Integrated Chemical and Biochemical Treatments to Produce Protein and Microbial Lipid Food Ingredients from Ryegrass

Pretreatment methods play a pivotal role in the efficient breakdown of lignocellulosic biomass to produce highly digestible solids. Incorporating multistage or combined pretreatments provides increased efficiency and further carbohydrate depolymerization. Unlike other biomass feedstocks, ryegrass is a promising nutritional plant protein source with a protein content of 6-16%. In this study, protein extraction was combined with a mechanochemical pretreatment and biochemical fermentation stage to maximize the value of products from the system. To this end, the effectiveness of cold press, ball mill, and mild alkaline combined treatments on protein extraction was investigated, and the efficiency of enzymatic saccharification of the resulting solid material and its use as a medium for the oleaginous yeast Metschnikowia pulcherrima was explored. The cold press allowed the extraction of approximately 39% of the crude protein. Ball milling assisted with 5% Na₂CO₃ provided a drastic increase in the surface area and decrease in the particle size, albeit it did not significantly alter the structure in favor of enzymatic hydrolysis. A subsequent 0.5% NaOH pretreatment achieved enhanced fermentable sugar production with 45.6 g/L total sugars realized, notably 11-fold and 3.8-fold higher compared to untreated and mechanochemical-treated samples, respectively. M. pulcherrima efficiently metabolized all monosaccharides presented in supplemented ryegrass hydrolysates, yielding 0.20 (Y _m/m) biomass with a 38.7% lipid content, similarly in a synthetic medium. Apart from being a lignocellulosic feedstock-based fermentable sugar production system serving the microbial lipid bioprocess, cold-press integrated mechanochemical biorefinery was shown to be a promising approach in the extraction of plant protein, while preventing robust separation operations before the severe chemical biorefinery stages of ryegrass.