Collaborative Research: Continuous Biomanufacturing using Decoupled Growth and Production Stages for Efficient Production and Recovery

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The range and scale of biomanufacturing operations continue to grow, sustainably and locally producing fuels, chemicals, materials, foods, beverages, and medicines. Currently, most biomanufacturing facilities operate as batch processes, a chemical manufacturing technology developed over 70 years ago that has effectively reached its limits in productivity gains and cost reductions achievable through process optimization. Over this same period, significant progress has been made in the ability to synthetically engineer microbial cells to enhance their desirable traits and, ultimately, their ability to produce valuable bioproducts. This motivates revisiting the design of current batch and fed-batch manufacturing processes, technologies that have fallen behind the fast pace of modern metabolic engineering advances, limiting the true conversion potential of the newly rewired cells. This project aims to establish a novel continuous biomanufacturing platform that overcomes the major limitations of conventional (fed)-batch processes, leading to significant increases in reactor system productivity and a substantial reduction in manufacturing costs. The engineering knowledge and scientific discoveries generated from this project will enable the transformation of most current (fed)-batch biomanufacturing facilities to cost-effective continuous processes for large-scale production of fuels, chemicals, and other high-value products, strengthening the U.S. global lead in biomanufacturing. This interdisciplinary, multi-university project will engage a team of researchers with a diverse set of skills, experiences, and educational backgrounds, providing a unique research environment for high-school, undergraduate, and graduate students.This project integrates advanced technologies in process system engineering and synthetic biology to develop a novel continuous biomanufacturing platform for production of hydrophobic products such as lipids or lipid-derived high-value products from cellulosic feedstocks. Intracellular lipids and extracellular free fatty acids (FFAs) from Yarrowia lipolytica and fatty acid ethyl esters (FAEEs) from Saccharomyces cerevisiae will be used as archetype bioproducts to develop the continuous biomanufacturing platform. First, a two-stage continuous fermentation process equipped with a smaller growth bioreactor and a larger production bioreactor will be developed so that cell growth and product formation can be decoupled and where process operating conditions can be independently optimized to maximize the production of both biomass and target product simultaneously. Second, the two representative yeasts, Y. lipolytica and S. cerevisiae, will be engineered to enable distinct growth and production phases controlled by environmental and/or genetic switching. Y. lipolytica will be engineered to use both C5 and C6 sugars derived from cellulosic biomass to produce either intracellular lipids or extracellular FFAs with greater than 80% dry cell weight (DCW) or equivalent. S. cerevisiae will also be engineered to produce high levels of FAEEs. Due to the low density and buoyant force of the lipid products, the product recovery can be easily achieved via simple phase separation. A computational fluid dynamics (CFD) study on lipid distribution in bioreactors will be conducted to help further guide the design and optimization of the continuous biomanufacturing process, coupling product formation and in-situ product removal.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.