viability of the end-to-end production pipeline, encompassing sugarcane feedstock processing, fermentation, and product recovery. The analyses considered several scenarios including laboratory batch, laboratory fed-batch, and pilot-scale batch fermentation, incorporating parameter uncertainty and sensitivity analyses. 13C metabolic flux analysis (MFA) was conducted to investigate carbon fluxes and NADH utilization in the engineered strains. Real-time PCR was also employed to assess the transcriptional levels of key genes involved in SA production and the TCA cycle.

The metabolic engineering efforts resulted in significant improvements in SA production. Strain SA/MAEI, with the addition of *SpMAE1*, showed a substantial increase in SA titer compared to the parent strain. While deleting the ethanol and glycerol pathways initially yielded similar results, further genetic manipulations resulted in improved SA titers. Co-fermentation of glucose and glycerol showed better SA titers and yields, especially under aerobic conditions. Deleting the dicarboxylic acid importer (g3473) and the external NADH dehydrogenase (NDE) further enhanced SA production. Fed-batch fermentations at pH 3 yielded exceptionally high SA titers, reaching 109.5 g/L in minimal medium and 104.6 g/L in sugarcane juice medium. Pilot-scale batch fermentation in a 300 L fermenter produced 63.1 g/L SA, which could be directly crystallized with a 64.0% yield. Techno-economic analysis revealed a minimum product selling price (MPSP) of $1.37/kg for the pilot batch scenario, which is below the market price range and highly competitive with other bio-based production methods. Life cycle assessment demonstrated a significant reduction in greenhouse gas emissions (34–90%) compared to fossil-based production. Sensitivity analysis highlighted fermentation yield and titer as key drivers for the economic and environmental performance of the production pipeline. The study also emphasized the advantages of low-pH fermentation in reducing DSP costs and improving sustainability, and also demonstrated its feasibility at pilot scale.

The study successfully addresses the research question by demonstrating a viable and sustainable end-to-end pipeline for SA production using *I. orientalis*. The high titers and yields achieved at low pH significantly reduce DSP costs compared to traditional bacterial-based methods. The use of sugarcane juice as a feedstock further enhances the economic and environmental benefits. The TEA and LCA results confirm the financial viability and superior sustainability of this approach compared to fossil-based production and many existing bio-based methods. The sensitivity analysis provides insights for future optimization, highlighting the importance of fermentation yield and titer improvements. While bacterial systems might exhibit superior performance in terms of titer, yield and productivity, the low-pH fermentation in yeast offers significant advantages in terms of cost and environmental impact, making it a more attractive option for industrial application.

This study presents a successful metabolic engineering strategy leading to a high-yielding, low-pH succinic acid production process using *I. orientalis*. The results of the TEA and LCA demonstrate the economic and environmental advantages over traditional approaches. Future work should focus on further enhancing yield and titer, potentially by integrating the glyoxylate shunt pathway or optimizing substrate utilization with crude glycerol. This platform technology has the potential to be extended for economical and sustainable production of other organic acids.

The study focused primarily on *I. orientalis* and may not be directly transferable to other microorganisms. The pilot-scale fermentation was conducted as a batch process, and the performance of a full-scale fed-batch operation might differ. While sugarcane juice was used as a real-world substrate, the variability of its composition could impact SA production. The purity of crystallized SA needs further optimization to reach commercial-grade standards. The techno-economic and life cycle assessments rely on certain model assumptions and parameters that introduce uncertainty into the results. Further optimization of the downstream processing to improve succinic acid recovery is necessary to reduce production costs.