Micromorphological changes and mechanism associated with wet ball milling of Pinus radiata substrate and consequences for saccharification at low enzyme loading Alankar A. Vaidya ⇑ , Lloyd A. Donaldson, Roger H. Newman, Ian D. Suckling, Sylke H. Campion, John A. Lloyd, Karl D. Murton Scion, Te Papa Tipu Innovation Park, 49 Sala Street, Rotorua 3046, New Zealand highlights  Wet ball milling can provide high cellulose digestibility at low enzyme loadings.  Micromorphological changes with milling time are characterized by SEM and 13 C NMR.  A mechanism of ball milling proposed based on actual micromorphological changes.  Proposed mathematical model supports the mechanism of wet ball milling. graphical abstract article info Article history: Received 19 January 2016 Received in revised form 15 April 2016 Accepted 16 April 2016 Available online 20 April 2016 Keywords: Ball milling Enzymatic saccharification Microscopy Pretreatment Softwood abstract In this work, substrates prepared from thermo-mechanical treatment of Pinus radiata chips were vibra- tory 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 elec- tron 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 glob- ular 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. Ó 2016 Elsevier Ltd. All rights reserved. 1. Introduction Lignocellulosic biomass accounts for approximately half of the global biomass production, and is therefore an attractive feedstock for future bio-refineries (Zhao et al., 2012). Enzymatic saccharifica- tion of wheat straw, corn stover, switchgrass and energy crops such as Arundo has been implemented at the scale of a demonstra- tion and/or commercial plant (Brown and Brown, 2013; Janssen et al., 2013; Larsen et al., 2012). Large-scale enzymatic processing of other, more recalcitrant, lignocellulosic biomass such as soft- wood will require advances in pretreatment methods (Zhao et al., 2012). In plant biomass, cellulose is embedded in a matrix of hemi- celluloses and lignin. Pretreatment is required, in order to make http://dx.doi.org/10.1016/j.biortech.2016.04.084 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. E-mail address: Alankar.vaidya@scionresearch.com (A.A. Vaidya). Bioresource Technology 214 (2016) 132–137 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech