(232b) Charting Development Pathways for Precision Fermentation through Agile System Design and Analyses

To address climate change by mitigating greenhouse gas emissions, there is a pressing need to develop technologies that can facilitate replacing crude oil with alternative, renewable feedstocks to manufacture fuels and chemicals. Precision fermentation has emerged as promising for the sustainable manufacturing of biofuels and bioproducts. Developments in metabolic engineering of microbial candidates have enabled the biological production of commercially significant chemicals including succinic acid (e.g., by Issatchenkia orientalis), acrylic acid (via 3-hydroxypropionic acid, 3-HP; e.g., by I. orientalis or Corynebacterium glutamicum), and sorbic acid (via triacetic acid lactone, TAL; e.g., by Yarrowia lipolytica), among others. We leveraged BioSTEAM—an open-source platform—to design, simulate, and evaluate under uncertainty (via techno-economic analysis, TEA, and life cycle assessment, LCA) biorefineries producing these chemicals by fermentation of substrates obtained from renewable 1st-generation (e.g., sugarcane, sweet sorghum) and 2nd-generation feedstocks (e.g., corn stover, miscanthus). Further, we simulated and evaluated entire theoretical fermentation performance landscapes (e.g., formed by all potential combinations of fermentation yield, titer, and productivity under neutral and low-pH regimes) in each of these biorefineries, revealing fermentation development pathways to achieve system-wide sustainability targets. We showed recent fermentation developments could enable financially viable and environmentally beneficial production of bio-based succinic and acrylic acids, and identified targeted improvements in TAL production that could enable sustainable bio-based sorbic acid production. Finally, we showcase AutoSynthesis, a new open-source platform in Python to automate key aspects of process synthesis and design for biorefineries. We show how AutoSynthesis could be used to explore potentially synergistic developments across technologies and to enable end-to-end frameworks for computationally guided discovery of promising metabolic pathways.

For example, recent efforts to engineer I. orientalis strains to produce 3-HP at low pH can enable acrylic acid production via 3-HP with an estimated minimum product selling price (MPSP) of $1.29−1.52·kg^-1 [5th-95th percentiles, hereafter in brackets], below the market price range of succinic acid in ~88% of Monte Carlo simulations. The biorefinery’s estimated 100-year global warming potential (GWP₁₀₀) of 3.00 [2.53−3.38] kgCO₂-eq·kg^-1 and fossil energy consumption (FEC) of 39.9 [31.6−45.1] MJ·kg^-1 were both significantly below those of fossil-derived acrylic acid.

Further, I. orientalis strains have recently been engineered to produce succinic acid under low-pH conditions at pilot scale. We showed biorefineries leveraging these developments could produce succinic acid with an MPSP of 1.37·kg^-1 [$1.23–1.54·kg^-1], which was market-competitive in 100% of Monte Carlo simulations. The biorefinery’s GWP₁₀₀ of 1.67 kgCO₂-eq·kg^-1 (1.22–2.17 kgCO₂-eq·kg^-1) and FEC of −0.21 MJ·kg^-1 (−7.08 to 6.47 MJ·kg^-1) were also consistently below those of fossil-derived succinic acid.

To design biorefineries that could produce high-purity TAL using engineered Y. lipolytica strains, we experimentally characterized TAL solubility in water at various temperatures. We calibrated a solubility model to these experimental data and used it to design a process to separate TAL from fermentation broths through fractional crystallization. The biorefinery could produce TAL (>95.0 dry wt% purity) at an MPSP of $6.13·kg^-1 [$5.12−8.38·kg^-1], comparable to the maximum viable TAL price range for sorbic acid production (approximately $5.99−7.74·kg^-1), with net displacement of fossil energy consumption (FEC) in 87.7% of simulations by displacing marginal grid electricity. Advancements in fermentation TAL yield and titer could greatly enhance the biorefinery’s financial viability (MPSP of 4.36·kg^-1 [$3.69−5.87·kg^-1], lower than the maximum viable price range in 96.1% of simulations) and environmental benefits (GWP₁₀₀ of 3.02 [0.905−5.22] kg CO₂-eq·kg^-1 and FEC of -21.8 [-51.7 to -3.26] MJ·kg^-1, with net displacement of FEC in 97.4% of simulations).

This work showcases how agile and robust system design and analyses can elucidate key drivers of system cost and environmental impacts across technological landscapes, chart roadmaps to navigate the opportunity space for precision fermentation, screen promising designs, navigate sustainability tradeoffs, and prioritize research, development, and deployment needs in biofuels and bioproducts development.