Micromorphological changes and mechanism associated with wet ball milling of Pinus radiata substrate and consequences for saccharification at low enzyme loading

•Wet ball milling can provide high cellulose digestibility at low enzyme loadings.•Micromorphological changes with milling time are characterized by SEM and 13C NMR.•A mechanism of ball milling proposed based on actual micromorphological changes.•Proposed mathematical model supports the mechanism of wet ball milling.

In this work, substrates prepared from thermo-mechanical treatment of Pinus radiata chips were vibratory ball milled for different times. In subsequent enzymatic hydrolysis, percent glucan conversion passed through a maximum value at a milling time of around 120 min and then declined. Scanning electron microscopy revealed breakage of fibers to porous fragments in which lamellae and fibrils were exposed during ball milling. Over-milling caused compression of the porous fragments to compact globular particles with a granular texture, decreasing accessibility to enzymes. Carbon-13 NMR spectroscopy showed partial loss of interior cellulose in crystallites, leveling off once fiber breakage was complete. A mathematical model based on observed micromorphological changes supports ball milling mechanism. At a low enzyme loading of 2 FPU/g of substrate and milling time of 120 min gave a total monomeric sugar yield of 306 g/kg of pulp which is higher than conventional pretreatment method such as steam exploded wood.

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