Characterization of Engineered Living Hydrogel Membranes for Modular SynBio Manufacturing

Abstract

In recent years, advancements in metabolic engineering have vastly increased the capabilities of synthetic biology (SynBio) manufacturing, leading to new avenues of exploration in the design of processing units. SynBio manufacturing offers many advantages over traditional synthetic manufacturing, particularly in the production of complex and fragile compounds like pharmaceutical drugs; however, for bio-based production to reach a scale competitive with traditional chemical manufacturing, continuous processes are a necessity. Shifting SynBio manufacturing from batch to continuous processing faces numerous challenges, including cell survival, production efficiency, and complex downstream purification steps. One approach to solving this issue is through the use of ELMs, or engineered living materials. Designed to emulate the natural extracellular matrices (ECMs) microorganisms thrive in, ELMs encapsulate the cells within a solid matrix, such as a hydrogel. These hydrogels are a class of soft materials consisting of three-dimensional networks of hydrophilic polymers that are known for their water retention, multi-scale permeability, and tunability. ELM-based processing units allow for continuous synthesis and can offer more complex stratification of engineered microorganisms than free-floating batch reactors do, further expanding synthesis opportunities. However, three-dimensional constructs of ELMs still face challenges with production efficiency and still require complex downstream purification steps. Here, we propose an alternative form factor for ELM-based processing units: in place of a three-dimensional construct, a two-dimensional membrane design that can act simultaneously as an ELM for SynBio production and as a product separations membrane. Because hydrogels have been investigated extensively for both ELM and membrane applications, combining the two features into a single engineered living membrane could allow for continuous bio-manufacturing with in situ product separations, reducing the number of downstream membrane-based separations needed for product purification. However, the features of a hydrogel that make it ideal for cell encapsulation are not necessarily ideal for membrane applications. In this work, I study process-structure-property relationships parameters in hydrogel materials to enable their use as ELM-based membrane processing units. In particular, I explore two hydrogel systems commonly used in ELMs: a functionalized form of F127, and poly(ethylene glycol) diacrylate (PEGDA). After identifying the features of F127 that make it unsuitable for membrane applications in this context, I investigate PEGDA-based hydrogels. Through the use of automated workflows, high-throughput characterization methods, and explainable AI (XAI) tools, my thesis investigates the expansive design space of PEGDA-based hydrogels and assesses how hydrogel composition and the use of glycerol as a plasticizer affects microstructure, permeability, and swelling behavior. With this work, I aim to characterize the foundational material requirements for engineered living hydrogel membranes and advance the methods of tailoring hydrogel microstructure to accommodate both cell encapsulation and separations membrane technologies.