Enhancing protein production and quality of Auxenochlorella protothecoides cultivated in side-stream hydrolysates via nitrogen supplementations and bioreactor scale-up

To advance sustainable production of high-quality alternative proteins, this study investigated nitrogen supplementation of a mixed hydrolysate medium (H) derived solely from brewer’s spent grain and soy whey to increase protein accumulation through scalable cultivation of heterotrophic Auxenochlorella protothecoides. Yeast extract (YE) supplementation to the hydrolysate medium (H+YE) achieved the highest biomass (YXmax: 24.19 g/L) and protein yield (PY: 7.21 g/L), followed by chicken carcass hydrolysate (YXmax: 19.95 g/L; PY: 4.95 g/L), arginine (YXmax: 16.76 g/L; PY: 5.05 g/L), and ammonium (YXmax: 15.43 g/L; PY: 3.95 g/L). With abundant endogenous nutrients, H+YE required 33% lower YE supplementation while achieving greater YXmax and PY than Bold’s basal medium (YXmax: 18.51 g/L; PY: 6.37 g/L), which contained equivalent glucose (∼50 g/L) and nitrogen (∼929 mg/L). Upscaled A. protothecoides cultivation in H+YE from flask to a 6.5-L bioreactor doubled biomass productivity (PXmax: 10.19 g/L/day), tripled specific growth rates (µ: 1.61 day-1), and reduced doubling time by three-fold (0.44 day), while maintaining (YXmax: 22.26 g/L). The resultant biomass exhibited high protein content and quality, with 28.67 % protein, 43.00 % essential amino acids (% total amino acids), 80.39 % protein bioaccessibility and 66.72 % potential protein bioavailability. Overall, the hydrolysate medium represents a promising organic basal medium for producing targeted biomolecules and demonstrates scalability. Moreover, the observed sequential nitrogen uptake in H+YE revealed the potential to replace YE with side-streams rich in preferential nitrogen species (NH4+, arginine), supporting a circular bioeconomy approach in alternative protein production