Additive manufacturing allows three-dimensional printing of polymeric materials together with cells, creating living materials for applications in biomedical research and biotechnology. However, an understanding of the cellular phenotype within living materials is lacking, which is a key limitation for their wider application. Herein, we present an approach to characterize the cellular phenotype within living materials. We immobilized the budding yeast Saccharomyces cerevisiae in three different photo-cross-linkable triblock polymeric hydrogels containing F127-bis-urethane methacrylate, F127-dimethacrylate, or poly(alkyl glycidyl ether)-dimethacrylate. Using optical and scanning electron microscopy, we showed that hydrogels based on these polymers were stable under physiological conditions, but yeast colonies showed differences in the interaction within the living materials. We found that the physical confinement, imparted by compositional and structural properties of the hydrogels, impacted the cellular phenotype by reducing the size of cells in living materials compared with suspension cells. These properties also contributed to the differences in immobilization patterns, growth of colonies, and colony coatings. We observed that a composition-dependent degradation of polymers was likely possible by cells residing in the living materials. In conclusion, our investigation highlights the need for a holistic understanding of the cellular response within hydrogels to facilitate the synthesis of application-specific polymers and the design of advanced living materials in the future.

Biotechnology requires efficient microbial cell factories. The budding yeast Saccharomyces cerevisiae is a vital cell factory, but more diverse cell factories are essential for the sustainable use of natural resources. Here, we benchmarked non-conventional yeasts Kluyveromyces marxianus (KM) and Rhodotorula toruloides (RT) against S. cerevisiae, CEN.PK and W303, for their responses to potassium and sodium salt stress. We found an inverse relationship between the maximum growth rate and the median cell volume that was responsive to salt stress. The supplementation of K+ to CEN.PK cultures reduced Na+ toxicity and increased the specific growth rate by four-fold. The higher K+ and Na+ concentrations impaired ethanol and acetate metabolism in CEN.PK, acetate in W303. In RT cultures, these salts' supplementation induced a trade-off between glucose utilization and cellular aggregates formation. Their combined use increased the beta-carotene yield by 60% compared with the reference. Neural network-based image analysis of exponential phase cultures showed that the vacuole to cell volume ratio increased with increased cell volume for W303 and KM, but not for CEN.PK and RT in response to salt stress. Our results provide insights into common salt stress responses in yeasts and will help design efficient bioprocesses. Importance Characterization of microbial cell factories in industrially relevant conditions is crucial for designing efficient bioprocesses. Salt stress, typical in industrial bioprocesses, impinges upon cell volume and affects productivity. This study presents an open-source neural network-based analysis method to evaluate volumetric changes using yeasts’ optical microscopy images. It allows quantification of cell and vacuole volumes relevant to cellular physiology. On applying salt stress in yeasts, we found that the combined use of K+ and Na+ improves the cellular fitness of CEN.PK and increases the beta-carotene productivity in R. toruloides, a commercially important antioxidant and a valuable additive in foods.

Additive manufacturing allows three-dimensional printing of polymeric materials together with cells, creating living materials for applications in biomedical research and biotechnology. However, understanding the cellular phenotype within living materials is lacking and a key limitation for their wider application. Herein, we present an approach to characterize the cellular phenotype within living materials. We immobilized the budding yeast Saccharomyces cerevisiae in three different photocross-linkable triblock polymeric hydrogels containing F127-bis-urethane methacrylate, F127-dimethacrylate, or poly(alkyl glycidyl ether)-dimethacrylate. Using optical and scanning electron microscopy, we showed that hydrogels based on these polymers were stable under physiological conditions, but yeast colonies showed differences in the interaction within the living materials. We found that the physical confinement, imparted by compositional and structural properties of the hydrogels, impacted the cellular phenotype by reducing the size of cells in living materials compared with suspension cells. These properties also contributed to the differences in immobilization patterns, growth of colonies, and colony coatings. We observed that a composition-dependent degradation of polymers was likely possible by cells residing in the living materials. In conclusion, our investigation highlights the need for a holistic understanding of the cellular response within hydrogels to facilitate the synthesis of application-specific polymers and the design of advanced living materials in the future.

Biotechnology processes rely on the efficiency of microbial cell factories. The budding yeast Saccharomyces cerevisiae is an important cell factory but shows a limited native substrate-product spectrum. Non−conventional yeasts with diverse origins can potentially broaden this spectrum. Here, we benchmarked non−conventional yeasts Kluyveromyces marxianus (KM) and Rhodotorula toruloides (RT) against S. cerevisiae CEN.PK and W303 strains. We developed a computational method for quantification of cellular/organellar volumes and applied it for evaluating K+ and Na+ cations impact on yeasts. We observed an inverse relationship between the maximal growth rate and cell volume that was responsive to K+/Na+ cationic interventions. We found that the addition of certain K+ concentration to CEN.PK cultures containing 1.0 M of Na+ increased the specific growth rate by four−fold with a parabolic shift in cell and vacuole volumes. An impairment of ethanol and acetate utilization in CEN.PK, acetate in W303, at increased cation concentrations implied that K+−Na+ interactions interceded over the metabolic pathways required for consumption of respiratory substrates. The addition of cations induced trade−off in glucose utilization but alleviated cellular aggregates formation in RT and K+−Na+ interactions increased beta−carotene yield by 60% compared with the reference. Comparison of cell and vacuole volumes in the exponential phase showed that volumes decreased the most for KM and least for RT in response to K+/Na+ cations. Noteworthy for the implication in aging research using yeasts, vacuole to cell volume ratio increased with the increase in cell volume for W303 and KM, but not for CEN.PK and RT strains.