(529g) Self-Sustaining Hybrid Bioinorganic Chassis for Improved Fermentation to Produce Complete Flavored Food

Achieving zero hunger goals by 2030 encourage self sufficient, uninterrupted and net zero food production system that promises global food security [1]. Recent technological progress in genome design, synthesis, and construction is capable of creating such microbial chassis with better fermentation performances [2]. More improvement, in the microbial chassis, is possible through tools such as Saturation Mutagenesis and Adaptive Laboratory Evolution (ALE) [3]. However, to produce complete food, the edited microbe must contain all the nutrients such as protein, carbohydrates, lipids, fats, and flavor in the required proportions along with text and texture [3,4]. This requires too much genome engineering and prompts a high cellular burden, ultimately leading to failed process scale-up [2, 3, and 5]. Thus, from the single-cell microbial chassis, the manufacturing of completely customizable food with the taste, texture, and nutrients on demand with minimal inputs and physical footprint is a long shot. However, it is solved by formulating a synthetic consortia chassis of which biomass is qualified as a complete food [6]. Further, to achieve the net zero goal, the utilization of C2 compounds (acetate, ethanol, glycols, etc.) as feedstock of microbial fermentation is gaining acceptance [7]. But, for an economical and efficient food production system, the challenges of C2 compound toxicity, lower energy content, slow growth, and low yield need to be resolved [7, 8]. It can solved via strain engineering followed by SM and ALE tools [3,4]. However, the real challenge is breaking through the barrier to attain cheap acetate in adequate amounts [7]. Besides, another challenge is to reach a high C2 utilization rate and high biomass yield coefficient so that short-duration fermentation produces complete food (biomass) [7, 8]. To mitigate these challenges and to make the hybrid bioinorganic chassis-based fermentation system sustainable; the hydrolysate, obtained from the produced biomass is supplemented with acetate and utilized as growth media in the subsequent fermentation.

Here, a sustainable hybrid bioinorganic system is conceptualized and proposed as an efficient route to transform acetate (model C2 compound) into complete food. We apply the mixture design of experiments (mixture DoE) to formulate the synthetic consortium comprising engineered yeast, fungus, and bacteria to generate balanced food-grade biomass with desired flavors. For ALE experiments, shake flask studies are done with the pure strains to improve their performance to grow at 15g.L^-1 acetate. The DoE-based synthetic consortium chassis is tested for 48h in a 1.5 to 300 L fermenter followed by downstream processing to recover biomass. During fermentation, the growth media is minimal salt media supplemented with yeast extract. Later, the recovered biomass is hydrolyzed at pH 5.5, temperature 50 °C, and agitation of 1×g. The produced hydrolysate serves as growth media for fermentation to utilize and assimilate acetate via synthetic consortium chassis into biomass. Before fermentation studies, the hydrolysate is diluted with de-ionized (DI) water in a ratio of 1:1, supplemented with 15g.L^-1 acetate, adjusted to pH 6, and autoclaved. This is the demo of sustainable hybrid bioinorganic chassis-based fermentation technology to produce complete food from C2 compound.

References

[1] http://www.un.org/ sustainabledevelopment.

accessed on: 3/20/2024

[2] Kallscheuer, N. (2018). Engineered Microorganisms for the Production of Food Additives Approved by the European Union-A Systematic Analysis. Front. Microbiol. 9, 1746. doi: 10.3389/fmicb.2018.01746

[3] Ramírez, Rojas., A.,A., Swidah, R., Schindler, D. (2022). Microbes of traditional fermentation processes as synthetic biology chassis to tackle future food challenges. Front. Bioeng. Biotechnol. 10, 982975. doi: 10.3389/fbioe.2022.982975

[4] Graham, A., E., Ledesma-Amaro, R. (2023). The microbial food revolution. Nat Commun 14, 2231. doi: 10.1038/s41467-023-37891-1

[5] Fox, R. (2019). Too Much Compromise in Today's CRISPR Pipelines. CRISPR J. 2(3),143-145. doi: 10.1089/crispr.2019.0015.

[6] Qian, X., Chen, L., Sui, Y., Chen, C., Zhang, W., Zhou, J., Dong, W., Jiang, M., Xin, F., Ochsenreither, K. (2020). Biotechnological potential and applications of microbial consortia. Biotechnol. Adv., 40, 107500

[7] Kim, Y., Lama, S., Agrawal, D., Kumar, V., Park, S. (2021). Acetate as a potential feedstock for the production of value-added chemicals: Metabolism and applications, Biotechnol. Adv., 49, 107736

[8] Chen, R-P., Xia, F-P. (2024). Carbon recycling with synthetic CO2 fixation pathways, Curr. Opin. Biotechnol., 85, 103023