Single-cell protein production from alfalfa biomass using Candida utilis and Komagataella pastoris

Alfalfa biomass contains significant carbohydrate fractions underutilized in animal feed. This research optimized the enzymatic hydrolysis of Alfalfa biomass and evaluated single-cell protein (SCP) production using Candida utilis and Komagataella pastoris . Hydrolysis kinetics in this study showed biphasic sugar release, with enzymatic optimization increasing monomeric sugar yield from 18.5% to a maximum of 33.0% (a 78% relative increase) at 22 mg protein/g biomass enzyme loading. C. utilis demonstrated superior metabolic versatility, consuming 23% more total sugars than K. pastoris , with particularly enhanced pentose utilization showing 45% greater xylose consumption. K. pastoris achieved 51% faster growth rates than C. utilis , while both yeasts produced comparable protein yields per unit sugar consumed. SCP production enriched protein content 2.1-fold compared to raw Alfalfa biomass, reaching approximately 40% crude protein. Essential amino acid profiling (tryptophan excluded) showed that fermentation substantially improved several essential amino acids relative to FAO/WHO reference values, particularly lysine, threonine, and methionine; however, methionine + cysteine remained below the FAO reference ratio in both yeasts ( K. pastoris : 0.99; C. utilis : 0.87), indicating that sulfur-containing amino acids remain a limiting factor despite overall nutritional improvement. The integrated bioprocess achieved 27.6% carbohydrate-to-biomass conversion efficiency and 11.3% carbohydrate-to-protein conversion efficiency based on measured monomeric sugars (oligosaccharide utilization during fermentation was not independently quantified). This work demonstrates a promising lab-scale strategy for alfalfa valorization through enzymatic hydrolysis and yeast fermentation, yielding a nutritionally improved protein product; techno-economic analysis, feeding trials, and scale-up studies are required before cost-effectiveness or industrial readiness can be established.