- Email: [email protected]
Production of cellulose nanofibers using phenolic enhanced surface oxidation
Accepted Manuscript Title: Production of cellulose nanofibers using phenolic enhanced surface oxidation Authors: Iman Beheshti Tabar, Ximing Zhang, Jeffrey P. Youngblood, Nathan S. Mosier PII: DOI: Reference:
S0144-8617(17)30695-1 http://dx.doi.org/doi:10.1016/j.carbpol.2017.06.058 CARP 12446
To appear in: Received date: Revised date: Accepted date:
31-3-2017 13-6-2017 15-6-2017
Please cite this article as: Tabar, Iman Beheshti., Zhang, Ximing., Youngblood, Jeffrey P., & Mosier, Nathan S., Production of cellulose nanofibers using phenolic enhanced surface oxidation.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.06.058 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Production of cellulose nanofibers using phenolic enhanced surface oxidation Iman Beheshti Tabara, Ximing Zhanga, Jeffrey P. Youngbloodb, Nathan S. Mosier*a a Laboratory of Renewable Resources Engineering and Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, Indiana, United States b School of Materials Engineering, Purdue University, West Lafayette, Indiana, United States. * To whom correspondence should be addressed. Email: [email protected]; Tel.: +1 (765) 494-7022, FAX: +1 (765) 494-7023 Author emails: Iman Beheshti Tabar: [email protected]; Jeffrey P. Youngblood: [email protected]; Ximing Zhang: [email protected];
2
Highlights
Use of a wood by-product with ozone offers a novel pathway to cellulose nanofibers. Syringic acid as lignin model compound enhanced ozone cellulose surface oxidation. Syringic acid, a byproduct of wood pulping, is a biorenewable oxidation enhancer. Enzyme treatment increased cellulose surface reactivity toward oxidation. New cellulose nanofiber pathway could have significant process advantages.
ABSTRACT. In this study we demonstrate that lignin monomers formed as byproducts of pulping or bioprocessing of lignocellulosic biomass is an effective enhancer to oxidize cellulose surfaces with ozone for the production of cellulose nanofibers (CNF). Never dried softwood pulp with minimum mercerization was enzymatically treated leading to a homogeneous pulp slurry with a higher reactivity. The slurry was oxidized by ozone gas in the presence of syringic acid, a lignin degradation model compound, as an oxidation enhancer at room temperature and pH 11. Transmission electron microscopy (TEM) observations showed that stable CNF bundles with 310 nm widths and lengths >100 nm were obtained after ultrasonication of the oxidized product in water. Extensive characterization of the new CNF films revealed the nanofibers had carboxylate content similar to conventional carboxylated cellulose prepared by TEMPO-mediated oxidation. Based on NMR spectra, chemical conversion of the syringic acid during oxidation is proposed.
KEYWORDS: Cellulose nanofibers; phenolics; surface oxidation; carboxyl groups Chemical compounds studied in this article Syringic Acid (PubChem CID: 10742); Ozone (PubChem CID: 24823); Bicinchoninic Acid Disodium Salt Hydrate (PubChem CID: 71308346); Sodium hydroxide (PubChem CID: 14798); TEMPO (PubChem CID: 2724126); Sodium hypochlorite (PubChem CID: 23665760); Sodium
3
bromide (PubChem CID: 253881); Uranyl acetate (PubChem CID: 10915); Sodium carbonate (PubChem CID: 10340) 1. Introduction Co-production of cellulose nanofibers (CNF) in paper mills and cellulosic ethanol plants can add value and diversity to the product portfolio (Zhu, Sabo, & Luo, 2011). Preceding any disintegration step, native celluloses need some form of surface modification to prevent aggregation of the fibrils. The common, extensively studied method of surface modification for CNF is TEMPO-mediated oxidation for selective conversion of primary C6 hydroxyl groups to charged caboxylates (Isogai, Saito, & Fukuzumi, 2011; Okita, Saito, & Isogai, 2010; Saito, Nishiyama, Putaux, Vignon, & Isogai, 2006). However, alternative methods of CNF oxidation without the use of hard-to-recycle corrosive chemicals such as TEMPO, NaBr, NaClO, while maintaining similar properties, are desirable. A lower cost oxidation process, using an easily produced and safer oxidant like ozone gas, could spur the development of new applications and markets for CNF. While ozone is a strong oxidant, reaction of ozone with cellulose leads to a minor oxidation reaction mostly at the C-6 position (Kato et al., 1999; Lemeune, Jameel, Chang, & Kadla, 2004). It is well known that advanced oxidation processes (AOPs) leading to formation of hydroxyl radicals have a higher oxidation potential than the conventional oxidizing agents alone. Ozone gas in combination with other oxidation enhancers such as UV light have successfully been used to oxidize the surface of cellulose (Gashti, Pournaserani, Ehsani, & Gashti, 2013; Kato et al., 1999). However, effective irradiation distance and UV light attenuation in large scale concentrated pulp processing can be problematic (Son, Lim, Khim, & Ashokkumar, 2012). Previous studies have reported that the presence of small amounts of lignin degradation products in pulp during Total Chlorine Free
4
(TCF) ozone bleaching enhances the formation of hydroxyl radicals, which in turn form carbonyl bonds on the cellulose surface (Pouyet, Chirat, Potthast, & Lachenal, 2014). The increased formation of radicals is attributed to the phenolics in lignin and other unsaturated compounds that are highly reactive toward ozone. The causes for the formation of hydroxyl and other radicals have been a matter of discussion and several mechanisms are proposed (Chirat & Lachenal, 1997; Pouyet et al., 2014; M. Ragnar, Eriksson, & Reitberger, 1999). Both paper mills and cellulosic ethanol plants have waste streams containing lignin degradation products which could be utilized in CNF surface oxidation. The most prominent fractions of these lignin waste streams in pulp black liquor include methoxyphenols such as vanillic acid, syrinigic acid and guaiacol (Löwendahl, Petersson, & Samuelson, 1978; Sun & Tomkinson, 2001). This study investigates the production of CNF by taking advantage of formation of carbonyl groups on cellulose by ozone gas in the presence of lignin-derived phenolic compounds. Moreover, reactivity of cellulose is a key parameter when modifying the surface of native cellulose. Due to the steric hindrance in the cellulose macromolecule, the primary reaction of oxidants is cleavage of the glycosidic linkage at the C1 position or oxidation of the primary C6 hydroxyl (–OH) groups (Hirota, Tamura, Saito, & Isogai, 2010; Lemeune et al., 2004; Okita et al., 2010). Mild enzyme treatment, using a pure endoglucanase, is known to reduce fibrillation energy and increase the surface reactivity of pulp to oxidation by increasing the availability of primary –OH groups (Östberg, Håkansson, & Germgård, 2012). Successful attempts to increase the reactivity of pulp for the production of rayon and other derivatives using endoglucanase enzymes have been reported (Engström, Ek, & Henriksson, 2006; Henriksson, Christiernin, & Agnemo, 2005). In this study we also investigate the efficacy of the cleaving action of endoglucanases in increasing the number of reducing ends leading to increased availability of
5
primary hydroxyl groups for oxidation. Increased surface reactivity is expected to translate into an increased degree of substitution, thereby an improved electrostatic repulsion after surface modifications. In this paper we investigate enzymatic treatment coupled with surface oxidation enhanced by lignin-derived phenolics to maximize the surface charge imparted on cellulose. We build upon previous findings regarding the substantially lower amount of mechanical energy required to prepare CNF after an enzymatic treatment and complement it with surface oxidation to enhance CNF quality (Pääkkö et al., 2007). Enzyme treated samples are oxidized by ozone gas in the presence of syringic acid and the characteristics of the resulting suspension (S-CNF) is compared with TEMPO mediated CNF (T-CNF).The goal of the study is to demonstrate that lignin-derived phenolics can be used in conjunction with ozone gas to produce CNF from enzyme treated cellulose. 2. Experimental 2.1. Materials Never dried DED bleached Kraft hardwood pulp with a water content of 60% was complimentary provided by the Forest Products Laboratory (U.S. Forest Service, Madison, WI). The pulp was stored at -18 ˚C before use. The chemical composition of the pulp was 93.2% glucan, 5.9% xylan, 0.8% Klason lignin as determined using the procedures described by National Renewable Energy Laboratory, Laboratory Analytical Procedures (LAP) (Sluiter et al., 2008; 2012; 2005). Depol 692L enzyme (Cellulase 800 u/g) and cellulase 13P were purchased from Biocatalyst, UK. Syringic acid, TEMPO, sodium bromide, 15% sodium hypochlorite solution, as well as chemicals for the cellulose reactivity measurement, disodium 2,2’bicinchoninate, Na2CO3, NaHCO3 and L-serine were of laboratory grade (Sigma Aldrich,
6
Urbana, IL). The enzyme and chemicals were used as received. Ozone gas was generated using a corona tube ozone generator (O3Co, Idaho Falls, ID, USA) and GC grade compressed O2 (Indiana Oxygen Co, Lafayette, IN, USA) and ozone concentration was measured using a UV106H ozone analyzer (Ozone Solutions, IA, USA). 2.2. Cellulose Surface Reactivity Measurement The amount of available reducing ends on the cellulose was measured by the spectrometric 2,2’-bicinchoninate (BCA) method as suggested by Zhang and Lynd (2005). Replicates of two different loadings of commercial endoglucanase Depol 692 and 13P with high endoglucanase C and low β-glucosidase activity were tested. Standard glucose solution was made within the BCA assay range of 0-50 µM glucose equivalent and BCA reagent was made fresh daily from the working solution. Enzyme treatment was carried out as described below. Pulp was recovered by centrifugation for 2 mins, (10,000 g) before absorbance measurement at 560 nm to eliminate interference. Background absorbance from a BCA blank reading was deducted from sample absorbance measurements. 2.3. Enzymatic Fractionation A stock 1% w/v solution of dispersed pulp was made by blending 10 g (dry mass) of never dried pulp in 1000 ml of deionized water for 5 min using a Ninja blender (Ninja NJ600 Professional Blender). The blending increased the fiber swelling in water, making the cellulose more accessible to the enzymes (Praskalo et al., 2009). Enzymatic pretreatment was carried out as follows: 150 ml of the stock solution was mixed with an equal amount of 50mM citrate buffer to maintain enzyme activity optimum pH of 4.8, bringing the solids loading to 0.5% w/v. Enzyme loading (Depol 692 and 13P) was 0.25 ml/g cellulose and the solution was incubated at 50 °C for 24 hours with agitation provided by a rotary shaker at 200 rpm. Enzyme treatment was
7
terminated by incubating the samples for 30 minutes at 80 °C using a beaker on heated stir plate. The pH of the 300 ml solution was then lowered to ~2 using 2M HCl and kept at room temperature for 2 hours to demineralize the pulp and remove excess charges adsorbed to the pulp surface during the pulping process (Rodionova et al., 2013; Shinoda, Saito, Okita, & Isogai, 2012). The high volume of enzyme loading was to eliminate the effect of other variables on the increase in pulp reactivity when comparing syringic acid/ozone treatment to TEMPO/hydrogen peroxide treatment. After treatment the fiber suspension was washed with excess deionized (DI) water until the pulp suspension reached pH 7 exhibiting a white color (Isogai et al., 2011). The fibers were then sonicated for 10 minutes with an ultrasound microtip (Branson, USA). After redispersion of the fibers, pH was increased to 11 and ozone oxidation was carried out. 2.4. Catalyzed Ozone Oxidation Prior to oxidation, the proteins and HCl were removed by washing the enzyme treated/acid soaked pulp using a vacuum filter. Syringic acid powder (0.25g) was added to 150 ml of 1% w/v enzyme treated pulp. The solution pH was raised to 11 using 1 M NaOH to increase the reactivity of the cellulose hydroxyl group. Ozone-containing gas was applied at a flow rate of 0.25 L/min with a concentration of 18 mg/L (1.26 wt. %). The pulp suspension was constantly stirred on a stir plate and the reaction was carried out in a fume hood at room temperature (20°C). Solubility of ozone in water (saturation concentration of ozone to applied gaseous ozone concentration) decreases at higher pH as the rate of decomposition increases. Therefore, solubility coefficient can be expected to be even lower than 0.35 as reported in the literature (Gurol & Singer, 1982). Neglecting decomposition, the amount of ozone inserted into the solution over the 8 hour experiment time is about 0.76 grams. As with the enzyme loading the amount of ozone applied to the experiment has not been optimized and is in excess to more
8
explicitly differentiate ozone treatment from the controls. After terminating the oxidation reaction the suspension was washed in a vacuum filter, re-suspended in 150 ml of DI water and sonicated for 5 minutes. 2.5. TEMPO-Mediated Oxidation Enzyme treated pulp (1.5 g) was suspended in water and TEMPO-mediated oxidation was carried out according to the methodology previously described with slight modifications as described below (Okita et al., 2010; Peyre, Paakkonen, Reza, & Kontturi, 2015; Rodionova et al., 2013; Saito, Kimura, Nishiyama, & Isogai, 2007; Shinoda et al., 2012). The TEMPO-pulp served as a control to compare with the catalyzed ozone oxidized CNF. Excessive addition of the TEMPO and NaBr catalysts causes severe degradation of the pulp, hence the amounts suggested in the literature (TEMPO 0.0025 g/g and NaBr 0.025 g/g cellulose) were followed. Some modifications to the procedure included a stepwise addition of 0.5 ml of NaClO (15% chlorine concentration) every 30 min, which was more efficient for oxidizing the cellulose surface. The slurry was under continuous stirring using a Coleman Parker stirrer and pH was maintained at 10.5 using 0.5M NaOH. The oxidized pulp was neutralized with 1 M HCl, 5 ml methanol was added to deactivate the NaClO and the solution was washed as described by Praskalo et al. (2009). 2.6. CNF Characterization 2.6.1. Zeta Potential Measurement and Particle Size Distribution The zeta potential (ζ, mV) of the CNF was measured using a Zetasizer Nano ZS equipped with a 4mW He–Ne laser operating at 633 nm (Malvern Instruments, UK). The oxidized sample supernatant was diluted to a 0.05 wt% suspension and the ζ-value was determined by the Smoluchowski equation from the measured mobility (Satyamurthy, Jain, Balasubramanya, &
9
Vigneshwaran, 2011). The carboxylate content resulting from oxidation of the cellulose surface is expected to result in a bipolar surface, higher charge and a stable suspension. Three measurements were taken for each suspension and an average charge was reported. The same instrument (Zetasizer Nano ZS) was used to determine the particle size distribution of cellulose nanofibril suspensions at 0.5% wt. % using a backscattering detection angle of 173º at 25 ºC. The viscosity was assumed to be equivalent to water for all measurements. The intensity particle size distributions are converted into volume in the instrument software. 2.6.2. Surface Charge Quantification Carboxylate content of the oxidized cellulose samples were determined by the conductometric titration method. The oxidized samples were washed thoroughly in a vacuum filter and freeze dried. The weight of the dried cellulose was recorded before re-suspending the samples in 50 ml of DI water resulting in solid loadings ranging from 0.14 to 0.2 %w/v. 0.01 M NaCl (5 ml) as a neutral ion was added to raise the baseline solution conductivity together with 0.01 M HCl (15 ml) to give a starting pH of <3 (Araki, Wada, & Kuga, 2001; da Silva Perez, Montanari, & Vignon, 2003; Hirota et al., 2010; Montanari, Roumani, Heux, & Vignon, 2005). The suspension was stirred and titrated with 0.01 M NaOH added at a rate of 100 μl/min. The conductivity of solution was measured using a conductivity probe (YSI Professional Plus, OH, USA). The surface charge density was measured as described in supporting information. 2.6.3. Fourier Transform Infrared Spectroscopy (FTIR) The supernatant of the oxidized pulp was dried in a rotary evaporator resulting in a clear film inside the flask. The film was peeled and surface composition was analyzed by ATR-FTIR using a Thermo-Nicolet Nexus 470 spectrophotometer with a Smart iTR ATR attachment (Thermo
10
Fisher Scientific, MA, USA) at a resolution of 4 cm-1 and accumulation of 36 scans. For untreated control samples it was not possible to make clear films due to complete settling of the pulp. Therefore a ground powder was analyzed for beam absorbance. Grinding did not affect the absorbance peaks as ground films from the T-CNF and S-CNF produced similar peaks to their respective clear films. The spectra baseline was corrected using the OMNIC software accompanying the FTIR. 2.6.4. Transmission Electron Microscopy (TEM) About 3 µl of the turbid solution supernatant after a 10 min sonication of the S-CNF, T-CNF and non-oxidized control was loaded onto a negatively charged 400-mesh carbon coated formvar copper grid (Electron Microscopy Sciences, Hatfield, PA, USA) using a micropipette. Water in the suspension on the carbon coated grid was removed and the samples were stained with 1% uranyl acetate. CNF coated grids were examined using Tecnai 20 transmission electron microscope at 200 kV. Image dimensions were determined by the TEM software. 2.6.5 Solution-state 13C NMR To determine the change in syringic acid structure that occurred during the oxidation reaction, separate experiments with the same reaction conditions minus the pulp were run and terminated when the solution color turned dark brown, but before the solution reverted to a colorless state. Water was evaporated using a rotary evaporator. The precipitant was solubilized in deuterated methanol (methanol-d4, CD3OD) and analyzed. Solution-state 13C NMR spectra of the syringic acid-ozone oxidation products dissolved in CD3OD were obtained using a Bruker ARX400-MHz NMR spectrometer (Bruker, Fremont, CA) equipped with a 5 mm QNP (H1, F19, P31, C13) probe and a sample temperature control unit. 3. Results and Discussion
11
3.1. Cellulose Reactivity Both endoglucanases (Depol 692 and 13P) have a high endoglucanase activity and a low βglucosidase activity as measured according to standard assays (Ghose, 1987; Mandels, Andreotti, & Roche, 1976 ). Depol 13P is a powdered enzyme and its activity was measured by diluting 25 g in 100 ml of water. The endocellulase activity of non-diluted Depol 692 and Depol 13P (250 g/l concentration) was measured to be 598 and 519 IU/ml, respectively using the CMCase assay. These enzymes had a much lower FPU and cellobiohydrolase (CBU) activity than common cellulases (with 100+ FPU activity) to cause minimal complete hydrolysis of the cellulose to soluble products. The pure endoglucanase also avoids hydrolysis of the hemicellulose content present in pulp which decreases the cell wall cohesion of the fibers, making cell wall disintegration easier. Both treatments using Depol 692 (81 CBU/ml, filter paper activity 18 FPU/ml) and Depol 13P (92 CBU/ml, 37 FPU/ml) resulted in a homogeneous pulp slurry with different degrees of sugar solubilization. The glucose and hemicellulose solubilization based on the initial composition of the pulp after each 24 hr enzyme treatment is shown in Table 1. As Paakko et al., reported (2007) much lower enzyme loading will lead to pulp homogenization (0.17 µL/g pulp (5 ECU/µL) therefore lower loadings could contribute to similar results. Indeed, they found that high loadings (30 µL/g) of endoglucanase decreased the refining and homogenization efficiency. The hydrolysis of the cellulose amorphous regions to monomeric glucose distorted the reducing end quantification, therefore the treated samples were washed in a vacuum filter using a 0.45 µm glass filter to remove solubilized sugars. Other methods of pulp reactivity measurement in the literature are laborious and often require toxic chemicals. Such methods include the intensive Fock method (Fock, 1959; Östberg et al., 2012), D2O-H2O exchange (Sepall & Mason, 1961), iodine absorption (Engström et al.,
12
2006) and acetylation or phosphitylation (Filpponen & Argyropoulos, 2008). For the measurement of the cellulose reactivity we quantified the available reducing ends of the cellulose using the BCA method. The BCA method with a sensitivity in the 0-50 µmoleq range proved more effective than the DNS method (Jiang & Hsieh, 2013). In fact, the DNS (3,5dinitrosalicylic acid) assay was incapable of detecting change in amount of reducing ends between various enzymatic treatments at the 0.5% w/v pulp loading. Figure 1 shows that there was a statistically significant difference between the Meq glucose of available reducing ends after enzyme hydrolysis. However the difference between enzyme type and loading was not significant. The Control and Blended Only samples shown in Figure 1 were analyzed with the same procedure without the enzyme treatment, with and without mechanical blending, respectively. The coagulation and heterogeneity of the non-enzyme untreated control sample made it difficult to remove 0.25 ml for analysis therefore an average of 6 readings were used for control. Enzyme treated samples were homogenous with a consistent absorbance reading. An increase in reducing ends correlates to a decrease in overall average cellulose polymer length as has been reported by (Rose, Babi, & Moran-Mirabal, 2015). Considering the insignificant difference between the amount of reducing ends at higher enzyme loadings, the similar homogeneity achieved with all treatments and favorability of a lower glucose release the 204 IU/g loading of Depol 692 was selected (i.e. 0.5 ml/g cellulose).
In order to verify the hypothesis that enzyme treatment increased the resulting oxidation of the cellulose surface, T-CNF was made and analyzed using enzyme and non-enzyme treated pulp. The z-potential, an indicator of the amount of surface oxidation, in the enzyme treated samples was -65 mV versus -47 mV in the T-CNF made from non-enzyme untreated pulp. This
13
could also be associated to the increased homogeneity and smaller particle size observed in the pulp after enzyme treatment. 3.2. Syringic Acid Oxidation With the oxidation of the syringic acid and formation of hydroxyl radicals, the enzyme treated pulp slurry changed color to dark brown within a few minutes after pumping ozone into the beaker. The solution pH dropped with formation of glucuronic acid which is made by oxidation and release of the –OH functional groups. Addition of 1M NaOH maintained the pH at 11. After two hours of ozone gas flow into the solution, the brown initial color paled; and after 8 hours the brown color completely disappeared resulting in a colorless suspension. At this point, solution pH drop was minimal and the reaction was stopped by ending gas flow into the solution. The change in the color of the pulp-syringic acid solution is attributed to the oxidation of the phenols by ozone, and as the syringic acid is further degraded, the solution color reverts to its original colorless state. The chemical structure of the syringic acid in the solution changed during ozone oxidation reaction (Compare NMR spectrum of syringic acid before and after a 2 hour oxidation in Figure 2). Presence of phenols is expected to enhance the formation of hydroxyl radicals similar to the mechanism proposed by Ragnar et al. (M. Ragnar et al., 1999; Martin Ragnar, Eriksson, Reitberger, & Brandt, 1999). It is evident that the hydroxyl group peak on the syringic acid (labeled 4) is missing from the oxidized solution spectrum. The degradation of the syringic acid structure is likely more complex than a simple cleavage of the hydroxyl group since peaks 5 and 6 have also shifted lower by 2 and 7 ppm, respectively appearing at 151 and 177 ppm. Our experiments with another lignin degradation product, guaiacyl, resulted in a lower surface charge on the cellulose. This is in line with previous reports confirming larger amounts of radical
14
release from syringyl compounds compared to guaiacyls (M. Ragnar et al., 1999). Unlike the TEMPO chemistry, it appears that the phenols do not act as a catalyst. The changing syringic acid structure during the reaction forms a series of intermediates that can lead to release of superoxides and hydroxyl radicals (Martin Ragnar et al., 1999). 3.3. Oxidation pH-dependence With cellulose oxidation carried out with a solution pH of 3, while maintaining other reaction conditions unchanged, no increase in the amount of cellulose oxidation was found as shown by CNF stability and a lower z-potential. Despite a mild color change in the acidic condition reaction, the NMR spectra of oxidized syringic acid under acidic conditions was not different from non-oxidized reagent. 3.4. Surface Charge Measurement and Quantification The never dried pulp had a high negative charge (~ -18 mV) after enzyme hydrolysis before being subjected to any oxidation. This charge is attributed to the pulp bleaching process when multivalent cations are picked up by the pulp which is consistent with values reported for bleached pulp in the literature (Buschle-Diller, Inglesby, & Wu, 2005). Acid soaking with 2 M HCl at room temperature neutralized the surface charge of pulp samples by demineralizing the pulp and removing unwanted cations, reducing the z-potential to -3 mV. Table 2 shows the negative charge on the oxidized pulp samples as compared to the control as well as the carboxyl content of the oxidized cellulose determined by conductometric titration method (see supporting information).
The negative charge on the surface of S-CNF confirms the oxidation of the hydroxyl groups on the cellulose surface to carbonyls and carboxylate groups. While this charge is significantly lower than T-CNF surface charges reported in the literature (between -70 to -85 mV in water), T-
15
CNF made by our method had a maximum surface charge of -65 mV. The surface charges on our CNF is higher than values reported for acid hydrolyzed CNC (Bondeson, Mathew, & Oksman, 2006; Jiang & Hsieh, 2013) however both S-CNF and T-CNF charges are lower than average values reported for CNF (Hirota et al., 2010; Peyre et al., 2015). 3.5. Chemical Structure of Oxidized CNF The FTIR spectra of non-oxidized control, S-CNF and T-CNF are shown in Figure 3. The dominant sharp absorbance peaks at 1600 cm-1 wavelength are carbonyl groups. When carbonyl peaks are accompanied by dominant aldehyde peaks in the region 2800 and 3600 cm-1, due to stretching vibrations of C-H and O-H range, it’s an indicator of carboxylic acids (Alemdar & Sain, 2008). The carbonyl peaks are very weak in non-oxidized pulp control as well as ozone only oxidized pulp. The absorbance is stronger for T-CNF as expected with the stronger chlorine-TEMPO oxidation reaction as compared to ozone/syringic acid for S-CNF oxidation. The peaks in the 1040 cm−1 region of all samples represents the C=C stretch of aromatic rings of lignin present in the original never dried pulp. The FTIR spectra confirm incapability of ozone gas alone in oxidizing the cellulose surface, which is also consistent with previous findings (Kato et al., 1999; Lemeune et al., 2004). It is known that in the oxidation of carbohydrates with ozone alone carboxylate groups form only as an intermediary reaction product and are subsequently decarboxylated to carbon dioxide (Lemeune et al., 2004). With the addition of phenolics, oxidation reactions involving hydroxyl radicals with cellulose dominates the minor reaction of ozone radicals with cellulose. 3.6. Size and Morphological Characterization Quantitative characterization of S-CNF size distributions in DI water suspension was performed on a 0.5 wt. % sample using dynamic light scattering (DLS). The hydrodynamic size
16
obtained is shown in Figure 4 by volume. There are two peaks observed at around 230 nm and 2.2 µm. With no mechanical mercerization, a volume of about 91% is in the particle size range of 700 nm to 6 µm while about 9% are in the size range of 130–397 nm. The yield of the S-CNF is estimated from the size distribution to be around 9%. The dimensions of S-NFC and T-CNF in suspension examined directly using a transmission electron microscope are shown in Figure 5. The images show that oxidation and enzymatic treatment have facilitated defibrillation and depolymerization of the cellulose fibers after a 10 min sonication. While similar images were obtained from enzyme treated non-oxidized fibers following sonication, the stability of those samples was limited and the fibers flocculated after a few hours. Sonication of non-enzyme treated oxidized and non-enzyme/non-oxidized samples failed to defibrillate the fibers. In addition, oxidation of non-enzyme treated pulp did not result in sufficient charges on the cellulose fibers to stay dispersed in the solution. TEM images of the flocculated samples were not taken because of the relatively large particle size (>1mm). The diameter of the fibers after the chemical and mechanical treatment was less than 20 nm with an average aspect ratio of 1:15. It is concluded from TEM images that sonication and the minimal defibrillation of the fibers applied in making our CNF is not efficiently breaking down the cellulose fibers. It is expected that due to the electroviscous effect, oxidation of cellulose C6 hydroxyl groups to carboxylates would lead to an increase in the observed viscosity of the suspension, however, this was not observed in our samples (Araki et al., 2001). This is likely due to the absence of mechanical treatment in the form of refining or homogenization as well as the likelihood of inadequacy of OH radicals to depolymerize cellulose fibers as efficiently as TEMPO mediated oxidation. Prior results reviewed by Coseri show significant reduction in the degree of
17
polymerization (DP) of cellulose upon TEMPO-mediated oxidation (Coseri, 2017). Change in the viscosity occurs after intensive refining of the pulp suspension accompanied by oxidation as a facilitator to the fiber breakdown process. While some level of surface oxidation is achievable, lower yields are expected in the absence of mechanical treatment. The low S-CNF yield (< 5% w/w of starting mass) can also be attributed to the lack of post oxidation mechanical treatment, as was also the case for the T-CNF produced in our lab. Sonication alone is not enough to defibrillate the cellulose fibers. As previously reported it merely disperses the particles without resulting in significant change in the size distribution (Peyre et al., 2015). Comparison of our TEM images to images taken from commercially available CNF confirm a greater amount of defibrillation in those samples. While fibrillation using disc refining or homogenizing are therefore essential to increase the CNF yield, we have not tested ozone/syringic acid oxidized pulps in such equipment (Brinchi, Cotana, Fortunati, & Kenny, 2013; Okita et al., 2010). 4. Conclusions An alternative to TEMPO-mediated oxidation of cellulose surface to produce CNF was presented in this study using ozone gas, a versatile oxidant, and lignin-derived phenolics to enhance oxidation reactions to modify the cellulose surface. We foresee compounds from degraded lignin in the pulping or cellulosic ethanol pretreatment process, such as syringic acid the model lignin compound used here, acting as oxidation enhancers for the production of CNFs. While ozone alone is not an efficient oxidizer of cellulose, in the presence of syringic acid reactions did occur with ozone which formed hydroxyl radicals that were able to effectively oxidize the cellulose surface. The results suggest that the reaction of hydroxyl radicals with cellulose C6 primary alcohol leads to formation of stable carboxylate groups on the celluloses. Unlike TEMPO, syringic acid does not act as a catalyst and is degraded during the reaction.
18
Nonetheless, complications in recycling TEMPO and the readily available lignin degradation products in large amounts from pulping may eliminate the economic necessity of its recovery. Additionally, enzyme treatment with a β-glucosidase proved to be effective in increasing the reactivity of pulp to chemical surface modification. While S-CNF may not completely match TCNF properties, the lower expected production cost by using a pulping byproduct (phenolics) coupled with a lower cost oxidant may lead to production of a similar grade yet less expensive CNF. Further study is needed to determine the efficacy of a mixture of lignin degradation compounds from pulping process in enhancing oxidation as compared to syringic acid alone. Excess amounts of reactants were used in the study as a proof of concept, and there is room for optimization of the amount of ozone, lignin degradation product and enzyme loading to achieve more economically favorable oxidation. S-CNF properties (such as tensile and thermal stability, crystallinity, film transparency, turbidity and water permeability) from other cellulosic sources also needs further study for potential applications. Acknowledgments This research was supported by USDA-NIFA Hatch Project IND010677 and the Purdue University Agricultural Research Station. The authors would like to acknowledge the support of Dr. Linda Mason for providing the ozone generator and Shane Peng for help with the FTIR. Valuable remarks from Dr. Alan W. Rudie, at the US Forest Service Research & Development and
Dr.
Galina
Rodionova
contributed
to
our
experimental
practices.
Author Contributions Iman Beheshti Tabar and Nathan S. Mosier conceived of the experimental approach. Iman Beheshti Tabar conducted the CNF production experiments. Ximing Zhang conducted the NMR analyses and the surface analyses in collaboration with Iman Behshti Tabar. Jeffrey P. Youngblood
19
contributed significantly to the CNF characterization. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
References Alemdar, A., & Sain, M. (2008). Isolation and characterization of nanofibers from agricultural residues – Wheat straw and soy hulls. Bioresource Technology, 99(6), 1664-1671. Araki, J., Wada, M., & Kuga, S. (2001). Steric Stabilization of a Cellulose Microcrystal Suspension by Poly(ethylene glycol) Grafting. Langmuir, 17(1), 21-27. Bondeson, D., Mathew, A., & Oksman, K. (2006). Optimization of the isolation of nanocrystals from microcrystalline cellulose by acid hydrolysis. Cellulose, 13(2), 171-180. Brinchi, L., Cotana, F., Fortunati, E., & Kenny, J. M. (2013). Production of nanocrystalline cellulose from lignocellulosic biomass: Technology and applications. Carbohydrate Polymers, 94(1), 154-169. Buschle-Diller, G., Inglesby, M. K., & Wu, Y. (2005). Physicochemical properties of chemically and enzymatically modified cellulosic surfaces. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 260(1–3), 63-70. Chirat, C., & Lachenal, D. (1997). Effect of Hydroxyl Radicals on Cellulose and Pulp and their Occurrence During Ozone Bleaching. Holzforschung - International Journal of the Biology, Chemistry, Physics and Technology of Wood (Vol. 51, p. 147). Coseri, S. (2017). Cellulose: To depolymerize… or not to? Biotechnology Advances, 35(2), 251266. da Silva Perez, D., Montanari, S., & Vignon, M. R. (2003). TEMPO-Mediated Oxidation of Cellulose III. Biomacromolecules, 4(5), 1417-1425. Engström, A.-C., Ek, M., & Henriksson, G. (2006). Improved Accessibility and Reactivity of Dissolving Pulp for the Viscose Process: Pretreatment with Monocomponent Endoglucanase. Biomacromolecules, 7(6), 2027-2031. Filpponen, I., & Argyropoulos, D. S. (2008). Determination of Cellulose Reactivity by Using Phosphitylation and Quantitative 31P NMR Spectroscopy. Industrial & Engineering Chemistry Research, 47(22), 8906-8910. Fock, W. (1959). Eine modifizierte method zur bestimmung der reaktivität von zellstoffen für viskosherstellung. Das Papier 13(3), 92-95. Gashti, M. P., Pournaserani, A., Ehsani, H., & Gashti, M. P. (2013). Surface oxidation of cellulose by ozone-gas in a vacuum cylinder to improve the functionality of fluoromonomer. Vacuum, 91, 7-13. Ghose, T. K. (1987). Measurement of cellulase activities. Pure and Applied Chemistry (Vol. 59, p. 257). Gurol, M. D., & Singer, P. C. (1982). Kinetics of ozone decomposition: a dynamic approach. Environmental Science & Technology, 16(7), 377-383. Henriksson, G., Christiernin, M., & Agnemo, R. (2005). Monocomponent endoglucanase treatment increases the reactivity of softwood sulphite dissolving pulp. Journal of Industrial Microbiology and Biotechnology, 32(5), 211-214.
20
Hirota, M., Tamura, N., Saito, T., & Isogai, A. (2010). Water dispersion of cellulose II nanocrystals prepared by TEMPO-mediated oxidation of mercerized cellulose at pH 4.8. Cellulose, 17(2), 279-288. Isogai, A., Saito, T., & Fukuzumi, H. (2011). TEMPO-oxidized cellulose nanofibers. Nanoscale, 3(1), 71-85. Jiang, F., & Hsieh, Y.-L. (2013). Chemically and mechanically isolated nanocellulose and their self-assembled structures. Carbohydrate Polymers, 95(1), 32-40. Kato, K., Vasilets, V. N., Fursa, M. N., Meguro, M., Ikada, Y., & Nakamae, K. (1999). Surface oxidation of cellulose fibers by vacuum ultraviolet irradiation. Journal of Polymer Science Part A: Polymer Chemistry, 37(3), 357-361. Lemeune, S., Jameel, H., Chang, H. M., & Kadla, J. F. (2004). Effects of ozone and chlorine dioxide on the chemical properties of cellulose fibers. Journal of Applied Polymer Science, 93(3), 1219-1223. Löwendahl, L., Petersson, G., & Samuelson, O. (1978). Phenolic compounds in kraft black liquor. Svensk Papperstidning, 81(12), 392-396. Mandels, M., Andreotti, R., & Roche, C. (1976 ). Measurement of saccharifying cellulase. Biotechnol. Bioeng. Symp (Vol. 6). United States: Army Natick Development Center, MA. Montanari, S., Roumani, M., Heux, L., & Vignon, M. R. (2005). Topochemistry of Carboxylated Cellulose Nanocrystals Resulting from TEMPO-Mediated Oxidation. Macromolecules, 38(5), 1665-1671. Okita, Y., Saito, T., & Isogai, A. (2010). Entire Surface Oxidation of Various Cellulose Microfibrils by TEMPO-Mediated Oxidation. Biomacromolecules, 11(6), 1696-1700. Peyre, J., Paakkonen, T., Reza, M., & Kontturi, E. (2015). Simultaneous preparation of cellulose nanocrystals and micron-sized porous colloidal particles of cellulose by TEMPOmediated oxidation. Green Chemistry, 17(2), 808-811. Pouyet, F., Chirat, C., Potthast, A., & Lachenal, D. (2014). Formation of carbonyl groups on cellulose during ozone treatment of pulp: Consequences for pulp bleaching. Carbohydrate Polymers, 109, 85-91. Praskalo, J., Kostic, M., Potthast, A., Popov, G., Pejic, B., & Skundric, P. (2009). Sorption properties of TEMPO-oxidized natural and man-made cellulose fibers. Carbohydrate Polymers, 77(4), 791-798. Pääkkö, M., Ankerfors, M., Kosonen, H., Nykänen, A., Ahola, S., Österberg, M., . . . Lindström, T. (2007). Enzymatic Hydrolysis Combined with Mechanical Shearing and HighPressure Homogenization for Nanoscale Cellulose Fibrils and Strong Gels. Biomacromolecules, 8(6), 1934-1941. Ragnar, M., Eriksson, T., & Reitberger, T. (1999). Radical Formation in Ozone Reactions with Lignin and Carbohydrate Model Compounds. Holzforschung (Vol. 53, p. 292). Ragnar, M., Eriksson, T., Reitberger, T., & Brandt, P. (1999). A New Mechanism in the Ozone Reaction with Lignin Like Structures. Holzforschung (Vol. 53, p. 423). Rodionova, G., Saito, T., Lenes, M., Eriksen, Ø., Gregersen, Ø., Kuramae, R., & Isogai, A. (2013). TEMPO-Mediated Oxidation of Norway Spruce and Eucalyptus Pulps: Preparation and Characterization of Nanofibers and Nanofiber Dispersions. Journal of Polymers and the Environment, 21(1), 207-214.
21
Rose, M., Babi, M., & Moran-Mirabal, J. (2015). The study of cellulose structure and depolymerization through single-molecule methods. Industrial Biotechnology, 11(1), 1624. Saito, T., Kimura, S., Nishiyama, Y., & Isogai, A. (2007). Cellulose Nanofibers Prepared by TEMPO-Mediated Oxidation of Native Cellulose. Biomacromolecules, 8(8), 2485-2491. Saito, T., Nishiyama, Y., Putaux, J.-L., Vignon, M., & Isogai, A. (2006). Homogeneous Suspensions of Individualized Microfibrils from TEMPO-Catalyzed Oxidation of Native Cellulose. Biomacromolecules, 7(6), 1687-1691. Satyamurthy, P., Jain, P., Balasubramanya, R. H., & Vigneshwaran, N. (2011). Preparation and characterization of cellulose nanowhiskers from cotton fibres by controlled microbial hydrolysis. Carbohydrate Polymers, 83(1), 122-129. Sepall, O., & Mason, S. G. (1961). Hydrogen Exchange Between Cellulose and Water: I. Measurement of Accessibility. Canadian Journal of Chemistry, 39(10), 1934-1943. Shinoda, R., Saito, T., Okita, Y., & Isogai, A. (2012). Relationship between Length and Degree of Polymerization of TEMPO-Oxidized Cellulose Nanofibrils. Biomacromolecules, 13(3), 842-849. Sluiter, A., Hames, B., Hyman, D., Payne, C., Ruiz, R., Scarlata, C., . . . Wolfe, J. (2008). Standard Procedures for Biomass Compositional Analysis - Biomass and Total Dissolved Solids in Liquid Process Samples. National Renewable Energy Laboratory. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., & Crocker, D. (2012). Standard Procedures for Biomass Compositional Analysis- Determination of Structural Carbohydrates and Lignin in Biomass. National Renewable Energy Laboratory. Sluiter, A., Ruiz, R., Scarlata, C., Sluiter, J., & Templeton, D. (2005). Standard Procedures for Biomass Compositional Analysis-Determination of Extractives in Biomass. National Renewable Energy Laboratory. Son, Y., Lim, M., Khim, J., & Ashokkumar, M. (2012). Attenuation of UV Light in Large-Scale Sonophotocatalytic Reactors: The Effects of Ultrasound Irradiation and TiO2 Concentration. Industrial & Engineering Chemistry Research, 51(1), 232-239. Sun, R., & Tomkinson, J. (2001). Fractional separation and physico-chemical analysis of lignins from the black liquor of oil palm trunk fibre pulping. Separation and Purification Technology, 24(3), 529-539. Zhang, Y. H. P., & Lynd, L. R. (2005). Determination of the Number-Average Degree of Polymerization of Cellodextrins and Cellulose with Application to Enzymatic Hydrolysis. Biomacromolecules, 6(3), 1510-1515. Zhu, J. Y., Sabo, R., & Luo, X. (2011). Integrated production of nano-fibrillated cellulose and cellulosic biofuel (ethanol) by enzymatic fractionation of wood fibers. Green Chemistry, 13(5), 1339-1344. Östberg, L., Håkansson, H., & Germgård, U. (2012). Some aspects of the reactivity of pulp intended for high-viscosity viscose. BioResources, 7(1), 743-755.
Reducing End in meq glucose/g cellulose
22
14 12
10 8 6 4 2 0 13P, Low 13P, High 692, Low 692, High Blended only pulp
Control
Various Depol® enzyme hydrolyzed samples
Figure 1. BCA assay Reducing end quantification of 0.005 g cellulose equivalent treated with two different Depol endoglucanase enzyme (13P and 692) with high (0.5 ml/g) and low (0.5 ml/g) loading. Control is non-enzyme treated 1% pulp dilution.
23
2
1
5 3
6
4
a
1 2
5
b
3
6
1 2
5
c
6
3 4
ppm
Figure 2. Carbon-13 NMR spectra in D-methanol (CD3OD): (a) Non oxidized syringic acid; (b) ozone oxidized syringic acid in alkaline condition (pH 11); (c) ozone oxidized syringic acid in acidic conditions (pH 3).
24
Figure 3. FTIR spectra of different cellulose nanofiber samples.
Figure 4. Size distribution (log10 nm) of 0.5 wt.% S-NFC by volume.
25
1
2
4
5
3
6
Figure 5. TEM images of uranyl acetate stained CNF. Images 1 and 2: 0.1% w/v S-CNF and image 3 is 0.001% w/v S-CNF. Images 4, 5 and 6 is 0.1% w/v T-CNF.
26
Table 1. Solubilized glucose and hemicellulose content after 24h enzymatic treatment of never dried pulp. Enzyme
Loading
Loading Amount
Glucose
Hemicellulose
(ml enzyme)
(IU/ g fiber)
(wt %)
(wt %)
Depol 692*
Low, 0.25
150
7.6
96
Depol 692
High, 0.5
300
13.2
98
Depol 13P**
Low, 0.25
130
7.76
49.6
Depol 13P
High, 0.5
260
15.3
74.5
*Activity: 598 IU/ml **Activity: 519 IU/ml
Table 2. z-potential of the various CNF samples at neutral pH. z-potential Carboxyl content Treatment (mV) (μmol/g) Blank DI
-3.6
-
S Control
-10
11
O3 Control
-13.4
14
Pulp Control
-7.5
19
S-CNF
-42.2
418
T-CNF
-65
687
S0144-8617(17)30695-1 http://dx.doi.org/doi:10.1016/j.carbpol.2017.06.058 CARP 12446
To appear in: Received date: Revised date: Accepted date:
31-3-2017 13-6-2017 15-6-2017
Please cite this article as: Tabar, Iman Beheshti., Zhang, Ximing., Youngblood, Jeffrey P., & Mosier, Nathan S., Production of cellulose nanofibers using phenolic enhanced surface oxidation.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.06.058 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Production of cellulose nanofibers using phenolic enhanced surface oxidation Iman Beheshti Tabara, Ximing Zhanga, Jeffrey P. Youngbloodb, Nathan S. Mosier*a a Laboratory of Renewable Resources Engineering and Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, Indiana, United States b School of Materials Engineering, Purdue University, West Lafayette, Indiana, United States. * To whom correspondence should be addressed. Email: [email protected]; Tel.: +1 (765) 494-7022, FAX: +1 (765) 494-7023 Author emails: Iman Beheshti Tabar: [email protected]; Jeffrey P. Youngblood: [email protected]; Ximing Zhang: [email protected];
2
Highlights
Use of a wood by-product with ozone offers a novel pathway to cellulose nanofibers. Syringic acid as lignin model compound enhanced ozone cellulose surface oxidation. Syringic acid, a byproduct of wood pulping, is a biorenewable oxidation enhancer. Enzyme treatment increased cellulose surface reactivity toward oxidation. New cellulose nanofiber pathway could have significant process advantages.
ABSTRACT. In this study we demonstrate that lignin monomers formed as byproducts of pulping or bioprocessing of lignocellulosic biomass is an effective enhancer to oxidize cellulose surfaces with ozone for the production of cellulose nanofibers (CNF). Never dried softwood pulp with minimum mercerization was enzymatically treated leading to a homogeneous pulp slurry with a higher reactivity. The slurry was oxidized by ozone gas in the presence of syringic acid, a lignin degradation model compound, as an oxidation enhancer at room temperature and pH 11. Transmission electron microscopy (TEM) observations showed that stable CNF bundles with 310 nm widths and lengths >100 nm were obtained after ultrasonication of the oxidized product in water. Extensive characterization of the new CNF films revealed the nanofibers had carboxylate content similar to conventional carboxylated cellulose prepared by TEMPO-mediated oxidation. Based on NMR spectra, chemical conversion of the syringic acid during oxidation is proposed.
KEYWORDS: Cellulose nanofibers; phenolics; surface oxidation; carboxyl groups Chemical compounds studied in this article Syringic Acid (PubChem CID: 10742); Ozone (PubChem CID: 24823); Bicinchoninic Acid Disodium Salt Hydrate (PubChem CID: 71308346); Sodium hydroxide (PubChem CID: 14798); TEMPO (PubChem CID: 2724126); Sodium hypochlorite (PubChem CID: 23665760); Sodium
3
bromide (PubChem CID: 253881); Uranyl acetate (PubChem CID: 10915); Sodium carbonate (PubChem CID: 10340) 1. Introduction Co-production of cellulose nanofibers (CNF) in paper mills and cellulosic ethanol plants can add value and diversity to the product portfolio (Zhu, Sabo, & Luo, 2011). Preceding any disintegration step, native celluloses need some form of surface modification to prevent aggregation of the fibrils. The common, extensively studied method of surface modification for CNF is TEMPO-mediated oxidation for selective conversion of primary C6 hydroxyl groups to charged caboxylates (Isogai, Saito, & Fukuzumi, 2011; Okita, Saito, & Isogai, 2010; Saito, Nishiyama, Putaux, Vignon, & Isogai, 2006). However, alternative methods of CNF oxidation without the use of hard-to-recycle corrosive chemicals such as TEMPO, NaBr, NaClO, while maintaining similar properties, are desirable. A lower cost oxidation process, using an easily produced and safer oxidant like ozone gas, could spur the development of new applications and markets for CNF. While ozone is a strong oxidant, reaction of ozone with cellulose leads to a minor oxidation reaction mostly at the C-6 position (Kato et al., 1999; Lemeune, Jameel, Chang, & Kadla, 2004). It is well known that advanced oxidation processes (AOPs) leading to formation of hydroxyl radicals have a higher oxidation potential than the conventional oxidizing agents alone. Ozone gas in combination with other oxidation enhancers such as UV light have successfully been used to oxidize the surface of cellulose (Gashti, Pournaserani, Ehsani, & Gashti, 2013; Kato et al., 1999). However, effective irradiation distance and UV light attenuation in large scale concentrated pulp processing can be problematic (Son, Lim, Khim, & Ashokkumar, 2012). Previous studies have reported that the presence of small amounts of lignin degradation products in pulp during Total Chlorine Free
4
(TCF) ozone bleaching enhances the formation of hydroxyl radicals, which in turn form carbonyl bonds on the cellulose surface (Pouyet, Chirat, Potthast, & Lachenal, 2014). The increased formation of radicals is attributed to the phenolics in lignin and other unsaturated compounds that are highly reactive toward ozone. The causes for the formation of hydroxyl and other radicals have been a matter of discussion and several mechanisms are proposed (Chirat & Lachenal, 1997; Pouyet et al., 2014; M. Ragnar, Eriksson, & Reitberger, 1999). Both paper mills and cellulosic ethanol plants have waste streams containing lignin degradation products which could be utilized in CNF surface oxidation. The most prominent fractions of these lignin waste streams in pulp black liquor include methoxyphenols such as vanillic acid, syrinigic acid and guaiacol (Löwendahl, Petersson, & Samuelson, 1978; Sun & Tomkinson, 2001). This study investigates the production of CNF by taking advantage of formation of carbonyl groups on cellulose by ozone gas in the presence of lignin-derived phenolic compounds. Moreover, reactivity of cellulose is a key parameter when modifying the surface of native cellulose. Due to the steric hindrance in the cellulose macromolecule, the primary reaction of oxidants is cleavage of the glycosidic linkage at the C1 position or oxidation of the primary C6 hydroxyl (–OH) groups (Hirota, Tamura, Saito, & Isogai, 2010; Lemeune et al., 2004; Okita et al., 2010). Mild enzyme treatment, using a pure endoglucanase, is known to reduce fibrillation energy and increase the surface reactivity of pulp to oxidation by increasing the availability of primary –OH groups (Östberg, Håkansson, & Germgård, 2012). Successful attempts to increase the reactivity of pulp for the production of rayon and other derivatives using endoglucanase enzymes have been reported (Engström, Ek, & Henriksson, 2006; Henriksson, Christiernin, & Agnemo, 2005). In this study we also investigate the efficacy of the cleaving action of endoglucanases in increasing the number of reducing ends leading to increased availability of
5
primary hydroxyl groups for oxidation. Increased surface reactivity is expected to translate into an increased degree of substitution, thereby an improved electrostatic repulsion after surface modifications. In this paper we investigate enzymatic treatment coupled with surface oxidation enhanced by lignin-derived phenolics to maximize the surface charge imparted on cellulose. We build upon previous findings regarding the substantially lower amount of mechanical energy required to prepare CNF after an enzymatic treatment and complement it with surface oxidation to enhance CNF quality (Pääkkö et al., 2007). Enzyme treated samples are oxidized by ozone gas in the presence of syringic acid and the characteristics of the resulting suspension (S-CNF) is compared with TEMPO mediated CNF (T-CNF).The goal of the study is to demonstrate that lignin-derived phenolics can be used in conjunction with ozone gas to produce CNF from enzyme treated cellulose. 2. Experimental 2.1. Materials Never dried DED bleached Kraft hardwood pulp with a water content of 60% was complimentary provided by the Forest Products Laboratory (U.S. Forest Service, Madison, WI). The pulp was stored at -18 ˚C before use. The chemical composition of the pulp was 93.2% glucan, 5.9% xylan, 0.8% Klason lignin as determined using the procedures described by National Renewable Energy Laboratory, Laboratory Analytical Procedures (LAP) (Sluiter et al., 2008; 2012; 2005). Depol 692L enzyme (Cellulase 800 u/g) and cellulase 13P were purchased from Biocatalyst, UK. Syringic acid, TEMPO, sodium bromide, 15% sodium hypochlorite solution, as well as chemicals for the cellulose reactivity measurement, disodium 2,2’bicinchoninate, Na2CO3, NaHCO3 and L-serine were of laboratory grade (Sigma Aldrich,
6
Urbana, IL). The enzyme and chemicals were used as received. Ozone gas was generated using a corona tube ozone generator (O3Co, Idaho Falls, ID, USA) and GC grade compressed O2 (Indiana Oxygen Co, Lafayette, IN, USA) and ozone concentration was measured using a UV106H ozone analyzer (Ozone Solutions, IA, USA). 2.2. Cellulose Surface Reactivity Measurement The amount of available reducing ends on the cellulose was measured by the spectrometric 2,2’-bicinchoninate (BCA) method as suggested by Zhang and Lynd (2005). Replicates of two different loadings of commercial endoglucanase Depol 692 and 13P with high endoglucanase C and low β-glucosidase activity were tested. Standard glucose solution was made within the BCA assay range of 0-50 µM glucose equivalent and BCA reagent was made fresh daily from the working solution. Enzyme treatment was carried out as described below. Pulp was recovered by centrifugation for 2 mins, (10,000 g) before absorbance measurement at 560 nm to eliminate interference. Background absorbance from a BCA blank reading was deducted from sample absorbance measurements. 2.3. Enzymatic Fractionation A stock 1% w/v solution of dispersed pulp was made by blending 10 g (dry mass) of never dried pulp in 1000 ml of deionized water for 5 min using a Ninja blender (Ninja NJ600 Professional Blender). The blending increased the fiber swelling in water, making the cellulose more accessible to the enzymes (Praskalo et al., 2009). Enzymatic pretreatment was carried out as follows: 150 ml of the stock solution was mixed with an equal amount of 50mM citrate buffer to maintain enzyme activity optimum pH of 4.8, bringing the solids loading to 0.5% w/v. Enzyme loading (Depol 692 and 13P) was 0.25 ml/g cellulose and the solution was incubated at 50 °C for 24 hours with agitation provided by a rotary shaker at 200 rpm. Enzyme treatment was
7
terminated by incubating the samples for 30 minutes at 80 °C using a beaker on heated stir plate. The pH of the 300 ml solution was then lowered to ~2 using 2M HCl and kept at room temperature for 2 hours to demineralize the pulp and remove excess charges adsorbed to the pulp surface during the pulping process (Rodionova et al., 2013; Shinoda, Saito, Okita, & Isogai, 2012). The high volume of enzyme loading was to eliminate the effect of other variables on the increase in pulp reactivity when comparing syringic acid/ozone treatment to TEMPO/hydrogen peroxide treatment. After treatment the fiber suspension was washed with excess deionized (DI) water until the pulp suspension reached pH 7 exhibiting a white color (Isogai et al., 2011). The fibers were then sonicated for 10 minutes with an ultrasound microtip (Branson, USA). After redispersion of the fibers, pH was increased to 11 and ozone oxidation was carried out. 2.4. Catalyzed Ozone Oxidation Prior to oxidation, the proteins and HCl were removed by washing the enzyme treated/acid soaked pulp using a vacuum filter. Syringic acid powder (0.25g) was added to 150 ml of 1% w/v enzyme treated pulp. The solution pH was raised to 11 using 1 M NaOH to increase the reactivity of the cellulose hydroxyl group. Ozone-containing gas was applied at a flow rate of 0.25 L/min with a concentration of 18 mg/L (1.26 wt. %). The pulp suspension was constantly stirred on a stir plate and the reaction was carried out in a fume hood at room temperature (20°C). Solubility of ozone in water (saturation concentration of ozone to applied gaseous ozone concentration) decreases at higher pH as the rate of decomposition increases. Therefore, solubility coefficient can be expected to be even lower than 0.35 as reported in the literature (Gurol & Singer, 1982). Neglecting decomposition, the amount of ozone inserted into the solution over the 8 hour experiment time is about 0.76 grams. As with the enzyme loading the amount of ozone applied to the experiment has not been optimized and is in excess to more
8
explicitly differentiate ozone treatment from the controls. After terminating the oxidation reaction the suspension was washed in a vacuum filter, re-suspended in 150 ml of DI water and sonicated for 5 minutes. 2.5. TEMPO-Mediated Oxidation Enzyme treated pulp (1.5 g) was suspended in water and TEMPO-mediated oxidation was carried out according to the methodology previously described with slight modifications as described below (Okita et al., 2010; Peyre, Paakkonen, Reza, & Kontturi, 2015; Rodionova et al., 2013; Saito, Kimura, Nishiyama, & Isogai, 2007; Shinoda et al., 2012). The TEMPO-pulp served as a control to compare with the catalyzed ozone oxidized CNF. Excessive addition of the TEMPO and NaBr catalysts causes severe degradation of the pulp, hence the amounts suggested in the literature (TEMPO 0.0025 g/g and NaBr 0.025 g/g cellulose) were followed. Some modifications to the procedure included a stepwise addition of 0.5 ml of NaClO (15% chlorine concentration) every 30 min, which was more efficient for oxidizing the cellulose surface. The slurry was under continuous stirring using a Coleman Parker stirrer and pH was maintained at 10.5 using 0.5M NaOH. The oxidized pulp was neutralized with 1 M HCl, 5 ml methanol was added to deactivate the NaClO and the solution was washed as described by Praskalo et al. (2009). 2.6. CNF Characterization 2.6.1. Zeta Potential Measurement and Particle Size Distribution The zeta potential (ζ, mV) of the CNF was measured using a Zetasizer Nano ZS equipped with a 4mW He–Ne laser operating at 633 nm (Malvern Instruments, UK). The oxidized sample supernatant was diluted to a 0.05 wt% suspension and the ζ-value was determined by the Smoluchowski equation from the measured mobility (Satyamurthy, Jain, Balasubramanya, &
9
Vigneshwaran, 2011). The carboxylate content resulting from oxidation of the cellulose surface is expected to result in a bipolar surface, higher charge and a stable suspension. Three measurements were taken for each suspension and an average charge was reported. The same instrument (Zetasizer Nano ZS) was used to determine the particle size distribution of cellulose nanofibril suspensions at 0.5% wt. % using a backscattering detection angle of 173º at 25 ºC. The viscosity was assumed to be equivalent to water for all measurements. The intensity particle size distributions are converted into volume in the instrument software. 2.6.2. Surface Charge Quantification Carboxylate content of the oxidized cellulose samples were determined by the conductometric titration method. The oxidized samples were washed thoroughly in a vacuum filter and freeze dried. The weight of the dried cellulose was recorded before re-suspending the samples in 50 ml of DI water resulting in solid loadings ranging from 0.14 to 0.2 %w/v. 0.01 M NaCl (5 ml) as a neutral ion was added to raise the baseline solution conductivity together with 0.01 M HCl (15 ml) to give a starting pH of <3 (Araki, Wada, & Kuga, 2001; da Silva Perez, Montanari, & Vignon, 2003; Hirota et al., 2010; Montanari, Roumani, Heux, & Vignon, 2005). The suspension was stirred and titrated with 0.01 M NaOH added at a rate of 100 μl/min. The conductivity of solution was measured using a conductivity probe (YSI Professional Plus, OH, USA). The surface charge density was measured as described in supporting information. 2.6.3. Fourier Transform Infrared Spectroscopy (FTIR) The supernatant of the oxidized pulp was dried in a rotary evaporator resulting in a clear film inside the flask. The film was peeled and surface composition was analyzed by ATR-FTIR using a Thermo-Nicolet Nexus 470 spectrophotometer with a Smart iTR ATR attachment (Thermo
10
Fisher Scientific, MA, USA) at a resolution of 4 cm-1 and accumulation of 36 scans. For untreated control samples it was not possible to make clear films due to complete settling of the pulp. Therefore a ground powder was analyzed for beam absorbance. Grinding did not affect the absorbance peaks as ground films from the T-CNF and S-CNF produced similar peaks to their respective clear films. The spectra baseline was corrected using the OMNIC software accompanying the FTIR. 2.6.4. Transmission Electron Microscopy (TEM) About 3 µl of the turbid solution supernatant after a 10 min sonication of the S-CNF, T-CNF and non-oxidized control was loaded onto a negatively charged 400-mesh carbon coated formvar copper grid (Electron Microscopy Sciences, Hatfield, PA, USA) using a micropipette. Water in the suspension on the carbon coated grid was removed and the samples were stained with 1% uranyl acetate. CNF coated grids were examined using Tecnai 20 transmission electron microscope at 200 kV. Image dimensions were determined by the TEM software. 2.6.5 Solution-state 13C NMR To determine the change in syringic acid structure that occurred during the oxidation reaction, separate experiments with the same reaction conditions minus the pulp were run and terminated when the solution color turned dark brown, but before the solution reverted to a colorless state. Water was evaporated using a rotary evaporator. The precipitant was solubilized in deuterated methanol (methanol-d4, CD3OD) and analyzed. Solution-state 13C NMR spectra of the syringic acid-ozone oxidation products dissolved in CD3OD were obtained using a Bruker ARX400-MHz NMR spectrometer (Bruker, Fremont, CA) equipped with a 5 mm QNP (H1, F19, P31, C13) probe and a sample temperature control unit. 3. Results and Discussion
11
3.1. Cellulose Reactivity Both endoglucanases (Depol 692 and 13P) have a high endoglucanase activity and a low βglucosidase activity as measured according to standard assays (Ghose, 1987; Mandels, Andreotti, & Roche, 1976 ). Depol 13P is a powdered enzyme and its activity was measured by diluting 25 g in 100 ml of water. The endocellulase activity of non-diluted Depol 692 and Depol 13P (250 g/l concentration) was measured to be 598 and 519 IU/ml, respectively using the CMCase assay. These enzymes had a much lower FPU and cellobiohydrolase (CBU) activity than common cellulases (with 100+ FPU activity) to cause minimal complete hydrolysis of the cellulose to soluble products. The pure endoglucanase also avoids hydrolysis of the hemicellulose content present in pulp which decreases the cell wall cohesion of the fibers, making cell wall disintegration easier. Both treatments using Depol 692 (81 CBU/ml, filter paper activity 18 FPU/ml) and Depol 13P (92 CBU/ml, 37 FPU/ml) resulted in a homogeneous pulp slurry with different degrees of sugar solubilization. The glucose and hemicellulose solubilization based on the initial composition of the pulp after each 24 hr enzyme treatment is shown in Table 1. As Paakko et al., reported (2007) much lower enzyme loading will lead to pulp homogenization (0.17 µL/g pulp (5 ECU/µL) therefore lower loadings could contribute to similar results. Indeed, they found that high loadings (30 µL/g) of endoglucanase decreased the refining and homogenization efficiency. The hydrolysis of the cellulose amorphous regions to monomeric glucose distorted the reducing end quantification, therefore the treated samples were washed in a vacuum filter using a 0.45 µm glass filter to remove solubilized sugars. Other methods of pulp reactivity measurement in the literature are laborious and often require toxic chemicals. Such methods include the intensive Fock method (Fock, 1959; Östberg et al., 2012), D2O-H2O exchange (Sepall & Mason, 1961), iodine absorption (Engström et al.,
12
2006) and acetylation or phosphitylation (Filpponen & Argyropoulos, 2008). For the measurement of the cellulose reactivity we quantified the available reducing ends of the cellulose using the BCA method. The BCA method with a sensitivity in the 0-50 µmoleq range proved more effective than the DNS method (Jiang & Hsieh, 2013). In fact, the DNS (3,5dinitrosalicylic acid) assay was incapable of detecting change in amount of reducing ends between various enzymatic treatments at the 0.5% w/v pulp loading. Figure 1 shows that there was a statistically significant difference between the Meq glucose of available reducing ends after enzyme hydrolysis. However the difference between enzyme type and loading was not significant. The Control and Blended Only samples shown in Figure 1 were analyzed with the same procedure without the enzyme treatment, with and without mechanical blending, respectively. The coagulation and heterogeneity of the non-enzyme untreated control sample made it difficult to remove 0.25 ml for analysis therefore an average of 6 readings were used for control. Enzyme treated samples were homogenous with a consistent absorbance reading. An increase in reducing ends correlates to a decrease in overall average cellulose polymer length as has been reported by (Rose, Babi, & Moran-Mirabal, 2015). Considering the insignificant difference between the amount of reducing ends at higher enzyme loadings, the similar homogeneity achieved with all treatments and favorability of a lower glucose release the 204 IU/g loading of Depol 692 was selected (i.e. 0.5 ml/g cellulose).
In order to verify the hypothesis that enzyme treatment increased the resulting oxidation of the cellulose surface, T-CNF was made and analyzed using enzyme and non-enzyme treated pulp. The z-potential, an indicator of the amount of surface oxidation, in the enzyme treated samples was -65 mV versus -47 mV in the T-CNF made from non-enzyme untreated pulp. This
13
could also be associated to the increased homogeneity and smaller particle size observed in the pulp after enzyme treatment. 3.2. Syringic Acid Oxidation With the oxidation of the syringic acid and formation of hydroxyl radicals, the enzyme treated pulp slurry changed color to dark brown within a few minutes after pumping ozone into the beaker. The solution pH dropped with formation of glucuronic acid which is made by oxidation and release of the –OH functional groups. Addition of 1M NaOH maintained the pH at 11. After two hours of ozone gas flow into the solution, the brown initial color paled; and after 8 hours the brown color completely disappeared resulting in a colorless suspension. At this point, solution pH drop was minimal and the reaction was stopped by ending gas flow into the solution. The change in the color of the pulp-syringic acid solution is attributed to the oxidation of the phenols by ozone, and as the syringic acid is further degraded, the solution color reverts to its original colorless state. The chemical structure of the syringic acid in the solution changed during ozone oxidation reaction (Compare NMR spectrum of syringic acid before and after a 2 hour oxidation in Figure 2). Presence of phenols is expected to enhance the formation of hydroxyl radicals similar to the mechanism proposed by Ragnar et al. (M. Ragnar et al., 1999; Martin Ragnar, Eriksson, Reitberger, & Brandt, 1999). It is evident that the hydroxyl group peak on the syringic acid (labeled 4) is missing from the oxidized solution spectrum. The degradation of the syringic acid structure is likely more complex than a simple cleavage of the hydroxyl group since peaks 5 and 6 have also shifted lower by 2 and 7 ppm, respectively appearing at 151 and 177 ppm. Our experiments with another lignin degradation product, guaiacyl, resulted in a lower surface charge on the cellulose. This is in line with previous reports confirming larger amounts of radical
14
release from syringyl compounds compared to guaiacyls (M. Ragnar et al., 1999). Unlike the TEMPO chemistry, it appears that the phenols do not act as a catalyst. The changing syringic acid structure during the reaction forms a series of intermediates that can lead to release of superoxides and hydroxyl radicals (Martin Ragnar et al., 1999). 3.3. Oxidation pH-dependence With cellulose oxidation carried out with a solution pH of 3, while maintaining other reaction conditions unchanged, no increase in the amount of cellulose oxidation was found as shown by CNF stability and a lower z-potential. Despite a mild color change in the acidic condition reaction, the NMR spectra of oxidized syringic acid under acidic conditions was not different from non-oxidized reagent. 3.4. Surface Charge Measurement and Quantification The never dried pulp had a high negative charge (~ -18 mV) after enzyme hydrolysis before being subjected to any oxidation. This charge is attributed to the pulp bleaching process when multivalent cations are picked up by the pulp which is consistent with values reported for bleached pulp in the literature (Buschle-Diller, Inglesby, & Wu, 2005). Acid soaking with 2 M HCl at room temperature neutralized the surface charge of pulp samples by demineralizing the pulp and removing unwanted cations, reducing the z-potential to -3 mV. Table 2 shows the negative charge on the oxidized pulp samples as compared to the control as well as the carboxyl content of the oxidized cellulose determined by conductometric titration method (see supporting information).
The negative charge on the surface of S-CNF confirms the oxidation of the hydroxyl groups on the cellulose surface to carbonyls and carboxylate groups. While this charge is significantly lower than T-CNF surface charges reported in the literature (between -70 to -85 mV in water), T-
15
CNF made by our method had a maximum surface charge of -65 mV. The surface charges on our CNF is higher than values reported for acid hydrolyzed CNC (Bondeson, Mathew, & Oksman, 2006; Jiang & Hsieh, 2013) however both S-CNF and T-CNF charges are lower than average values reported for CNF (Hirota et al., 2010; Peyre et al., 2015). 3.5. Chemical Structure of Oxidized CNF The FTIR spectra of non-oxidized control, S-CNF and T-CNF are shown in Figure 3. The dominant sharp absorbance peaks at 1600 cm-1 wavelength are carbonyl groups. When carbonyl peaks are accompanied by dominant aldehyde peaks in the region 2800 and 3600 cm-1, due to stretching vibrations of C-H and O-H range, it’s an indicator of carboxylic acids (Alemdar & Sain, 2008). The carbonyl peaks are very weak in non-oxidized pulp control as well as ozone only oxidized pulp. The absorbance is stronger for T-CNF as expected with the stronger chlorine-TEMPO oxidation reaction as compared to ozone/syringic acid for S-CNF oxidation. The peaks in the 1040 cm−1 region of all samples represents the C=C stretch of aromatic rings of lignin present in the original never dried pulp. The FTIR spectra confirm incapability of ozone gas alone in oxidizing the cellulose surface, which is also consistent with previous findings (Kato et al., 1999; Lemeune et al., 2004). It is known that in the oxidation of carbohydrates with ozone alone carboxylate groups form only as an intermediary reaction product and are subsequently decarboxylated to carbon dioxide (Lemeune et al., 2004). With the addition of phenolics, oxidation reactions involving hydroxyl radicals with cellulose dominates the minor reaction of ozone radicals with cellulose. 3.6. Size and Morphological Characterization Quantitative characterization of S-CNF size distributions in DI water suspension was performed on a 0.5 wt. % sample using dynamic light scattering (DLS). The hydrodynamic size
16
obtained is shown in Figure 4 by volume. There are two peaks observed at around 230 nm and 2.2 µm. With no mechanical mercerization, a volume of about 91% is in the particle size range of 700 nm to 6 µm while about 9% are in the size range of 130–397 nm. The yield of the S-CNF is estimated from the size distribution to be around 9%. The dimensions of S-NFC and T-CNF in suspension examined directly using a transmission electron microscope are shown in Figure 5. The images show that oxidation and enzymatic treatment have facilitated defibrillation and depolymerization of the cellulose fibers after a 10 min sonication. While similar images were obtained from enzyme treated non-oxidized fibers following sonication, the stability of those samples was limited and the fibers flocculated after a few hours. Sonication of non-enzyme treated oxidized and non-enzyme/non-oxidized samples failed to defibrillate the fibers. In addition, oxidation of non-enzyme treated pulp did not result in sufficient charges on the cellulose fibers to stay dispersed in the solution. TEM images of the flocculated samples were not taken because of the relatively large particle size (>1mm). The diameter of the fibers after the chemical and mechanical treatment was less than 20 nm with an average aspect ratio of 1:15. It is concluded from TEM images that sonication and the minimal defibrillation of the fibers applied in making our CNF is not efficiently breaking down the cellulose fibers. It is expected that due to the electroviscous effect, oxidation of cellulose C6 hydroxyl groups to carboxylates would lead to an increase in the observed viscosity of the suspension, however, this was not observed in our samples (Araki et al., 2001). This is likely due to the absence of mechanical treatment in the form of refining or homogenization as well as the likelihood of inadequacy of OH radicals to depolymerize cellulose fibers as efficiently as TEMPO mediated oxidation. Prior results reviewed by Coseri show significant reduction in the degree of
17
polymerization (DP) of cellulose upon TEMPO-mediated oxidation (Coseri, 2017). Change in the viscosity occurs after intensive refining of the pulp suspension accompanied by oxidation as a facilitator to the fiber breakdown process. While some level of surface oxidation is achievable, lower yields are expected in the absence of mechanical treatment. The low S-CNF yield (< 5% w/w of starting mass) can also be attributed to the lack of post oxidation mechanical treatment, as was also the case for the T-CNF produced in our lab. Sonication alone is not enough to defibrillate the cellulose fibers. As previously reported it merely disperses the particles without resulting in significant change in the size distribution (Peyre et al., 2015). Comparison of our TEM images to images taken from commercially available CNF confirm a greater amount of defibrillation in those samples. While fibrillation using disc refining or homogenizing are therefore essential to increase the CNF yield, we have not tested ozone/syringic acid oxidized pulps in such equipment (Brinchi, Cotana, Fortunati, & Kenny, 2013; Okita et al., 2010). 4. Conclusions An alternative to TEMPO-mediated oxidation of cellulose surface to produce CNF was presented in this study using ozone gas, a versatile oxidant, and lignin-derived phenolics to enhance oxidation reactions to modify the cellulose surface. We foresee compounds from degraded lignin in the pulping or cellulosic ethanol pretreatment process, such as syringic acid the model lignin compound used here, acting as oxidation enhancers for the production of CNFs. While ozone alone is not an efficient oxidizer of cellulose, in the presence of syringic acid reactions did occur with ozone which formed hydroxyl radicals that were able to effectively oxidize the cellulose surface. The results suggest that the reaction of hydroxyl radicals with cellulose C6 primary alcohol leads to formation of stable carboxylate groups on the celluloses. Unlike TEMPO, syringic acid does not act as a catalyst and is degraded during the reaction.
18
Nonetheless, complications in recycling TEMPO and the readily available lignin degradation products in large amounts from pulping may eliminate the economic necessity of its recovery. Additionally, enzyme treatment with a β-glucosidase proved to be effective in increasing the reactivity of pulp to chemical surface modification. While S-CNF may not completely match TCNF properties, the lower expected production cost by using a pulping byproduct (phenolics) coupled with a lower cost oxidant may lead to production of a similar grade yet less expensive CNF. Further study is needed to determine the efficacy of a mixture of lignin degradation compounds from pulping process in enhancing oxidation as compared to syringic acid alone. Excess amounts of reactants were used in the study as a proof of concept, and there is room for optimization of the amount of ozone, lignin degradation product and enzyme loading to achieve more economically favorable oxidation. S-CNF properties (such as tensile and thermal stability, crystallinity, film transparency, turbidity and water permeability) from other cellulosic sources also needs further study for potential applications. Acknowledgments This research was supported by USDA-NIFA Hatch Project IND010677 and the Purdue University Agricultural Research Station. The authors would like to acknowledge the support of Dr. Linda Mason for providing the ozone generator and Shane Peng for help with the FTIR. Valuable remarks from Dr. Alan W. Rudie, at the US Forest Service Research & Development and
Dr.
Galina
Rodionova
contributed
to
our
experimental
practices.
Author Contributions Iman Beheshti Tabar and Nathan S. Mosier conceived of the experimental approach. Iman Beheshti Tabar conducted the CNF production experiments. Ximing Zhang conducted the NMR analyses and the surface analyses in collaboration with Iman Behshti Tabar. Jeffrey P. Youngblood
19
contributed significantly to the CNF characterization. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
References Alemdar, A., & Sain, M. (2008). Isolation and characterization of nanofibers from agricultural residues – Wheat straw and soy hulls. Bioresource Technology, 99(6), 1664-1671. Araki, J., Wada, M., & Kuga, S. (2001). Steric Stabilization of a Cellulose Microcrystal Suspension by Poly(ethylene glycol) Grafting. Langmuir, 17(1), 21-27. Bondeson, D., Mathew, A., & Oksman, K. (2006). Optimization of the isolation of nanocrystals from microcrystalline cellulose by acid hydrolysis. Cellulose, 13(2), 171-180. Brinchi, L., Cotana, F., Fortunati, E., & Kenny, J. M. (2013). Production of nanocrystalline cellulose from lignocellulosic biomass: Technology and applications. Carbohydrate Polymers, 94(1), 154-169. Buschle-Diller, G., Inglesby, M. K., & Wu, Y. (2005). Physicochemical properties of chemically and enzymatically modified cellulosic surfaces. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 260(1–3), 63-70. Chirat, C., & Lachenal, D. (1997). Effect of Hydroxyl Radicals on Cellulose and Pulp and their Occurrence During Ozone Bleaching. Holzforschung - International Journal of the Biology, Chemistry, Physics and Technology of Wood (Vol. 51, p. 147). Coseri, S. (2017). Cellulose: To depolymerize… or not to? Biotechnology Advances, 35(2), 251266. da Silva Perez, D., Montanari, S., & Vignon, M. R. (2003). TEMPO-Mediated Oxidation of Cellulose III. Biomacromolecules, 4(5), 1417-1425. Engström, A.-C., Ek, M., & Henriksson, G. (2006). Improved Accessibility and Reactivity of Dissolving Pulp for the Viscose Process: Pretreatment with Monocomponent Endoglucanase. Biomacromolecules, 7(6), 2027-2031. Filpponen, I., & Argyropoulos, D. S. (2008). Determination of Cellulose Reactivity by Using Phosphitylation and Quantitative 31P NMR Spectroscopy. Industrial & Engineering Chemistry Research, 47(22), 8906-8910. Fock, W. (1959). Eine modifizierte method zur bestimmung der reaktivität von zellstoffen für viskosherstellung. Das Papier 13(3), 92-95. Gashti, M. P., Pournaserani, A., Ehsani, H., & Gashti, M. P. (2013). Surface oxidation of cellulose by ozone-gas in a vacuum cylinder to improve the functionality of fluoromonomer. Vacuum, 91, 7-13. Ghose, T. K. (1987). Measurement of cellulase activities. Pure and Applied Chemistry (Vol. 59, p. 257). Gurol, M. D., & Singer, P. C. (1982). Kinetics of ozone decomposition: a dynamic approach. Environmental Science & Technology, 16(7), 377-383. Henriksson, G., Christiernin, M., & Agnemo, R. (2005). Monocomponent endoglucanase treatment increases the reactivity of softwood sulphite dissolving pulp. Journal of Industrial Microbiology and Biotechnology, 32(5), 211-214.
20
Hirota, M., Tamura, N., Saito, T., & Isogai, A. (2010). Water dispersion of cellulose II nanocrystals prepared by TEMPO-mediated oxidation of mercerized cellulose at pH 4.8. Cellulose, 17(2), 279-288. Isogai, A., Saito, T., & Fukuzumi, H. (2011). TEMPO-oxidized cellulose nanofibers. Nanoscale, 3(1), 71-85. Jiang, F., & Hsieh, Y.-L. (2013). Chemically and mechanically isolated nanocellulose and their self-assembled structures. Carbohydrate Polymers, 95(1), 32-40. Kato, K., Vasilets, V. N., Fursa, M. N., Meguro, M., Ikada, Y., & Nakamae, K. (1999). Surface oxidation of cellulose fibers by vacuum ultraviolet irradiation. Journal of Polymer Science Part A: Polymer Chemistry, 37(3), 357-361. Lemeune, S., Jameel, H., Chang, H. M., & Kadla, J. F. (2004). Effects of ozone and chlorine dioxide on the chemical properties of cellulose fibers. Journal of Applied Polymer Science, 93(3), 1219-1223. Löwendahl, L., Petersson, G., & Samuelson, O. (1978). Phenolic compounds in kraft black liquor. Svensk Papperstidning, 81(12), 392-396. Mandels, M., Andreotti, R., & Roche, C. (1976 ). Measurement of saccharifying cellulase. Biotechnol. Bioeng. Symp (Vol. 6). United States: Army Natick Development Center, MA. Montanari, S., Roumani, M., Heux, L., & Vignon, M. R. (2005). Topochemistry of Carboxylated Cellulose Nanocrystals Resulting from TEMPO-Mediated Oxidation. Macromolecules, 38(5), 1665-1671. Okita, Y., Saito, T., & Isogai, A. (2010). Entire Surface Oxidation of Various Cellulose Microfibrils by TEMPO-Mediated Oxidation. Biomacromolecules, 11(6), 1696-1700. Peyre, J., Paakkonen, T., Reza, M., & Kontturi, E. (2015). Simultaneous preparation of cellulose nanocrystals and micron-sized porous colloidal particles of cellulose by TEMPOmediated oxidation. Green Chemistry, 17(2), 808-811. Pouyet, F., Chirat, C., Potthast, A., & Lachenal, D. (2014). Formation of carbonyl groups on cellulose during ozone treatment of pulp: Consequences for pulp bleaching. Carbohydrate Polymers, 109, 85-91. Praskalo, J., Kostic, M., Potthast, A., Popov, G., Pejic, B., & Skundric, P. (2009). Sorption properties of TEMPO-oxidized natural and man-made cellulose fibers. Carbohydrate Polymers, 77(4), 791-798. Pääkkö, M., Ankerfors, M., Kosonen, H., Nykänen, A., Ahola, S., Österberg, M., . . . Lindström, T. (2007). Enzymatic Hydrolysis Combined with Mechanical Shearing and HighPressure Homogenization for Nanoscale Cellulose Fibrils and Strong Gels. Biomacromolecules, 8(6), 1934-1941. Ragnar, M., Eriksson, T., & Reitberger, T. (1999). Radical Formation in Ozone Reactions with Lignin and Carbohydrate Model Compounds. Holzforschung (Vol. 53, p. 292). Ragnar, M., Eriksson, T., Reitberger, T., & Brandt, P. (1999). A New Mechanism in the Ozone Reaction with Lignin Like Structures. Holzforschung (Vol. 53, p. 423). Rodionova, G., Saito, T., Lenes, M., Eriksen, Ø., Gregersen, Ø., Kuramae, R., & Isogai, A. (2013). TEMPO-Mediated Oxidation of Norway Spruce and Eucalyptus Pulps: Preparation and Characterization of Nanofibers and Nanofiber Dispersions. Journal of Polymers and the Environment, 21(1), 207-214.
21
Rose, M., Babi, M., & Moran-Mirabal, J. (2015). The study of cellulose structure and depolymerization through single-molecule methods. Industrial Biotechnology, 11(1), 1624. Saito, T., Kimura, S., Nishiyama, Y., & Isogai, A. (2007). Cellulose Nanofibers Prepared by TEMPO-Mediated Oxidation of Native Cellulose. Biomacromolecules, 8(8), 2485-2491. Saito, T., Nishiyama, Y., Putaux, J.-L., Vignon, M., & Isogai, A. (2006). Homogeneous Suspensions of Individualized Microfibrils from TEMPO-Catalyzed Oxidation of Native Cellulose. Biomacromolecules, 7(6), 1687-1691. Satyamurthy, P., Jain, P., Balasubramanya, R. H., & Vigneshwaran, N. (2011). Preparation and characterization of cellulose nanowhiskers from cotton fibres by controlled microbial hydrolysis. Carbohydrate Polymers, 83(1), 122-129. Sepall, O., & Mason, S. G. (1961). Hydrogen Exchange Between Cellulose and Water: I. Measurement of Accessibility. Canadian Journal of Chemistry, 39(10), 1934-1943. Shinoda, R., Saito, T., Okita, Y., & Isogai, A. (2012). Relationship between Length and Degree of Polymerization of TEMPO-Oxidized Cellulose Nanofibrils. Biomacromolecules, 13(3), 842-849. Sluiter, A., Hames, B., Hyman, D., Payne, C., Ruiz, R., Scarlata, C., . . . Wolfe, J. (2008). Standard Procedures for Biomass Compositional Analysis - Biomass and Total Dissolved Solids in Liquid Process Samples. National Renewable Energy Laboratory. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., & Crocker, D. (2012). Standard Procedures for Biomass Compositional Analysis- Determination of Structural Carbohydrates and Lignin in Biomass. National Renewable Energy Laboratory. Sluiter, A., Ruiz, R., Scarlata, C., Sluiter, J., & Templeton, D. (2005). Standard Procedures for Biomass Compositional Analysis-Determination of Extractives in Biomass. National Renewable Energy Laboratory. Son, Y., Lim, M., Khim, J., & Ashokkumar, M. (2012). Attenuation of UV Light in Large-Scale Sonophotocatalytic Reactors: The Effects of Ultrasound Irradiation and TiO2 Concentration. Industrial & Engineering Chemistry Research, 51(1), 232-239. Sun, R., & Tomkinson, J. (2001). Fractional separation and physico-chemical analysis of lignins from the black liquor of oil palm trunk fibre pulping. Separation and Purification Technology, 24(3), 529-539. Zhang, Y. H. P., & Lynd, L. R. (2005). Determination of the Number-Average Degree of Polymerization of Cellodextrins and Cellulose with Application to Enzymatic Hydrolysis. Biomacromolecules, 6(3), 1510-1515. Zhu, J. Y., Sabo, R., & Luo, X. (2011). Integrated production of nano-fibrillated cellulose and cellulosic biofuel (ethanol) by enzymatic fractionation of wood fibers. Green Chemistry, 13(5), 1339-1344. Östberg, L., Håkansson, H., & Germgård, U. (2012). Some aspects of the reactivity of pulp intended for high-viscosity viscose. BioResources, 7(1), 743-755.
Reducing End in meq glucose/g cellulose
22
14 12
10 8 6 4 2 0 13P, Low 13P, High 692, Low 692, High Blended only pulp
Control
Various Depol® enzyme hydrolyzed samples
Figure 1. BCA assay Reducing end quantification of 0.005 g cellulose equivalent treated with two different Depol endoglucanase enzyme (13P and 692) with high (0.5 ml/g) and low (0.5 ml/g) loading. Control is non-enzyme treated 1% pulp dilution.
23
2
1
5 3
6
4
a
1 2
5
b
3
6
1 2
5
c
6
3 4
ppm
Figure 2. Carbon-13 NMR spectra in D-methanol (CD3OD): (a) Non oxidized syringic acid; (b) ozone oxidized syringic acid in alkaline condition (pH 11); (c) ozone oxidized syringic acid in acidic conditions (pH 3).
24
Figure 3. FTIR spectra of different cellulose nanofiber samples.
Figure 4. Size distribution (log10 nm) of 0.5 wt.% S-NFC by volume.
25
1
2
4
5
3
6
Figure 5. TEM images of uranyl acetate stained CNF. Images 1 and 2: 0.1% w/v S-CNF and image 3 is 0.001% w/v S-CNF. Images 4, 5 and 6 is 0.1% w/v T-CNF.
26
Table 1. Solubilized glucose and hemicellulose content after 24h enzymatic treatment of never dried pulp. Enzyme
Loading
Loading Amount
Glucose
Hemicellulose
(ml enzyme)
(IU/ g fiber)
(wt %)
(wt %)
Depol 692*
Low, 0.25
150
7.6
96
Depol 692
High, 0.5
300
13.2
98
Depol 13P**
Low, 0.25
130
7.76
49.6
Depol 13P
High, 0.5
260
15.3
74.5
*Activity: 598 IU/ml **Activity: 519 IU/ml
Table 2. z-potential of the various CNF samples at neutral pH. z-potential Carboxyl content Treatment (mV) (μmol/g) Blank DI
-3.6
-
S Control
-10
11
O3 Control
-13.4
14
Pulp Control
-7.5
19
S-CNF
-42.2
418
T-CNF
-65
687