Plasma–freeze pretreatment reduces recalcitrance and enhances enzymatic reducing-sugar release from flax bast biomass

Flax bast biomass is a phloem-derived lignocellulosic substrate whose enzymatic conversion to reducing sugars is limited by a compact non-cellulosic matrix and restricted interfacial accessibility. A sequential plasma-freeze pretreatment (PF) was developed to reduce this recalcitrance by combining plasma-induced surface modification with a subsequent aqueous freezing followed by freeze-drying sequence. Under optimized plasma conditions (215 W, 52 sccm O2, and 4 min), the mass loss was 8.690%. At 72 h, PF achieved a cumulative reducing-sugar release of 174.83 ± 5.90 mg glucose equivalents/g initial dry fiber, 95.8% higher than that of raw flax bast biomass (RF) and 40.2% higher than that of the low-temperature plasma-only control (LTP); its mean value also exceeded those of ultrasonic and supercritical CO2 pretreatments. Comparisons with LTP and the aqueous freezing followed by freeze-drying control (FFD) indicated that plasma mainly increased the exposure of oxygen-containing functional groups and generated fine-scale etching, whereas the subsequent sequence promoted fiber-bundle separation and pore enlargement. PF increased the relative cellulose proportion from 62.53% to 71.08%, while decreasing pectin, lignin, and wax contents to 6.94%, 3.41%, and 2.93%, respectively. The surface O/C ratio increased from 0.2687 to 0.4700, and PF produced the highest specific surface area and total pore volume of 1.433 m2/g and 0.003394 cm3/g, respectively. The cellulose I crystalline structure was retained. PF showed higher Bacillus subtilis OD600 than RF and a larger late-stage rebound in liquid-phase reducing-sugar concentration during Aspergillus niger cultivation. PF offers a low-chemical-input pretreatment route for enhancing enzymatic reducing-sugar release from flax bast biomass.