Canola meal protein hydrolysate as a sustainable medium for heterotrophic cultivation of Euglena gracilis

Euglena gracilis, a Generally Recognized as Safe (GRAS) microorganism, is of growing biotechnological interest due to its ability to accumulate diverse cellular metabolites under heterotrophic conditions. This study evaluated canola meal protein hydrolysate (CMPH) as a protein-derived organic nitrogen source to partially substitute conventional complex supplements (yeast extract and tryptone) in heterotrophic E. gracilis cultivation. CMPH was produced by enzymatic hydrolysis of canola meal using trypsin (3000 mL^-1, pH 8.0, 37 °C, 24 h). The hydrolysate contained 74.28 ± 5.92 % protein (micro-Kjeldahl), and X-ray fluorescence analysis indicated phosphorus (6.8 %), calcium (16.7 %), potassium (1.7 %), and magnesium (1.2 %) as major mineral components. A Box-Behnken design coupled with response surface methodology (BBD-RSM) was applied to optimize growth conditions in CMPH-supplemented medium. Cultures inoculated at 1.0 × 10⁵ cells mL^-1 were incubated at 25 °C in the dark for 168 h. RSM identified pH 6.8, 1 g L^-1 CMPH, and 120 h as statistically optimal conditions within the tested design space, yielding 9.8 × 10⁵ cells mL^-1 in the absence of added glucose, with a specific growth rate of 0.69 ± 0.23 d^-1. Higher cell densities were obtained at increased CMPH concentrations and upon glucose supplementation, reaching levels comparable to standard Hutner's medium. These results indicate that the RSM-derived optimum reflects efficient nitrogen utilization rather than maximal biomass accumulation. Overall, CMPH demonstrates potential as a functional organic nitrogen supplement for heterotrophic E. gracilis cultivation, supporting the valorization of canola meal as a protein-rich agricultural by-product.