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The production of volatile fatty acids (VFA) is gaining momentum due to their central role in the emerging carboxylate platform. Particularly, the production of the longest VFA (from butyrate to caproate) is desired due to their increased economic value and easier downstream processing. While the use of undefined microbial cultures is usually preferred with organic waste streams, the use of defined microbial co-culture processes could tackle some of their drawbacks such as poor control over the process outcome, which often leads to low selectivity for the desired products. However, the extensive experimentation needed to design a co-culture system hinders the use of this technology. In this work, a workflow based on the combined use of mathematical models and wet experimentation is proposed to accelerate the design of novel bioprocesses. In particular, a co-culture consisting of Pediococcus pentosaceus and Megaphaera cerevisiae is used to target the production of high-value odd- and even‑carbon VFA. An unstructured kinetic model was developed, calibrated and used to design experiments with the goal of increasing the selectivity for the desired VFA, which were experimentally validated. In the case of even‑carbon VFA, the experimental validation showed an increase of 38 % in caproate yield and, in the case of enhanced odd‑carbon VFA experiments, the yield of butyrate and caproate diminished by 62 % and 94 %, respectively, while propionate became one of the main end products and valerate yield value increased from 0.007 to 0.085 gvalearte per gconsumed sugar. The workflow followed in this work proved to be a sound tool for bioprocess design due to its capacity to explore and design new experiments in silico in a fast way and ability to quickly adapt to new scenarios This work was financially supported by the project BIOCHEM (ERA-IB-2 7th call, ERA-IB-16-056) funded by MINECO (PCIN 2016-102) and Academy of Finland (311738). A. Regueira would like to acknowledge the support of the Xunta de Galicia through a postdoctoral fellowship (ED481B-2021-012). A. Regueira, M. Carballa and M. Mauricio-Iglesias belong to the Galician Competitive Research Group ED431C-2021/37, co-funded by Xunta de Galicia and ERDF (EU) SI

This study focuses on verifying the occurrence of elongation processes during protein mixed culture fermentation, without the supplementation of electron donor compounds. During casein mixed-culture fermentation at pH 5, it was observed that longer chain volatile fatty acid production increased, which could not be justified by the associated amino acid consumption. Consequently, the occurrence of chain elongation processes was hypothesized. To verify this hypothesis, three casein batch tests, with and without acetic acid initial supplementation, were performed at pH 5. The results suggest that acetic and propionic acids are indeed consumed to selectively generate n-valeric acid through the coupling with electron donor amino acids, whose consumption was further verified through the amino acid analysis. Prolonged simultaneous availability of suitable amino acids and short chain volatile fatty acids and an acid equivalent concentration threshold were identified as key parameters for the occurrence of chain elongation. The supplementation of acetic acid at the beginning of the test changed the selectivity of the elongation process, promoting n-butyric and iso-valeric production. The associated mechanisms were preliminary conceptualized, constituting a first step for further studies on the subject. To the best of our knowledge, this is the first study demonstrating the feasibility of chain elongation processes during protein mixed culture fermentation without electron donor supplementation This project has received funding from the Spanish Government through BIOCHEM project (PCIN-2016-102, ERA-IB-2 7th call, ERA-IB-16-052) SI