NADES pretreatment of ryegrass pressed cake for biosugar and γ–PGA production

Ryegrass is a key feedstock for European grass biorefineries, producing ryegrass pressed cake (RGPC) as a major but underutilized co-product. Valorising RGPC into high-value bioproducts such as poly-γ-glutamic acid (γ–PGA) requires an efficient pretreatment to enhance saccharification and fermentation. In this study, citric acid and glycerol were selected as NADES components because of their capacity to be utilised as carbon source by γ–PGA-producing Bacillus strains and act as hydrogen bond acceptors and donors in the formation of NADES. Four aqueous citric acid–glycerol NADES (molar ratios 1:1–1:4, with 10 % water) were evaluated for RGPC pretreatment. The influence of molar ratio and pretreatment time was optimized using a Taguchi orthogonal array design. The NADES pretreatment solubilised 72–84 % hemicellulose and 59–82 % lignin, while retaining 50–71 % cellulose. These changes enhanced enzymatic sugar yield by approximately 3.5-fold compared to untreated RGPC. Structural disruption in NADES-pretreated RGPC involving the cleavage of ether and glycosidic linkages support the compositional data. Taguchi analysis showed that both molar ratio and time significantly influenced sugar and total inhibitor yield. The predicted optimal pretreatment condition of Ca:Gly 1:2 and 2 h was validated and resulted in 95 % enzymatic sugar yield. Subsequent fermentation of the non-detoxified NADES hydrolysate produced a γ–PGA titre of 7.73 g/L. This study demonstrates the effectiveness of these tailored NADES pretreatment for lignocellulosic γ-PGA production. Further studies are needed to improve the γ–PGA titre from the NADES hydrolysate and extend the applicability of this pretreatment to other lignocellulosic biomasses.