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

Significance Statement

In the recent article of Dr. Alankar Vaidya and colleagues from Scion which is a Crown Research Institute dedicated to improving the international competitiveness of the New Zealand forest industry. The authors describe an investigation of wet vibratory ball milling as an effective pretreatment, before the enzymatic saccharification of the most recalcitrant lignocellulosic substrates such as Pinus radiata softwood using relatively low enzyme inputs. In this study, wet ball milling mechanism is proposed (see the Figure), based on the observed micromorphological changes and changes in the cellulose crystallinity with different ball milling time. A mathematical model validates the proposed ball milling mechanism. This research work is now published in the journal, Bioresource Technology. According to the team, radiata pine wood chips obtained from a sawmill were treated with steam under pressure and then refined to produce substrate that was diluted with water prior to the ball milling.

One third of the glucan was converted to glucose at enzyme loadings as low as 2 FPU/g of dry substrate, after 60 minutes of ball milling. They attributed this rapid enzymatic conversion of cellulose exposed in fibrils torn from cell walls. They use scanning electron microscopy to characterize micromorphological changes in the substrates that were ball milled for different times. Based on the observed micromorphological changes, the cellulose present in the substrate is assigned to different categories. Carbon-13 NMR spectroscopy was also used in their study to investigate whether exposure of cellulose in the crystallites changed during the ball milling process.

Alankar A. Vaidya the first author in the paper said, ball milling performed for less than 120 min showed extensive fiber breakage and defibrillation of the broken fibers exposing more and more cellulose to enzymatic hydrolysis. However, over-milling caused compression of the porous fragments to compact globular particles with a granular texture, decreasing accessibility of enzymes to cellulose. From the carbon-13 NMR spectroscopy investigations, partial loss of interior cellulose in crystallites occur when the fiber breakage was completed. The authors found that wet ball milling performed for optimum time can provide highly digestible softwood substrate at low enzyme loadings. At a low enzyme loading of 2 FPU/g of substrate and milling time of 120 min a total monomeric sugar yield of 306 g/kg of substrate was obtained which is higher than conventional pretreatment method such as steam exploded wood.

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

About the author

Dr Alankar Vaidya completed Masters in Biochemistry in 1993 and then Masters in Biotechnology from Indian Institute of Technology, Kharagpur in 1996.  His doctoral work was on Design, synthesis and evaluation of affinity polymers for separation of enzymes under the guidance of Dr R. A. Mashelkar (FRS, FNAS) in the year 2000. His entire doctoral work was protected under 4 US patents.  He went to AIST Tsukuba, Japan in 2000 on ITIT fellowship where he worked on synthesis and evaluation of stimuli sensitive polymers for nucleotide separation. Thereafter, he was awarded two separate postdoctoral fellowships first in 2002 to work on Crosslinked Imprinted Proteins (CLIP’s) in the Lab. of Prof. Lutz Fischer at University of Hohenheim, Stuttgart, Germany and thereafter he secured DARPA postdoctoral fellowship award in 2004 from DOE to work with Prof. Richard Gross at Polytechnic University of New York, Brooklyn, USA where he worked on immobilized enzyme mediated polymer synthesis.

In 2009 he joined Scion (formerly Forest Research Institute) as a Scientist working on interdisciplinary fields such as biochemical route to biofuels, forest biorefinery, microbial biopolymers and production, isolation and application of enzymes in biomass hydrolysis or biomass valorisation.  He is author of 40 + scientific international publications including 3 book chapters. He was also granted 6 US patents to his credit.   

About the author


Ian Suckling received his PhD in Chemistry from the University of British Columbia in 1983. He is presently the Research Leader, Biofuels and Bioenergy at Scion and prior to this has held a number of leadership and managerial roles within Scion.

Dr. Suckling is currently the New Zealand representative on IEA Bioenergy Task 39 – Commercializing Liquid Biofuels. His research interests focus on wood and wood processing chemistry in both the biofuels and pulp and paper industries, with a particular interest in lignin. He is author of 100 + scientific publications.  

About the author

Dr Lloyd Donaldson, senior scientist, received his Master’s degree with honours in Plant Science in 1986 from the University of Canterbury and his doctorate in Wood Science in 2002 also from the University of Canterbury, New Zealand. 

He has worked at Scion (previously the New Zealand Forest Research Institute) since 1980 as scientist on wood anatomy and wood quality. In 2003 he was elected a fellow of the International Academy of Wood Science and since 2004 has served as associate editor for the International Association of Wood Anatomists Journal. From 2007 to 2012 he was president of the New Zealand Microscopy Society, and from 2013 has been the president of the Rotorua Branch of the Royal Society of New Zealand. In 2011 he received the Charles Fleming senior scientist award, and at various times has been visiting scientist at Swedish University of Agricultural Sciences, Beijing Forestry University, Institute of Multidisciplinary Research, University of Belgrade, and INRA, Reims, France.

His main research interests include wood structure/quality/properties relationships with emphasis on lignin topo-chemistry and microfibril angle, wood formation especially lignification, developing new techniques for quantifying wood structure and properties using confocal and electron microscopy, microscopy of genetically modified tissue/plants for phenotype evaluation, microscopy of biomaterials, fluorescence & electron microscopy, molecular microscopy, digital image processing & analysis, and wood identification. He is author of 111 scientific publications including 89 journal articles and 13 book chapters. 

About the author

John Lloyd, a scientist at Scion, received his master’s degree with honours in chemistry from the University of Auckland, New Zealand in 1971.  He has worked as a scientist in the fields of wood chemistry and wood and fibre processing since that time. His contribution to the present paper was in attrition, thermochemical pretreatment and analytical chemistry.

Wood extractives and their impact on fibre processing was his initial research interest before concentrating on research into the preparation of chemical wood pulp fibres and their processing.  During his career he has led or contributed to a large number of projects requiring both fundamental and applied research and industrial troubleshooting.  He has worked closely with pulp and paper companies in helping them understand their processes better and to improve product quality.

He is the author of more than 30 peer reviewed scientific publications as well as numerous conference papers and presentations. In recent years his research interests include exploring opportunities for wood as a source of chemicals, bio-based products and biofuels.

About the author

Sylke Campion received her MS in Chemistry and Environmental Protection in Neustadt / Weinstrasse in Germany in 1982 and worked at the University of Munich at the Institute for Water Research for 5 years.

She joined Scion (formally known Forest Research Institute) in Rotorua, New Zealand in 1993 as a technical assistant. She started in the paper and pulp division, PAPRO, and was intensively involved with the application of enzymes in the bleaching and strengthening of wood fibre pulp. Later, as a Senior Technician, she was involved in the manufacturing and testing of fibre cement boards for five years and from 2009 she is actively participated in lignocellulosic bioethanol initiative programme where she is involved in the production of ball milled pulp samples for subsequent enzymatic hydrolysis and testing of their digestibility into simple sugars for further ethanol production.

She is author of 19 scientific peer-reviewed publications and 194 technical papers.

Journal Reference

Alankar A. Vaidya, Lloyd A. Donaldson, Roger H. Newman, Ian D. Suckling, Sylke H. Campion, John A. Lloyd, Karl D. Murton, Micromorphological Changes and Mechanism Associated with Wet Ball Milling of Pinus Radiata Substrate and Consequences for Saccharification at Low Enzyme Loading, Bioresource Technology 214 (2016) 132–137.

Scion, Te Papa Tipu Innovation Park, 49 Sala Street, Rotorua 3046, New Zealand.


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