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ABSTRACT Hydrophobic feedstocks such as waste cooking oil have recently been considered for microbial biotransformation due to their abundance, low cost, and unique advantage for lipid‐derived fermentation products. Most fermentations with hydrophobic substrates are conducted at the tube or flask scale (less than 1 L total volume) or with the hydrophobic substrate comprising a small fraction of the media. Low substrate concentrations require additional feeding. Alternatively, high concentrations do not require significant dilution of the oil feedstock, which reduce volumetric requirements for larger scale fermentations. However, high‐oil‐density fermentations complicate efficient mixing and mass transfer challenges which are exacerbated at larger scales. To address this, computational fluid dynamics (CFD) models were explored to simulate three‐phase (hydrophobic, hydrophilic, and gaseous) bench (3 L) and pilot scale (4000 L) bioreactors, highlighting challenges and potential considerations. Bioreactor fermentations ofYarrowia lipolyticastrain L36DGA1 with substrate loadings of 5%, 10%, 20%, 30%, 40%, and 50% (v/v) waste cooking oil were also conducted, representing one of the highest concentrations in the reported literature. This work supports future research into and implementation of high‐oil‐density fermentations at the bench and pilot bioreactor scale. 

Abstract Engineered living materials (ELMs) have broad applications for enabling on‐demand bioproduction of compounds ranging from small molecules to large proteins. However, most formulations and reports lack the capacity for storage beyond a few months. In this study, we develop an optimized procedure to maximize stress resilience of yeast‐laden ELMs through the use of desiccant storage and 10% trehalose incubation before lyophilization. This approach led to over 1‐year room temperature storage stability across a range of strain genotypes. In particular, we highlight the superiority of exogenously added trehalose over endogenous, engineered production in yielding robust preservation resilience that is independent of cell state. This simple, effective protocol enables sufficient accumulation of intracellular trehalose over a short period of contact time across a range of strain backgrounds without requiring the overexpression of a trehalose importer. A variety of microscopic analysis including µ‐CT and confocal microscopy indicate that cells form spherical colonies within F127‐BUM ELMs that have variable viability upon storage. The robustness of the overall procedure developed here highlights the potential for widespread deployment to enable on‐demand, cold‐chain independent bioproduction.