and Cellulose Accessibility Measurements To Better Elucidate the

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The application of fibre quality analysis (FQA) and cellulose accessibility measurements to better elucidate the impact of fibre curls and kinks on the enzymatic hydrolysis of fibres Richard P Chandra, Jie Wu, and Jack (John) Nicholas Saddler ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.9b00783 • Publication Date (Web): 17 Apr 2019 Downloaded from http://pubs.acs.org on April 20, 2019

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ACS Sustainable Chemistry & Engineering

Title: The application of fibre quality analysis (FQA) and cellulose accessibility measurements to better elucidate the impact of fibre curls and kinks on the enzymatic hydrolysis of fibres Authors: Richard P. Chandra*, Jie Wu, Jack N. Saddler *Corresponding author: Richard P. Chandra, [email protected] Affiliation: Forest Products Biotechnology/Bioenergy Group, Department of Wood Science, Faculty of Forestry, University of British Columbia, 2424 Main Mall, Vancouver BC, V6T 1Z4, Canada

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Abstract Fibre curls, kinks, micro-compressions, nodes, crimps and dislocations have been frequently associated with weak points in biomass fibres that exhibit increased accessibility to enzymes and chemicals. Rapid measurements using fibre quality analysis (FQA) showed that the curl and kink indices were increased by 300% when processing fibres at high solids loadings, but were readily reversed by applying a “straightening” treatment to the fibres. The curlation of fibres increased their susceptibility to shortening when they were exposed to endoglucanases and hydrochloric acid. Increased fibre curl also enhanced cellulose accessibility as measured by Simons staining (SS) and water retention value (WRV), and resulted in the formation of fibre networks with increased bulk. Upon straightening the fibres, these effects were reversed, with the exception of the increases in cellulose accessibility measured by SS and WRV. Inducing fibre curls and kinks also resulted in irreversible increases in cellulose hydrolysis yields of up to 17% that were more pronounced at higher solids loadings. The better hydrolysis at higher solids loadings was likely due to the well known tendency of curled fibres to form bulkier fibre networks with decreased fibre bonding. The results suggest that it should be possible to simultaneously increase cellulose accessibility when performing hydrolysis at high solids loadings by applying the appropriate physical treatments. It was apparent that the increased cellulose accessibility measured by SS and WRV and reflected by the enhancement in enzymatic hydrolysis yields, was a by-product of curl and kink induction and thus was not directly measurable using an FQA. Keywords: Fibre, Curl, Kink, Dislocation, Micro-compression Cellulose, Hydrolysis


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Introduction Lignocellulosic biomass is composed of fibres that are a highly recalcitrant natural composite of cellulose, hemicellulose and lignin. The high recalcitrance of biomass is primarily a result of its arrangement of substructures ranging from fibre cells that form tissues down to the molecular organization of cellulose, hemicelluloses and lignin 1-3. Even within each of the cellulose, hemicelluloses and lignin polymers, considerable variation and arrangement exists, including the crystalline and amorphous regions of cellulose, the side groups such as acetyl groups that decorate hemicellulose and the relative abundance of ether and carbon-carbon linkages that constitute the lignin macromolecule. Increasing the ease of enzymatic hydrolysis of carbohydrate components within the lignocellulosic substrate will be critical if we are to achieve the economical production of sugars from the cellulose and hemicellulose components. It is generally acknowledged that this recalcitrance originates from the various levels of arrangement from the fibre down to the elementary fibril. Due to the significant role that they play in final product quality, the properties of woody and plant fibres such as length, width, kink and curl have been studied for over a century in the pulp and paper industry4,5. The importance of these properties were the main impetus for the development of high throughput optical measurement techniques capable of measuring the characteristics of 10-50 fibres per second such as the fibre quality analyzer (FQA)6. However, the effects of fibre characteristics on the ease of enzymatic hydrolysis of cellulose contained within fibres are less clearly defined. Properties such as fibre length, which are typically measured in millimeters, are several orders of magnitude larger than the suggested rate-limiting-



pore-size for cellulose hydrolysis, which has been estimated to be in the 37-51 angstrom range7. However, there are several examples where it has been shown that shorter fibre lengths are more amenable to hydrolysis by cellulases8-10. Earlier work suggested that fibres with a smaller size had a higher surface area, thus the cellulose contained in the smaller fibres was easier to hydrolyze8-11,12. However, later studies attempting to maintain all other fibre characteristics constant while varying fibre size suggested that fibre length does not play a role in the ease of enzymatic hydrolysis13. Instead, It was proposed that the hydrolytic enzymes reduced the length of longer fibres to a constant size regardless of the initial length of the fibres 13,,14. In addition, the hydrolysis of biomass substrates at the initial stages was associated with the cleavage of fibres at points that were referred to as natural “dislocations”, nodes or “slip planes” in the fibre wall15-17. These areas of disruption were also observed in early pulp and paper research using polarized light microscopy 18-20. Several studies have surmised that these dislocations, nodes or slip planes observed at the fibre level could either be a manifestation of amorphous zones of the cellulose itself at the molecular level or areas of fibre where the cellulose had higher accessibility especially to enzymes and chemicals15,16. Early studies estimated that areas of so-called dislocation in fibre cells vary in length from 5-30um,21 thus they should be readily accessible by cellulases. These areas of disruption bear a close resemblance to the curls, kinks and micro-compressions that have been visualized in pulp fibres by polarized light microscopy and measured devices such as the fibre quality analyzer 18. These fibre defects exist naturally and can be readily induced by applying a mechanical action to fibres at high solids loadings18,22 . The presence of curly, microcompressed or kinked fibres has been directly associated with a decrease in fibre and thus paperstrength, as these zones of “dislocation” are associated with discontinuous defects in the


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fibre that act as weak points during the application of tensile force. However, the curl or micro compressions occurring in fibers can also be exploited, as curly fibres have increased extensibility, bulk, flexibility and tensile energy absorption.23,24 As mentioned above, the development of curls, kinks and micro-compressions usually results from the physical processing of fibres at high solids loadings (>20%). In the case of lignin containing fibres, the curl can be subsequently removed by stirring at low solids loadings at temperatures above 100oC which is in the range of the glass transition temperature of lignin18. It is hypothesized that the softening of the lignin allows the release of the tension contained in the micro-compressions18,25. In the case of fibres with a low lignin content, the generation of curls and kinks when applying physical action at high solids loadings can be released by “fibre straightening”. This is accomplished by the application of mechanical treatment at lower solids loadings16,26. Thygesen and others have undertaken numerous informative studies on measuring and linking natural fibre dislocations measured with confocal and polarized light microscopy, to enzymatic hydrolysis15,16,19,27-29. However, it is unclear if these “dislocations” which improve enzymatic accessibility to cellulose are represented by the rapid kink and curl measurement provided by instruments such as the FQA that typically analyze 20-40 fibres per second. Although, fibre defects are detrimental to fibre strength18,25,30, curl and kink induction duringhigh solids processing may be beneficial during enzymatic hydrolysis, if the induced kinks and curls can improve the ease of hydrolysis of the resulting biomass slurry. For the past decade, bioconversion research has aimed to subject fibres to enzymatic hydrolysis at the highest possible solids loadings, with the goal of increasing sugar concentrations to maximize ethanol concentrations, thereby decreasing distillation costs31,32. Therefore, it may be beneficial to



hydrolyze substrates at high solids loadings while simultaneously inducing curl to improve enzymatic hydrolysis yields. As well as the induction of curl at high solids loadings, the influence of straightening “curly” fibres on the ease of hydrolysis of the resulting cellulose has yet to be assessed. Therefore, the objective of this study was to determine whether the kinks and curls induced when processing biomass at high solids that are readily measurable using the FQA were indicative of the ease of hydrolysis of the fibre cellulose component. Since fibre defects are assumed to be areas of higher cellulose accessibility to chemicals and enzymes, typically fibre defects such as kinks and curls can also be quantified by analyzing fibre length using an FQA after a mild acid or enzymatic hydrolysis. Therefore, we also wanted to relate these measurements to cellulose accessibility measurements such as Simons staining and water retention value. As described below, the induction of kink and curl at high solids loadings resulted in increased cellulose accessibility and cellulose hydrolysis yields at both low and high solids loadings. Materials and Methods Pulp, enzymes and chemicals: Unbleached softwood kraft pulp was obtained from an industrial source in British Columbia, Canada. Direct Orange 15 dye was purchased from Pylam Products. Novozymes Ctec 3 cellulase and Fibrecare R endoglucanase enzyme preparations were generous donations from Novozymes. Hydrochloric acid was purchased from Fisher Scientific. Pulp chemical composition: The pulp substrate was analyzed according to the klason protocol from TAPPI standard method T222. Acid soluble lignin was measured through determining absorbance at 205 nm using UV-vis. Sugars were determined by Dionex (Sunnyvale, CA) HPLC (ICS-3000) equipped with an anion exchange column (Dionex CarboPac PA1). The pulp had a


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chemical composition of 86% cellulose, 5.9% galactan, 5.1% mannan, 0.3% xylan, 0.3% arabinan and 4,1% acid insoluble lignin,. The pulp was stored at 4oC. Curlation and Straightening: Pulp curlation was performed using the Unbleached kraft pulp suspended at 20% solids. The pulp was placed in the mixing bowl of a Kitchen Aid mixer (KitchenaidTM professional 6000 hd mixer) equipped with a conventional mixing head and allowed to mix on speed setting “5” for 1 hour stopping every 20 minutes to use a rubber spatula to remove any material from the sidewall of the mixing bowl. The pulp was subsequently placed in a sealed bag and stored at 4oC. A second curlated sample that was prepared as specified above was then diluted to 10% solids and subjected to PFI milling for 5000 revolutions according to previous work on fibre straightening18. Pulp tests: Standard pulp handsheets were formed from the fibres using Technical Association of the Pulp and Paper Industry Standard (TAPPI) T-220. Zero Span and Apparent Density tests were performed according to TAPPI Standard tests 231 and T-220 respectively. Fibre quality analysis (FQA). Fibre dimensions and fines content were determined using a high resolution fibre quality analyzer (FQA) (LDA02. OpTest Equipment, Inc., Hawkesbury, ON, Canada). The functioning and principles of the FQA is described in detail by Olson et al 6. The fibre quality analyzer combines image analysis using a polarizer and CCD camera with a flow cell to orient fibres hydro-dynamically in order to rapidly measure fibre properties. The FQA measures “curl index” by estimating the fibres’ “tip to tip” distance if the fibre were straightened without stretching. Multiple kink locations and the kink angle along the length of each fibre are measured using algorithms to calculate a kink index. The parameters on FQA were pre-set to



measure particles down to 0.07mm. Fines were defined as fibre lengths in the range of 0.07mm to 0.20mm. Fibres shortening by endoglucanase hydrolysis: The cellulolytic activity of the endoglucanase was expressed in Endo Cellulase Units (ECU). The endoglucanase hydrolyzes hydroxyethylcellulose, and the reducing sugars produced are assayed spectrophotometrically using dinitrosalicylic acid. One Unit of endo-cellulase activity was defined as the amount of enzyme required to release one μmole of glucose reducing-sugar equivalents per minute from hydroxyethylcellulose (10 mg/mL) in phospate buffer (pH 6, 100 mM). For the fibre shortening test, one hundred and fifty milligrams each of the starting curled and straightened fibres were suspended in water and allowed to shake in an orbital shaker at 50oC at 150 rpm. After 30 minutes of shaking, 20 ECU of endoglucanases were added to each fibre suspension and the reaction was allowed to proceed in the incubator at 50oC and 150rpm for 4 hours. After the reaction, the fibre suspension was placed in a boiling water bath for 30 minutes and then allowed to cool. The fibre suspensions were stored in the refrigerator prior to measurement on the FQA. The number of cleavages per fibre was calculated as described below for the hydrochloric acid hydrolysis. Fibre Shortening by Hydrochloric acid hydrolysis: Hydrochloric acid hydrolysis was used to measure the decrease in fibre length that resulted from acid hydrolysis of the curled and kinked regions of fibres. The reaction was performed using a slightly modified version of the method developed by Ander and Daniel33. In brief, 150 mg of fibres were swollen for 15 minutes in water using stir bar. The samples were subsequently filtered on a Buchner funnel without suction and then incubated in 40 mL 1 N HCl in a 50mL sealed falcon tube at 80oC for 4 hours in a rack that was placed in an 80oC water bath. The tubes were then placed in a shaker at 150 rpm at


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room temperature and allowed to cool for 30 minutes. The samples were then washed on a 10 cm Buchner funnel with water and then 100 mL 0.1N Phosphate buffer (ph 7.5) and resuspended in new 50 mL plastic tubes in 40 mL buffer solution with subsequent testing of the pH to ensure pH neutrality. The number of cleavages per fibre after either endoglucanase or acid treatment was calculated according to Ander and Daniel33 using the following equation: Equation 1: Cleavage per fibre = Lo/(L-1) Where Lo= length weighted fibre length for the control in water in mm L= length weighted fibre length for the endoglucanase or acid treated fibres in mm Simons’ Stain: The >100 kDa fraction of a Direct Orange 15 (DO) dye was used to measure the accessibility of cellulose, according to previous studies by Chandra et al34. In brief, a set of 10 mg (oven-dry basis) of material were mixed with PBS buffer and increasing concentration of DO dye in 2 mL screw cap tubes overnight. The tubes were then moved to a shaking incubator at 60 °C and speed of 180 rpm overnight. The tubes were subsequently centrifuged and the absorbance of supernatant at 450 nm was measured by a spectrophotometer. Water Retention Value (WRV): WRV was measured (in triplicate) using TAPPI Useful Method-256. In brief, approximately 0.5 g (oven-dry basis) of the pulp was soaked in 50 mL of water overnight prior to filtration through 200-mesh screen in the WRV unit. The resulting pulp pad was then centrifuged at 900g for 30 minutes at 25 °C. The sample was subsequently weighed and dried in the oven at 105°C overnight. WRV was calculated by the equation: Equation 2: WRV = (Wet mass – Dry mass) / (Dry mass)



Where Wet mass is the weight of wet sample after the centrifugation and Dry mass is the weight of the dried sample. Enzymatic hydrolysis: The enzymatic hydrolysis of Kraft pulps was performed in duplicate at 2% solids (w/v) in acetate buffer (50 mM, pH 5.0), using an enzyme loading of 10 mg of CTec 3 enzymes(commercial enzyme mixture from Novozymes) per gram of cellulose at 50oC and agitation at 150 rpm. Released sugars were measured on a glucose analyzer (YSI 2700 Select Biochemistry Analyzer) after 6, 24, 48 and 72 hours of hydrolysis. A second round of enzymatic hydrolysis was performed at a 10% solids loading (w/v) in 50 ml glass septa bottles. In brief, 0.5 g (oven dry basis) of each substrate was suspended in acetate buffer (50 mM, pH 5.0) with subsequent addition of 10 mg cellulase enzymes per gram of cellulose (commercial enzyme mixture Ctec 3). The mixture was incubated in a shaking incubator at 50°C and speed of 150 rpm for 48 hours.

Results and Discussion Fibre defects in pulp and pretreated biomass have been described using numerous overlapping terminology including “dislocations”,” slip planes”, “micro compressions”, “curl” “nodes” and “kinks”15,16,18-21.

These defects have been associated with weak points in fibres where either

enzymes or chemicals may have increased access or where fibres undergo tensile failure19,30. Instruments such as the FQA provide rapid measurements of curl and kink measuring as many as 40 fibres per second. However, it is unclear if the curl and kink quantified by the FQA represent changes at the nano-scale where enzymes would be acting to degrade cellulose. Pulp and paper studies have mostly focused on the removal of kinks and curls that are a consequence of fibre


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processing at high solids loadings.. Processing biomass at high solids is also critical for enzymatic hydrolysis during biological conversion, as hydrolysis at high solids loadings increases sugar concentrations to increase ethanol concentrations thereby reducing distillation costs31,32. The induction of fibre defects by processing biomass at high solids may also provide added benefits of increasing the susceptibility of the substrate to enzymatic hydrolysis. Therefore, the goal of this study was to use measurement tools such as the fibre quality analyzer, fibre shortening due to acid and endoglucanase hydrolysis, pulp strength tests and cellulose accessibility measurements. These tools were used to assess whether the induction of curls and kinks in fibres and their subsequent reversal through fibre straightening would affect cellulose accessibility and thus the susceptibility of the fibres to enzymatic hydrolysis. The FQA was used to measure the curlation and kinks of an unbleached softwood kraft pulp that contained each of the lignin, cellulose and hemicellulose components. The longer fibres of the softwood pulp furnish would also facilitate the visualization of the induced fibre curl and kinks using the FQA. Similar to previous work using an industrial baking mixer18, a household baking mixer was used to provide a compressive action to process the fibres at high consistency (20% solids) in order to avoid fibre cutting. The physical treatment at high solids almost quadrupled the curl index of the pulp (Table 1). Consequently, the measured length of the fibres also decreased after inducing fibre curl (Table 1). The induction of fibre curl was accompanied by only limited fibre cutting as the fines content (fibres with lengths in the range of 0.07mm to 0.20mm) remained the same for the pulp substrate samples. The high solids treatment also increased the amount of fibre kinks (Table 1). Low solids (10%) physical treatment was performed using a PFI mill to straighten the pulp fibres as described previously18. The lower consistency physical treatment has been thought to exert tensile forces on the fibres thereby



“pulling” the curls and kinks to restore fibre straightness35. Similar to previous work25,30, it was apparent that both the curl and kink indices decreased and thus were at least partially reversed after the application of the straightening treatment (Table 1). As mentioned in the Introduction, fibre kinks, curls and dislocations have been for the most part undesirable for pulp and paper applications due to their negative effects on fibre strength18,22. The zero-span tensile test is the established method to estimate the strength of individual fibres within a paper sheet. It was anticipated that the curlation treatment would decrease the zero span strength of the sheets due to the kinks and weaker points formed in the fibre during the curlation treatment. It was evident that the curlation treatment decreased the zero span tensile strength of the sheets, indicating a decrease in fibre strength. However, the removal of kinks and curls reversed the fibre strength loss, thus confirming the ability of the applied treatment to reduce the load bearing capability of the fibres (Table 1). The decreased density of fibre networks composed of curled or kinked fibres has been shown to impart “bulk”, swelling, accessibility and softness to tissue paper 18, 23,30. The bulk of the fibre networks (inverse of the sheet density) formed from the three pulps indicated that the curled fibres formed a more “bulky” network with more voids and a lower amount of fibre bonding (Table 1). As discussed below, when considering enzymatic hydrolysis, we hypothesized that curly fibres may have a lower association and thus may be easier to mix when enzymatic hydrolysis is performed at higher solids loadings. Similar to the results of zero-span and the FQA, straightening the fibres increased the apparent density. It is likely that the straightening of fibres using mechanical refining caused a greater fibre association and increased fibre bonding upon the removal of water from the fibre network26.


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Table 1. Initial fibre characteristics of unbleached softwood kraft pulp before and after curlation and straightening treatment as measured by the Fibre Quality Analyzer Sample

Starting pulp Curlated Straightened

Fibre lengtha Arithmetic Length mean weighted (mm) mean (mm) 1.87 1.53 1.70

2.62 2.22 2.49

Fines (%) 32 32 32

Curl index

Kink Index

Fibre Width (µm)

Zero Span Tensile (N/cm)

Sheet bulk (cm3/g)

0.11 0.39 0.13

1.1 3.4 1.5

28 28 28

27.4 (0.9)b 23.4 (0.8) 29.1 (1.0)

2.7 (0.1)c 3.1 (0.2) 1.8 (0.2)

a The

measurements of fibre length, width, fines content, curl and kink index using the fibre quality analyzer are the average measurement of 10,000 fibres. b

Standard deviation obtained from 5 measurements

c Standard

deviation from 5 measurements

As well as reducing fibre strength, fibre defects such as curls, kinks etc. are known to increase the susceptibility of cellulose to both chemical and enzymatic treatments16,17,28,29. Therefore, previous work by Ander and Daniel, compared the ability of hydrochloric acid and a cellulase cocktail (Endoglucanase 1 and Cellobiohydrolase 1) to characterize the dislocations of industrial and laboratory pulps33. This previous work showed that hydrochloric acid was more effective for characterizing the dislocations in pulp fibres. It was hypothesized that the protons released by the hydrochloric acid were able to readily access the dislocated regions of the fibres, while the cellulase cocktail acted via a delaminating action at the fibre surface33. In this study, rather than a full cellulase cocktail, an endoglucanase rich cocktail was applied. Previous work has shown that locations of fibre defects are highly attractive binding sites for endoglucanases during the initial stages of enzymatic hydrolysis 15.16. Therefore, the action of the endoglucanase was compared to that of the hydrochloric acid. The decrease in fibre length as a result of either the acid or the enzyme treatment was used to estimate of the amount of fibre defects contained in each pulp substrate. These values were used to calculate the cleavage per fibre33 after either the hydrochloric acid or endoglucanase treatment was applied.



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As anticipated, the curlated fibres were more responsive to the acid and endoglucanase treatments. Upon reversal of the curl and kink in the fibres using the mild mechanical treatment in the PFI mill, the cleavages per fibre decreased, indicating that effects of the curlation treatment could be at least partially reversible (Table 2). Unlike previous work that indicated that the application of a cellulase cocktail was ineffective in differentiating between fibres with varying amounts of fibre defects33, the endoglucanase hydrolysis seemed to exhibit comparable trends to the hydrochloric acid treatment (Table 2). As discussed above, it is likely that the protons released by the hydrochloric acid (100 kDa fraction of the Direct Orange (DO) dye component of the Simons’ stain to a given substrate has been shown to be highly specific for cellulose and reflects the ease of enzymatic hydrolysis of diverse sets of substrates34. Yu and Attalla36 hypothesized that the dye has an average size similar to the rate limiting substrate pore size required for enzymatic hydrolysis. In contrast to the >100 kDa molecular weight fraction of the Direct Orange dye, the water retention value technique (WRV), used frequently in pulp and paper measurements to characterize recycled pulp fibres37, utilizes water, which has a diameter of 0.275 nm. Regardless, the WRV test has also been shown to be an effective predictor of the ease of hydrolysis of several pretreated substrates38. It was quite apparent from the data that both the WRV and the DO dye adsorption indicated an increased accessibility of the curlated fibres (Table 2). Unlike the results of FQA and the fibre shortening using hydrochloric acid or the endoglucanases that reversed when the fibres were straightened, the WRV and Simons stain showed the fibres maintained their increased accessibility. These results may be due to duration of the tests as the endoglucanase and hydrochloric acid treatments were performed over a short period of 4 hours while the pulp was soaked for the WRV and incubated with the DO dye overnight, likely allowing the probes to reach all of the accessible points in the fibres.

The enzymatic hydrolysis of the starting pulp substrate, curlated and straightened fibres was initially performed at a solids loading of 2% to limit the interaction between the fibres as entanglement of the curled fibres may compromise the mixing of the fibre suspension. The



curlation of the fibres resulted in increased cellulose hydrolysis yields of up to 10%. Upon straightening the fibres, the increased hydrolysis yields of the fibres were maintained and increased even further (Figure 1). It is likely that the mechanical refining treatment that was used to straighten the fibres may have also increased the cellulose accessibility, which was also shown by the increased adsorption of the Direct Orange dye and the WRV (Table 2). These results were in contrast to the reversal of curl and kink that was measured for the straightened fibres using the FQA, which corresponded with decreased fibre shortening after treatment using the hydrochloric acid and endoglucanases. It is evident that the acidic and enzymatic fibre shortening are effective measurements for assessing fibre defects, but these tests only reflect a portion of the increased cellulose accessibility that resulted from the curlation treatment. The overall cellulose accessibility of the fibres provided by curlation treatment was more effectively estimated by tests such as the Simons stain and WRV. Regardless, when performing the enzymatic hydrolysis at 2% solids, it was apparent that the induction of kinks and curls in fibres as a result of mechanical treatment at high solids loadings provided nearly a 17% benefit in hydrolysis yields which was increased further to nearly 20% after the straightening treatment. Increased fibre association that occurs as a result of the removal of inter-fibre or “free” water has been well known to compromise the mixing of biomass fibre suspensions due to an increase in fibre-fibre interactions39,,40. The decrease in inter-fibre water is analogous to the formation of a sheet of paper where inter-fibre hydrogen bonds are progressively increased. The induction of fibre curls and kinks are well established to increase “bulk” in paper products23 which, as described above, are indicative of a lower density fibre network. Therefore, the solids loading was raised to 10% during enzymatic hydrolysis in order to assess whether the curlation treatment was beneficial at a higher solids loading. It was apparent that the approximate 17% increase in


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hydrolysis yield after curlation was maintained when the solids loading was raised to 10% (Figure 1). However, unlike the results observed at 2% solids, the high solids hydrolysis yields of the straightened fibres were lower than the curlated fibres. The results suggested that the straightened fibres may have formed a fibre network with a higher density that compromised the mixing of the fibre suspension. These results may be indicative of the higher “bulk” and lower fibre-fibre interactions known to be provided by networks of curled fibres41. The results also indicate the potential to simultaneously provide improvements in enzymatic hydrolysis yields by appropriate processing at high solids, without the need for additional chemical treatments.

Starting pulp Curlated Straightened

Cellulose hydrolysis yield (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


90.0

10% Solids Starting pulp

80.0

10% solids curlated 10% solids straightened

70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 0

24

Time (h)

48

72

Figure 1. Enzymatic hydrolysis of the starting, curlated and straightened fibres at solids loadings of 2% and 10% (w/v). Hydrolysis was performed at a pH of 5 in 50 mM sodium acetate buffer over a duration of 72 hours and 48 hours for the reactions at 2 and 10% solids respectively. (The average standard deviation between hydrolysis yields observed between replicates of hydrolysis experiments performed at 2 and 10% solids was 1.8% and 1.2% respectively)



Conclusions Fibre quality analysis, endoglucanase and acid hydrolysis were used to measure the effects of applying mechanical treatment at high solids to biomass fibres to induce curls and kinks. Although these methods can readily measure the induction of fibre defects that can be visualized by the FQA and light microscopy, they do not seem to exhibit the capability to estimate the increase in overall cellulose accessibility to cellulases that result from inducing fibre curl. This was demonstrated by the fact that the reversal of curls and kinks in fibres reduced the amount fibre defects, but the overall cellulose accessibility measured by Direct Orange dye adsorption (Simon staining) and the water retention value remained the same. The results also indicated that the inducing fibre damage may be an effective method of increasing hydrolysis yields of cellulose especially at high solids when mechanical treatment and enzymatic hydrolysis can be performed simultaneously. It was apparent that the combination of the bulkiness of the curled fibre network and its increased cellulose accessibility may be beneficial to the hydrolysis of biomass fibre suspensions. Acknowledgements The authors like to acknowledge the financial support from the Natural Science and Engineering Council of Canada (NSERC). The authors would also like to thank Novozymes (Davis, CA) for the provision of enzymes for this study.


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TOC Graphic

Curl and kink induction can enhance enzymatic hydrolysis for the production of sustainable sugars for conversion to fuels and chemicals.


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and Cellulose Accessibility Measurements To Better Elucidate the

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