Impact of Mechanical Downsizing on the Physical Structure and

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Impact of Mechanical Downsizing on the Physical Structure and Enzymatic Digestibility of Pretreated Hardwood J. Dennis Fougere, Megan Lynch, Jie Zhao, Ying Zheng, and Kecheng Li* Chemical Engineering Department, University of New Brunswick, 2 Garland Court, Fredericton, New Brunswick E3B 1X3, Canada ABSTRACT: When pretreating woody biomass for the production of cellulosic ethanol, a mechanical downsizing step is commonly included to ensure an appropriate particle size for enzyme hydrolysis. Different methods of mechanical downsizing will result in wood particles with markedly different physical structures. Dry grinding methods, such as knife-milling, will produce a powder-like substrate, which consists of cut or truncated fiber bundles. The substrate will also have a reduced pore volume because of the required drying. Using a disc-refiner, wet wood chips are separated into single wood fibers and loosened fiber bundles, increasing available surface area and avoiding pore collapse because of drying. The following study compared knifemilled and disc-refined substrates produced from native and dilute-acid-pretreated wood chips to determine the impact of the mechanical-downsizing method on the enzyme digestibility and physical characteristics of a hardwood substrate. For dilute-acidpretreated aspen, disc-refining produced a substrate that was 58−80% digestible, while knife-milling produced a substrate that was 24−36% digestible. The difference in substrate digestibility was partially attributed to hornification during the drying step and also attributed to differences in physical structure because of the downsizing method. Analysis via microscopy indicated that disc-refined substrates had a greater length, smaller width, and greater fibrillation then the knife-milled substrates. The discrefined substrates also had a more exposed cellulose surface and a greater volume of accessible pores.

1. INTRODUCTION Bioethanol produced from lignocellulose is a renewable alternative to petroleum-based fuels, which has the potential to lower carbon dioxide emissions and increase energy security. Commercialization of this technology is dependent upon a successful enzyme hydrolysis step, wherein more than 90% of substrate cellulose should be hydrolyzed to monomer glucose.1 To increase the enzymatic digestibility of a substrate, pretreatment technology is employed. Pretreatment uses chemical or mechanical action to alter substrate characteristics, thereby increasing the rate and yield of the enzyme hydrolysis reaction. Altered characteristics include physical features, such as surface area, pore volume, particle size, crystallinity, and cellulose degree of polymerization, as well as chemical characteristics, which includes the chemicals present and their distribution within the cell wall.2,3 These features are highly interdependent; for instance, a pretreatment that removes hemicellulose will not only alter the chemical composition but also increase the pore volume by removing materials that were previously blocking said pores.4 Understanding how pretreatment technology alters the lignocellulosic structure and how that structure then impacts substrate digestibility can aid in developing new pretreatment methods and improving current pretreatment methods. A common feature of pretreatment technology designed for forest biomass is the inclusion of a mechanical downsizing step. The purpose of this step is to reduce wood chips to an appropriate size to ensure that enzyme hydrolysis and chemical treatment efficacy is not affected by mass- and heat-transfer effects.5,6 There are several methods of mechanical downsizing that can process forest biomass, including knife-milling, hammer-milling, ball-milling, and disc-refining.7,8 Size reduction of wood chips by mechanical downsizing is accomplished by the repetitive application of compressive and/or shear stresses © 2014 American Chemical Society

to the biomass structure. Each type of mill applies a unique pattern of mechanical stresses to break apart biomass. Therefore, when an identical wood sample is passed through different mills, the resulting physical structure can differ greatly. Because the physical structure of lignocellulose is important for maximizing enzyme accessibility and adsorption, understanding how different methods of mechanical downsizing impact physical structure is important when evaluating a specific milling method. While the particle size is the primary variable manipulated by the downsizing process, other features of physical structure that can be altered or created include substrate morphology, surface features, lumen accessibility, accessible pore volume, and accessible surface area. Because direct physical contact between the enzyme and the substrate is required for hydrolysis, the amount of surface area available for such contact is of primary importance to the reaction rate.9 In reality, the available surface area is determined by many of the features mentioned, because it encompasses both the external and internal pore structures of a substrate. Cowling and Brown10 described these two main types of surfaces as the gross capillaries (external surface) and the cell-wall capillaries (internal surface). The external surface includes the gross surface of the fiber, the lumen, and the pit aperture and pit-membrane pores that are visible under light microscopy. The internal surface of the fiber includes those spaces between the microfibrils and the ends between the crystallites. The total external surface area for cellulose fibers is generally on the magnitude of 1 m2/g.9 In comparison, the internal surface area is generally 1−2 orders of magnitude Received: January 22, 2014 Revised: March 20, 2014 Published: March 23, 2014 2645

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greater that the outer surface area, ranging from 300 to 600 m2/ g for fully swelled fibers. However, enzyme accessibility to the pore structure is limited. The pore size of the capillaries can be as large as 20 nm, but many are smaller than 3 nm.9,11 Thus, the accessible surface in the pore structure is of particular importance when evaluating physical structure and substrate digestibility. Two common methods of mechanical downsizing are knifemilling and disc-refining. Knife-milling, much like hammermilling, is a common method of dry grinding for wood. Dry grinding is extensively used for many chemical pretreatments, where wood samples are ground to powder prior to chemical processing. For instance, in the comparative study of pretreatment technology for hardwood biomass by the Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI), wood samples were size-reduced using a Mitts and Merrill knife-mill prior to pretreatment. Because of the cutting action of the knife-mill, the produced substrate will generally consist of cut or truncated fiber bundles. Disc-refining is a method of mechanical downsizing, which is more common within the pulp and paper industry, where it is the predominant method of producing high-yield mechanical pulp. The substrate produced by disc-refining is generally more fibrous than that produced by grinding and consists of loosened fiber bundles and single fibers. Whether or not wood chips need to be dried is a significant concern for a pretreatment process using mechanical downsizing. Drying wood chips allows for a wider range of downsizing equipment to be used, and less energy is required to downsize dry wood than wet wood.12 However, drying wood is an extra cost in itself and will also result in hornification of the lignocellulose substrate. Hornification occurs when the pore structure collapses during drying, allowing for new hydrogen bonds to form within the cell wall.13 These bonds do not break after rewetting, and there is a corresponding loss of pore volume. Because accessible pore volume has been correlated with high enzyme hydrolysis yields, hornification can greatly reduce enzyme hydrolysis yields.14 To develop pretreatment technologies for wood biomass that can be used in industry, further research into the mechanical downsizing step is required. In particular, more detailed analyses of the physical dimensions and structure of downsized substrates are needed to differentiate between milling methods. Furthermore, there has been little work analyzing the mechanical downsizing of chemically treated wood chips. Treatment of wood chips rather than wood powder can reduce the energy consumed during the mechanical-downsizing step and is common practice in the pulping industry. Because chemical treatment generally results in complex changes to lignocellulose structure and chemistry, it is likely that chemically treated wood chips will physically deconstruct in a different manner than native wood when mechanically downsized. The following work thus aims to provide a general survey of common physical characteristics for a combined chemimechanical pretreatment using either a knife-mill or discrefiner and how these characteristics can impact enzyme digestibility. The potential use of disc-refining for bioethanol production is of particular interest, because of the widespread use of this technology for mechanical pulping. Within Canada, there are a number of mechanical pulping plants that have shut down because of the decreased demand for newsprint and increased operating costs. The equipment in these plants could be co-

opted for bioethanol production at a reduced cost, e.g., using disc-refiner technology for the mechanical-downsizing step. This would help to reduce the high capital investment that would be required for a large-scale bioethanol production plant. Aside from the biological processes used for enzyme hydrolysis and fermentation, the handling and pretreatment steps used in cellulosic ethanol processing are very similar to common pulping practices. Using the technology and expertise of the pulping industry, both the biofuels and pulping industries could benefit. The following study determines the impact of two common mechanical downsizing methods, knife-milling and discrefining, on the physical structure and enzyme digestibility of a hardwood substrate. Both methods were used to prepare sizereduced substrates from native and dilute-acid-pretreated aspen wood chips. Common process parameters, such as final particle size and extent of drying, were also manipulated to determine any additional impact on physical structure and digestibility. Produced substrates were analyzed for accessible pore volume by the solute exclusion technique (SET) and for substrate morphology and surface features by light microscopy and scanning electron microscopy (SEM).

2. EXPERIMENTAL SECTION 2.1. Materials. Aspen wood chips were graciously donated by AV Nackawic, located in Nackawic, New Brunswick, Canada. Under- and oversized wood chips were removed using a chip classifier, and the remaining chips were less than 8 mm in thickness and between 1.3 and 3.2 cm in length and width. After separation, fractionated chips were sealed and stored at 4 °C. Native wood chip consistency ranged from 50 to 55%. Commercial enzyme mixtures Celluclast 1.5L and Novozyme 188 were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. 2.2. Sample Preparation. A comparison of knife-milling and discrefining was made using six sample groups. Half of the samples were prepared using native wood chips, and the other half were prepared using acid-pretreated wood chips. Each set of wood chips (native or acid-pretreated) was downsized via one of three procedures: knifemilling (dried), disc-refining (wet), and disc-refining (dried). 2.2.1. Dilute Acid Treatment. Substrates that were acid-pretreated prior to downsizing are labeled acid treated (AT). Acid treatment of aspen chips was performed using a rotating autoclave-type digester. The digester used was an approximately 20 L pressure vessel with electrical heaters located on the top and bottom of said vessel. During treatment, the wood sample plus sulfuric acid liquor was contained within separate bombs, which rotated within the heated pressure vessel. The wood sample was stewed in the acid liquor overnight prior to the digester run to ensure complete mass transfer of liquor into the chip structure. The digester could hold up to four bombs, with each bomb having a working volume of approximately 600 mL. Bombs were held in a cage that rotated around the central axis of the vessel. This corresponded to a wood chip batch weight of 60 g oven dried (od) per bomb and 240 g od per digester run. Each bomb was run at 1.2% (w/ w) acid charge using sulfuric acid, with a solids loading of 20%. The treatment temperature was set at 170 °C for 20 min, with a temperature ramp of 2.5 °C/min from 100 °C to the set temperature. Cooling to 100 °C took less than 10 min. Following acid treatment, solids were filtered using Fisher P8 filter paper and thoroughly washed. Washing consisted of 0.3 L/g od wood using hot tap water (∼40 °C). Solids were then sealed, moisture-tested, and stored at 4 °C prior to further processing. 2.2.2. Mechanical Downsizing. Untreated and AT wood chips were downsized using either knife-milling or disc-refining. Knifemilling was carried out using a Thomas Scientific Model 4 Wiley Mill (800 rpm at 60 Hz or 667 rpm at 50 Hz, Swedesboro, NJ). Processing of wet wood chips using a Wiley mill will result in blockage of the exit screen and overheating of the motor. Thus, prior to milling, wood 2646

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Table 1. Chemical Composition of Untreated and AT Aspen acid treatment conditions

chemical composition (%)

sample

acid charge (%, w/w)

temperature (°C)

time (min)

mass yield

cellulose

hemicellulose

lignin

untreated AT

n/a 1.2

n/a 170

n/a 20

100 65.7

47.0 70.5

22.6 1.4

26.8 27.9

chips were air-dried for 7 days to ensure a moisture value below 10%. During milling, chip samples were hand-fed into the mill chamber and had to pass a set screen to exit said chamber. For the following work, a sieve size of 4 mm was used to produce powders for acid treatment. Following knife-milling, substrates were rewetted by soaking in excess water overnight. Excess moisture was then removed by filtering, and the samples were sealed and stored at 4 °C. Wood chips were disc-refined using a Kumagai Riki Kogyo (KRK) high-consistency disc-refiner. This is a single-disc model with a plate size of 12 in. and rotational speed of 3000 rpm. The disc gap was set between 1.0 and 2.5 mm for AT wood chips and 0.5−1.0 mm for native wood chips. Disc-gap settings were chosen to achieve a similar degree of refining for native and AT wood chips. Consistency of wood chip samples was adjusted to 25% prior to refining. The refining rate ranged from 50 to 52 g od/min. During the refining process, chips were hand-fed to a screw feeder that supplied the refiner chamber according to the set refining rate. At the end of the set time, the refining chamber was flushed with excess water to clear out any wood material that was stuck. The flushed refined sample was gathered in a 20 L container, and excess moisture was removed by filtering through a cloth bag. Following filtering, the substrate was sealed and stored at 4 °C. Wood chips that underwent refining were either never dried or airdried for the same period of time as the knife-milled samples. The airdried chip samples were rewetted prior to refining to 25% consistency. The samples produced by each downsizing method include Mill-D from knife-milling, ref-ND from disc-refining, and ref-D from airdrying followed by disc-refining. For the described samples, the D and ND labels refer to dried and never dried, respectively. 2.2.3. Bauer-McNett Fractionation. Mechanically downsized substrates were fractionated using a Bauer-McNett fiber classifier. This is a wet-screening technique, wherein the wood sample is moved through a series of screens using a constant, well-mixed water flow. For the following work, substrates were separated using 14-, 30-, and 50mesh screens. The resulting fractions include R14, P14−R30, and P30−R50. During screening, approximately 25 g od of wood substrate was fed to the classifier, with a screening time of 25 min. Operation of the Bauer-McNett classifier followed TAPPI T33-cm06. Fractionation is only used here to better determine differences in physical structure by better controlling the particle size of the pretreated substrates, and the fractionation process used would not be intended for an industrial process. 2.3. Compositional Analysis. Compositional analysis of prepared substrates was carried out on an extractive-free basis. Extractives were removed as detailed in the TAPPI T204 standard method. The cellulose and hemicellulose components were dissolved using a twostage process, wherein 0.3000 g of sample underwent strong acid hydrolysis in 3 mL of 72% sulfuric acid at 30 °C for 1 h. Samples were then diluted with 84 mL of deionized water to 4% sulfuric acid and underwent dilute acid hydrolysis at 121 °C for 1 h in a Tuttnauer autoclave. Filtrate was suction-filtered through fine glass crucibles, and the filtrate was analyzed by a high-performance anion-exchange chromatography (HPAEC) system, providing component glucose, xylose, mannose, rhamnose, galactose, and arabinose. The HPAEC system used was a Dionex-300 (Dionex Corporation, Canada) with a pulsed amperometric detector (PAD) and CarboPacTM PA1 column. Total lignin was determined as the addition of Klason lignin and acidsoluble lignin. Acid-soluble lignin was determined by ultraviolet (UV) spectrometry using a wavelength of 240 nm. Klason lignin was determined as the mass remaining following acid hydrolysis, minus ash. Ash was determined as described in TAPPI standard T 413. 2.4. Enzyme Hydrolysis. Digestibility of prepared samples was tested using enzyme hydrolysis trials run for 0, 2, 24, and 48 h.

Samples were hydrolyzed using Celluclast 1.5L, a complete cellulase system, and supplemented with Novozyme 188, a β-glucosidase. Dosing was set at 15 filter paper units (FPU)/g of glucan of Celluclast 1.5L and 22.5 cellobiase units (CBU)/g of glucan of Novozyme 188. Hydrolysis conditions were consistent at 50 °C, pH 4.8, and 2% solids loading. Hydrolysis was ended by boiling for 5 min, to ensure denaturation of cellulase enzymes. Samples were then cooled to room temperature in an ice bath and suction-filtered using Whatman no. 4 filter paper (catalog number 1004185). Enzyme hydrolysis yields were reported as cellulose conversion, and percentages were calculated on the basis of grams of sugar released per gram of component cellulose in the pretreated substrate. Glucose concentrations were determined using a YSI 2700D SELECT biochemistry analyzer (Transition Technologies, model 2700, Toronto, Ontario). 2.5. Microscopy and Image Analysis. Digital images of the fractionated samples were taken using a Leica DM4000M microscope. Slides were prepared using sample−water mixtures of less than 5% consistency. At least three slides were prepared, and a minimum of 30 images were taken per sample. Analysis of microscopy images was performed to estimate the mean particle length and width for each fractionated sample. ImageJ (http://rsbweb.nih.gov/ij/) was used to take length and width measurements from a minimum of 120 separate particles per sample. The particle size was estimated as the projected area (PA), equal to the product of the length and width of a particle. Field-emission SEM images of prepared samples were obtained using a Hitachi SU-70 Field Emission Gun SEM operated at 5 kV. Prior to imaging, samples underwent sequential dehydration in 30− 100% ethanol, followed by drying in a critical point dryer. Samples were then mounted on carbon tape and carbon-coated. 2.6. SET. The accessible pore volume of pretreated substrates was determined using the SET first developed by Stone and Scallan. This method determines the pore volume accessible to a set of dextran probes ranging in size from 0.4 to 50 nm. Dextran fractions and α-Dglucose used were obtained from Sigma-Aldrich Canada, Ltd. (Oakville, Ontario, Canada). Changes in the dextran concentration following the mixture with the wet substrate were determined using a Rudolph Autopol II polarimeter with a resolution of 0.001°, a 589 nm filter, and an accuracy of ±0.01° (Rudolph Research Analytical, Hackettstown, NJ). On the basis of the change in the concentration, the volume of the pore water that was inaccessible to a specific dextran fraction was determined. It was assumed that the pore structure was wholly inaccessible to the largest dextran fraction (∼50 nm), allowing for the calculation of a total specific pore volume, termed the fiber saturation point (FSP). The pore volume accessible to specific dextran fractions could then be determined as the FSP minus the calculated inaccessible pore volume.

3. RESULTS AND DISCUSSION 3.1. Enzyme Digestibility of Disc-Refined and KnifeMilled Aspen. The impact of two common downsizing methods on substrate digestibility was investigated. Native and AT aspen wood chips were downsized by disc-refining and knife-milling, using three separate procedures. The chemical compositions of the native and AT substrates are presented in Table 1 as the percent of total extract-free mass. Prepared substrates include Mill-D, ref-D, and ref-ND. Mill-D was produced by air-drying wood chips followed by knife-milling; ref-ND was produced by disc-refining wet wood chips; and refD was produced by disc-refining wet wood chips that had been previously air-dried. Furthermore, each substrate type was 2647

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cellulose conversion is severe, with a 33−49% loss in overall cellulose conversion, while for untreated substrates, the effect of air-drying was relatively small, with a 0−8% loss in cellulose conversion. This is reasonable, because the pore structure of the untreated substrates is already relatively inaccessible to cellulase enzymes; thus, further reductions in pore accessibility are not significant. For AT substrates, the accessible pore volume has been increased by the removal of hemicellulose. The decrease in the accessible pore volume because of drying therefore results in a relatively larger loss of digestibility, although the overall digestibility of AT substrates is still higher than that of untreated substrates. Given that air-drying can have a major effect on substrate digestibility, the Mill-D and ref-D substrates were compared. Because both these substrates have undergone air-drying, any hornification effects should be minimal. For AT substrates, the AT ref-D substrate was 20−48% more digestible than AT MillD, and for untreated substrates, ref-D was 112−121% more digestible than Mill-D. The increased digestibility of the discrefined substrate could then be accounted for by either differences in the particle size or differences in the physical structure because of the downsizing method (e.g., morphology, pore volume, and surface features). Past work on dilute acid pretreatment of hardwood indicated cellulose conversion rates of 60−90% depending upon specific conditions.15−17 Differences in the digestibility of dilute-acidpretreated substrates are generally attributed to specific reaction conditions, the apparatus used, and the initial/final particle size. A comparison of these results to those outlined in Figure 1 indicates that the AT ref-ND substrates fall within the expected range of digestibility, while both the AT ref-D and AT Mill-D substrates have relatively poor digestibility. One possible reason for the difference in digestibility is that the pretreatment methodology used for the following work, where wood chips are chemically treated and then downsized, differs from common dilute acid pretreatment, where wood is ground to powder and then chemically treated. However, Tian et al.18 used a similar acid-based methodology to pretreat hardwood and achieved substrate digestibility of 75−85%, following 24− 48 h of enzyme hydrolysis. These results agree well with the hydrolysis yields for the AT ref-ND. The low digestibility of the AT ref-D and AT Mill-D substrates is most likely attributable to hornification effects, because these substrates were air-dried prior to enzyme hydrolysis. In the referenced work, wood is milled prior to acid treatment and any substrate hornification will occur prior to the acid treatment. The acid treatment is able to create a new pore volume and, in effect, overcome the hornification. 3.2. Light Microscopy and Particle Size Analysis of Mechanically Downsized Substrates. In its native form, lignocellulose biomass consists of multiple fiber cells arranged in parallel orientation. The majority of cellulose mass is contained within the fiber cell wall, with the internal lumen being empty and the extracellular matrix consisting of primarily lignin. When this structure is mechanically downsized, the accessibility of internal and external surfaces to cellulase enzymes is not just determined by the size but also the morphology of the resulting particles. While the resulting particle size can be adjusted during a downsizing process, the particle morphology is more dependent upon the type of process being used. Prior to testing, prepared substrates were separated using 14-, 30-, and 50-mesh screens, i.e., R14, R30, and R50 fractions.

separated into size fractions to better control the impact of the particle size on substrate digestibility. While a fractionation step would not be used in an industrial pretreatment process, the two methods of mechanical downsizing used in the following work produce a substrate with a relatively wide particle size distribution. Thereby using fractionation in the following work, we were better able to determine differences in the physical structure between the two substrate types, i.e., Mill and ref. The fractions compared in the following work include R14 (i.e., retained on 14-mesh screen), R30 (i.e., passed 14 mesh and retained on 30 mesh), and R50 (i.e., passed 30 mesh and retained on 50 mesh). Figure 1 shows the enzyme hydrolysis yields of aspen substrates that have been downsized by either disc-refining or

Figure 1. Cellulose conversion to glucose following enzyme hydrolysis of (a) AT and (b) untreated aspen substrates produced by discrefining (ref) or knife-milling (Mill). Substrates were never-dried (ND) or air-dried (D) during preparation.

knife-milling. A comparison of the standard disc-refiner and knife-milling processes indicates that disc-refined substrates are significantly more digestible than the knife-milled substrates, as demonstrated by the cellulose conversion following 48 h of hydrolysis. Considering the AT substrates specifically, AT refND had a total cellulose conversion of 58.2−80.3%, while AT Mill-D was lower at 24.5−36.3% conversion. Similar results were found for earlier hydrolysis time points, i.e., 2 and 10 h, indicating that the initial hydrolysis reaction rates for the AT ref-ND substrate were also higher those for AT Mill-D. To determine the degree to which air-drying affects the comparison of knife-milled and disc-refined substrates, discrefined substrates that were never-dried, i.e., ref-ND, and discrefined substrates that were air-dried, i.e., ref-D, were compared. For AT substrates, the effect of air-drying on 2648

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Figure 2. Light microscopy images of the (a) R14, (b) R30, and (c) R50 size fractions of AT Mill-D and the (d) R14, (e) R30, and (f) R50 size fractions of AT ref-ND.

Table 2. Particle Size Analysis of Ref-ND, Ref-D, and Mill-D Substrates Produced from Untreated and AT Aspen Wood ref-ND untreated substrates

R14

length (μm) width (μm) PA (×103, μm2) AR

2410 106 257 23

R30

ref-D

Mill-D

R50

R14

R50

R14

R30

R50

1364 71 98 19 AT ref-ND

694 31 21 22

1963 126 248 16

950 45 43 21

1643 471 775 3

1321 317 420 4 AT Mill-D

658 151 100 4

AT ref-D

AT substrates

R14

R30

R50

R14

R50

R14

R30

R50

length (μm) width (μm) PA (×103, μm2) AR

1680 199 335 8

1404 164 231 9

849 59 50 14

2260 223 505 10

743 40 40 19

1770 402 712 4

632 157 99 4

547 102 56 5

While fractionation of prepared substrates was used to minimize the impact of the particle size on substrate digestibility, this process was not ideal. Because the process uses mesh screens, substrate morphology will influence fractionation, where more spherical particles of a diameter D will more easily pass through a given mesh size than more cylindrical particles of the same diameter. To determine differences in particle size and morphology, light microscopy images of prepared substrates were gathered. Figure 2 includes light microscopy images of the R14, R30, and R50 fractions of knife-milled and disc-refined AT substrates to demonstrate relative size and morphology. The AT ref-D substrate was not included, because it was similar to AT ref-ND in size and morphology. For a quantitative analysis, length and width measurements of imaged particles were taken using ImageJ. This is a similar imaging technique to that used by Zhu et al.19 Table 2 outlines the mean length and width of each prepared substrate as well as the calculated aspect ratio (AR) and PA. The AR was calculated as the ratio of the length to width and is a measure of substrate morphology. The PA was calculated as the product of the length and width and is used as an estimate of the overall particle size. On the basis of Figure 2 and Table 2, it can be seen that the Bauer-McNett process was partially successful at fractionating the Mill and ref substrates. Figure 3 illustrates a decrease in the relative size from R14 to R50 for both the ref and Mill

substrates, which is in agreement with the length and width measurements found in Table 2. However, there are still two problems with comparing substrates from a specific size fraction, i.e., R14 fraction of ref-D and ref-ND. First is that the particle size distribution for each fraction was still relatively wide, and thus, there is some overlap in size between the separate fractions. The second is that the different substrates were fractionated to different sizes, and in particular, the Mill substrates are on average larger than the ref substrates for a given size fraction. It appears that fractionation was largely determined by the particle length rather than the width, which is reasonable, given the use of mesh sieves. The exception is the R50 fraction of the AT substrates, where the PA of Mill and ref substrates only varies from 40 to 56 × 103 μm2. Comparing the ref-ND and ref-D substrates to the Mill-D substrate at these conditions indicates that the refined substrates are still 122 and 47% more digestible, respectively. While these substrates are comparable in terms of PA, there are still large differences in substrate morphology that may account for the digestibility values. Figure 2 illustrates this difference, with the Mill substrates consisting of short fiber bundles at all three size fractions, while the ref substrate consists of longer fiber bundles at R14 and R30, which are partially separated into single fibers at the R50 fraction. This is in agreement with the AR data from Table 2, where the Mill substrates range from 3 to 5, while the ref substrates range from 2649

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surface area. Furthermore, enzyme mass transfer to the internal surface of lignocellulose will be inhibited by a large particle size. Sangseethong et al.,20 in their analysis of microcrystalline cellulose, posited that the impact of the particle size on enzyme hydrolysis rates was due to differences in the pore depth. Even though the substrates tested had a similar accessible pore volume, an increased pore depth restricted mass transfer of enzymes into the inner surface, decreasing the hydrolysis yield. For lignocellulose biomass, this pore depth corresponds to the width of the cell wall. For a single fiber that is accessible from both the lumen and fiber surface, the pore depth is effectively half of the cell wall thickness. However, for a fiber bundle, where multiple fiber walls are connected, the effective pore depth is significantly greater. Therefore, as particle width is decreased, there should be a corresponding decrease in the pore depth, allowing for easier mass transfer of enzymes into the pore space. Past studies investigating the impact of the particle size on the enzyme hydrolysis of various lignocellulosic feedstock have generally determined size by the use of a sieve, similar to BauerMcNett fractionation.7,8,12 The conclusions of these studies have often been in disagreement regarding whether particle size affects hydrolysis yields and the relative size of this effect. As mentioned, sieving of a particle sample will generally separate particles based on their largest dimension, which is normally length. As Figure 4 demonstrates though, the relationship between the particle length and digestibility differs based on the downsizing method and resulting particle morphology. Thus, for a primarily fibrous material, such as woody biomass, particle size analysis via screening is not ideal. The wet-imaging method used by Zhu et al.,19 which provides a more detailed analysis of size in multiple dimensions, is more likely to be useful in determining the optimal degree of downsizing needed for the enzyme hydrolysis process. 3.3. SEM of Mechanically Downsized Substrates. SEM was used to determine differences in detailed morphology and surface features between ref-ND and Mill-D substrates. Figure 4 contains digital images of the R30 fraction of AT ref-ND and Mill-D at 200×, 1000×, and 4000× magnifications. On the basis of the LM images in Figure 2, both of these substrates are still comprised of primarily fiber bundles. However, closer analysis via SEM indicates that, even within the fiber bundles, there are clear differences in morphology. A comparison of AT ref-ND and AT Mill-D show that the types of fiber bundles formed by disc-refining and knife-milling are different. The AT ref-ND substrate shows significant loosening of the fiber bundle structure in Figure 4a, with parts of a single fiber wholly detached at points. These cracks in the bundle structure increases the surface accessible to enzymes. The single fibers that comprise the fiber bundles also appear to be relatively whole, with few noticeable openings or cuts. The fiber bundle structure of the Mill-D substrate does not show this loosening effect, and the individual fibers are tightly ordered. The cutting action of the knife-mill can also be seen in Figure 4d, where the lumens of multiple fibers have been exposed. This cutting effect may benefit the enzyme hydrolysis reaction by increasing access to the lumen. Surface features of the downsized substrates can be seen in Figure 4. For the AT ref-ND substrate, small threads can be seen on the cell-wall surface of the three fibers in Figure 4b and in a section of Figure 4c. These threads are exposed cellulose microfibrils, and their presence suggests that these areas are the exposed cellulose surface. Similar surfaces can be seen for the

Figure 3. (a) Particle length and (b) width of AT ref-ND, AT ref-D, and AT Mill-D substrates and their relation to cellulose conversion following enzyme hydrolysis.

8 to 22. There is also a noticeable decrease in AR between the AT and untreated ref substrates. This is likely due to increased cutting action during refining, because of the effect of acid treatment on the wood cell structure. To better understand the impact of particle size and morphology, Figure 3 outlines the relationship of substrate digestibility with particle length and width for the AT ref and Mill substrates. From Figure 3a, it can be seen that a decrease in particle length results in an increase in substrate digestibility, with a separate correlation for each substrate type. In comparison, Figure 3b shows that, when particle width and substrate digestibility are compared, the two dried substrates, AT Mill-D and AT ref-D, fall on a similar trend line. Furthermore, the AT ref-ND substrate appears to follow a similar shaped trend line with a higher y intercept. To clarify, it appears that particle width accounts for the variation in enzyme hydrolysis within the substrate group, e.g., AT ref-D R14 versus AT ref-D R50, and also between the sample groups, i.e., AT Mill-D versus AT ref-D. On the basis of these results, it appears that differences in particle width between AT Mill-D and AT ref-D may be the cause for the differences in substrate digestibility that were observed. Note that further data would be needed to make a more solid conclusion of the effects of particle length and width on substrate digestibility, particularly because particle width and length covary. The importance of particle width for enzyme hydrolysis is reasonable based on lignocellulose and fiber structures. For a fiber bundle, a reduction in length or width will liberate different amounts of external surface area for enzyme adsorption. Cross-sectional cutting of a fiber bundle will decrease the length and increase lumen accessibility, but if the lumen is already accessible, further cutting will only expose the cross-sectional area. When the particle width is reduced, the fiber bundle will generally by split at the fiber−fiber interface or along the cell wall, resulting in a greater increase of the external 2650

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Figure 4. SEM images of AT ref-ND at (a) 200×, (b) 1000×, and (c) 4000× magnifications and AT Mill-D at (d) 200×, (e) 1000×, and (f) 4000× magnifications.

Mill-D substrate in Figure 4e, although there appears to be less exposed microfibrils. Two forms of surface lignin can also be seen in panels c and f of Figure 4, one in the form of small, clustered spheres and the other in the form of a smooth sheet. This lignin sheet is most likely the lignin-rich middle lamella, and it can be seen that it shows different degrees of contact with the cellulose-rich surface. In certain areas, the middle lamella completely covers the surface, while in others, the middle lamella has been completely or partially separated from the remainder of the cell wall. The lignin spheres are possibly due to the condensation and movement of lignin during the high-temperature acid treatment of the aspen wood chips.21 On the basis of the surface features seen in Figure 4, it appears that

the ref-ND substrate has a more exposed surface cellulose, although further quantitative analysis is needed. 3.4. Accessible Pore Volume of Mechanically-Downsized Substrates. Accessibility of cellulase enzymes to the pore structure of lignocellulose is considered to be an important factor impacting enzyme hydrolysis yields. In particular, the pore volume accessible to a 5.1 nm dextran has been correlated with the enzyme digestibility of a substrate.11,22 Pore accessibility was therefore tested using dextran probes via the SET.23 The total pore volume as well as the pore volume accessible to a 5.1 nm dextran is shown for each of the downsized substrates in Figure 5. As seen, there are significant differences in accessible pore volume depending 2651

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sample drying required for knife-milling resulted in a loss of accessible pore volume because of substrate hornification, with a corresponding decrease in enzyme digestibility. The discrefining process also produced a substrate with physical characteristics distinct from the knife-milled substrate, including better separation of individual wood fibers, smaller particle width, and more exposed surface cellulose. In particular, the measured particle width appeared to be an important factor for cellulose digestibility. With regard to pretreatment technology, disc-refining appears to be a better choice than dry grinding for mechanical downsizing, because it can handle native biomass without prior drying and produces a more digestible fiber substrate.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +1-506-451-6861. Fax: +1-506-453-4767. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support of the Strategic Project Grant of the National Sciences and Engineering Research Council of Canada (NSERC−SPG), Irving Paper, and Resolute Forest Products Canada is gratefully acknowledged.

Figure 5. Accessible pore volume of disc-refined (ref) and knife-milled (Mill) substrates produced from AT and untreated aspen wood chips.

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upon sample drying and whether a substrate was AT. Acid treatment of a substrate has been shown to increase pore volume of a substrate via the removal of hemicellulose. For the ref-ND substrate, this increase can be seen for both total pore volume and the larger pore volume, i.e., accessible to a 5.1 nm dextran. In comparison, the ref-D and Mill-D substrates do not show the same increase. This is due to the effect of sample drying on the pore structure. Following significant drying, there is a permanent reduction in the pore volume, even if the sample is rewetted.13 As seen, the total pore volume of the AT ref-D and Mill-D substrates does not differ from the untreated substrates. This decrease in the pore volume is in agreement with the decrease in substrate digestibility seen for dried samples. However, the pore structure of the R14 fraction of the ref-D substrate has a higher accessibility to the 5.1 nm dextran than that of the Mill-D substrate. This may account for the higher digestibility of the ref-D substrate. As mentioned previously, the disc-refiner process is better able to fracture wood at the fiber− fiber interface, resulting in a smaller particle width and the separating of fiber walls. When fiber walls were separated, a previously inaccessible pore volume may be revealed. Furthermore, the disc-refiner process will often result in internal fibrillation, where the physical stresses of refining will aid in breaking bonds within the cell wall. This can also increase the pore volume accessible to cellulase enzymes.

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NOMENCLATURE AT = acid treated ref = disc-refined substrate Mill = knife-milled substrate D = air dried ND = never dried R14 = retained on 14 mesh R30 = passed 14 mesh and retained on 30 mesh R50 = passed 30 mesh and retained on 50 mesh od = oven dried SET = solute exclusion technique REFERENCES

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4. CONCLUSION Native and dilute-acid-pretreated aspen wood chips were mechanically downsized using either a disc-refiner or a knifemill, with the respective substrates tested for enzyme digestibility and physical structure. Disc-refining of aspen wood chips produced a fibrous substrate that was more easily digested by cellulase enzymes than the substrate produced by knife-milling. One reason for the increased digestibility was that the disc-refiner could be directly fed with a wet feedstock. The 2652

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