BACKGROUND This work explores the feasibility of vinasse as an inexpensive feedstock for industrial biocatalyst production within the context of an integrated sugar beet biorefinery. As an exemplar, production of CV2025 omega-Transaminase (omega-TAm) in Escherichia coli BL21 was studied. RESULTS Characterisation of vinasse showed that it comprised mainly of glycerol along with several reducing sugars, sugar alcohols, acetate, polyphenols and protein. Preliminary results showed E. coli BL21 cell growth and CV2025 omega-TAm production were feasible in cultures using 17% to 25% (v/v) vinasse with higher concentrations demonstrating inhibitory effects. The d-galactose present in vinasse facilitated auto-induction of the pQR801 plasmid enabling CV2025 omega-TAm expression without addition of expensive Isopropyl-beta-d-thiogalactopyranoside (IPTG). Assessment of different vinasse pre-processing options confirmed simple dilution of the vinasse was sufficient to reduce the concentration of polyphenols to below inhibitory levels. Optimisation experiments, carried out using a controlled, 24-well microbioreactor platform, showed supplementation of diluted vinasse medium with 10 g L-1 yeast extract enabled enhancements of 2.8, 2.5, 5.4 and 3-fold in specific growth rate, maximum biomass concentration, CV2025 omega-TAm volumetric and specific activity, respectively. Investigation into the metabolic preferences of E. coli BL21 when grown in vinasse showed a preference for D-mannitol utilisation before simultaneous metabolism of glycerol, d-xylitol, d-dulcitol and acetate. Scale-up of optimised conditions for batch CV2025 omega-TAm production to a 7.5 L stirred tank reactor (STR) was demonstrated based on matched volumetric mass transfer coefficient (k(L)a). The results showed good comparability with respect to cell growth, substrate consumption and CV2025 omega-TAm production representing over a 700-fold volumetric scale translation. Further enhancements in CV2025 omega-TAm production were possible in the STR when operated at higher k(L)a values. CONCLUSION This work describes the promising application of vinasse for production of microbial enzymes and insights into carbon source utilisation in complex feedstocks. Exploitation of vinasse as a fermentation feedstock could be further extended to other processes involving different microorganisms and target enzymes. (c) 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.