Accepted Manuscript Lignin removal enhancement from prehydrolysis liquor of kraft-based dissolving pulp production by laccase- induced polymerization Qiang Wang, M. Sarwar Jahan, Shanshan Liu, Qingxian Miao, Yonghao Ni PII: DOI: Reference:
S0960-8524(14)00664-6 http://dx.doi.org/10.1016/j.biortech.2014.05.005 BITE 13413
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
21 March 2014 30 April 2014 2 May 2014
Please cite this article as: Wang, Q., Sarwar Jahan, M., Liu, S., Miao, Q., Ni, Y., Lignin removal enhancement from prehydrolysis liquor of kraft-based dissolving pulp production by laccase- induced polymerization, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.05.005
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Lignin removal enhancement from prehydrolysis liquor of kraftbased dissolving pulp production by laccase- induced polymerization Qiang Wanga,b*, M. Sarwar Jahanb,c, Shanshan Liu a,b, Qingxian Miao b,d, Yonghao Nib a
Key Laboratory of Pulp & Paper Science and Technology (Qilu University of Technology), Ministry of
Education, Jinan, Shandong, P. R. China 250353 b
Limerick Pulp and Paper Centre, University of New Brunswick, Fredericton, New Brunswick, Canada
E3B 5A3 c
Pulp and Paper Research Division, BCSIR Laboratories, Dhaka, Dr. Qudrat-i-Khuda Road, Dhaka
1205, Bangladesh d
College of Material Engineering, Fujian Agriculture and Forestry University, Fuzhou, P. R. China
350002
Abstract: Lignin removal is essential for value-added utilization of hemicelluloses and acetic acid present in the prehydrolysis liquor (PHL) of a kraft- based hardwood dissolving pulp production. In this paper, a novel process concept, consisting of laccase-induced lignin polymerization, followed by filtration/ flocculation, was developed to enhance the lignin removal. The results showed that the lignin removal increased from 11% to 46-61% at laccase concentration of 1-4 U mL-1. The GPC results showed that the molecular weight of the lignin from the laccase treated PHL was increased by 160% in comparison with the original one. The subsequent flocculation using singular Poly-DADMAC system or dual polymer system of Poly1
DADMAC/CPAM can further remove 10-15% lignin. The concentrations of hemicelluloses and acetic acid were negligibly affected during the laccase treatment, while flocculation caused 12-15% of total sugar loss. Additionally, the process incorporates this new concept into the kraft-based dissolving pulp production process was proposed. Keywords: Prehydrolysis liquor; Lignin removal; Laccase; Polymerization; Flocculation; Biorefinery. 1. Introduction Lignocellulose material, wood in particular, has the greatly potential to produce bio-fuel and bio-materials (Salehian et al., 2013). For this reason, the Forest biorefinery has gained much interest in the recent years (Yu et al., 2013). The kraft-based dissolving pulp production process fits well into the forest biorefinery concept (Shen et al., 2013). Prehydrolysis is an important step in the kraftbased dissolving pulp production process to remove hemicelluloses, which are not desired in the final product. In the prehydrolysis process, a part of lignin is also dissolved, along with hemicelluloses (Hage et al., 2010). The presence of lignin in the prehydrolysis liquor (PHL) is detrimental for its value-added utilization of hemicelluloses. For example, xylose/xylan fermentation for ethanol or xylitol production can be negatively affected (Lee, et al., 2013). The prehydrolysis process also releases acetic acid from the bound acetyl groups of hemicelluloses (Wei, et al., 2011), in particular if a mixture of hardwood was used as the raw material. The separation and concentration of acetic acid from the PHL is crucial in implementing 2
the biorefinery concept; however, the presence of lignin may hamper the separation and concentration of acetic acid from the PHL (Yang et al., 2013b). In the process of converting xylose/xylan in the PHL to furfural, the presence of lignin can cause side reactions (Liu et al., 2013). Therefore, lignin should be removed first in order for successful exploitation of hemicelluloses and acetic acid in the PHL. Once separated from the PHL, lignin itself can be raw material for many valueadded products (Schorr et al., 2014), e.g. phenols, biofuel, and plastics, which can generate additional revenues to the kraft-based dissolving pulp mills. A number of methods have been proposed for the lignin removal from PHL in the literature: (1) by acidification (Shi et al., 2011), although lowering its pH is an effective method for lignin precipitation/separation from the black liquor of kraft pulping process, only 3.8% of lignin can be removed from PHL; (2) by adding polymers such as polyethylene oxide (PEO) (Shi et al., 2011); (3) using adsorbent (Shen et al., 2013), e.g., lime mud and activated carbon, to remove lignin via adsorption; (4) nanofiltration or microfiltration (Hu et al., 2010). However, lignin in PHL (pH 3.6) is quite different from that in the black liquor: for example, the molecular weight and structure are much different (Yang et al., 2013a). Activated carbon performed well on the lignin removal (Shen et al. 2013); however, it still has practical challenges. Therefore, development of more effective/ efficient methods for removing/ separating lignin from the PHL is still desirable. The bio- and/ or enzyme technology has received much attention in the pulp and paper industry (Huttermann et al., 2001), because of (i) high selectivity, (ii) mild 3
reaction conditions, (iii) no formation of undesired by-product. For example, the aerobic/ anaerobic processes have been widely used in mill effluent treatment, xylanase for pulp bleaching, enzymatic deinking/ pitch control (Areskogh et al., 2010a). Laccase, a copper-containing oxidase, was of interest for detoxification and polymerization in lignocellulosic material utilization. Jurado et al (2009) reported the detoxification wheat straw from steam explosion process by laccase, and found that the toxic phenolic compounds were polymerized in the process, hence improving the enzymatic efficiency of the cellulose. Kolb et al (2012) using HS-SPME/GC-MS to monitor the removal of phenolic monomers during laccase treatment, and reported that they can be removed in the process. Ludwig et al (2013) using laccase and laccase-anion exchanger to improve the fermentability of cooked wheat straw from the ethanol- based organosolv process, and successfully enhanced the enzymatic efficiency. Gouveia et al (2012; 2013) employed laccase to polymerize different lignin fractionation isolated from black liquor through successive organic solvent extraction, the results showed that the initial lignin with higher molecular weight lead to the pronounce molecular weight increase. The objective of present study is to enhance the lignin removal from PHL by the laccase- treatment, followed by flocculation. The hypothesis was that laccase can induce polymerization of lignin present in the PHL, which can then facilitate the lignin removal in the subsequent step. The laccase treatment was optimized by varying pH, temperature, time and laccase concentration. The molecular weight of 4
laccase-treated lignin was determined based on the gel permeation chromatography (GPC) technique. The addition of coagulant (Poly-DADMAC) and/or flocculant (CPAM) on the lignin removal after the laccase treatment was also investigated. The effect on other dissolved organics, like hemicelluloses, acetic acid in the PHL were characterized.
2. Materials and Methods 2.1 Raw materials, Enzyme, and chemicals The prehydrolysis liquor (PHL) was collected from a commercial plant in Eastern Canada that produces dissolving pulp based on the kraft technology and uses a mixture of maple (70% wt.), poplar (20% wt.) and birch (10% wt.) as raw materials. The large particles of PHL were filtered using Whatman qualitative filter paper. The compositions of the original PHL are listed in Table 1, and pH is 3.63. The laccase sample (based on Trametes versicolor) and 2,2’-azino-bis (3ethylbenzthiazoline-6-sulfonate) (ABTS) were purchased from Sigma-Aldrich. Poly-diallyl dimethyl ammonium chloride (Poly-DADMAC) with a molecular weight of 400-500 kDa was purchased from Aldrich. Cationic polyacrylamide (CPAM) with a molecular weight of 1 MDa was received from BASF. Prior to use, solutions of 4 g L-1 and 1 g L-1 for Poly-DADMAC and CPAM were prepared, respectively. 2.2 Determination of laccase activity The laccase activity was determined spectrophotometrically at 420 nm (ε=36000 M-1 cm-1), as described by Mansfield (2002), at pH 4.5 and 20 oC with ABTS 0.5 mM, 5
as the substrate. One activity unit was defined as the amount of enzyme that oxidized 1µmol of ABTS per min. The activities were expressed in U mL-1. 2.3 Laccase treatments 60 mL PHL was added into 250 mL Erlenmeyer flask. Then, a required amount of 30 U mL-1 laccase solution was added to the flask, the reaction was carried out in shaker in a water bath. The treatment conditions were: Laccase concentration of 0-4 U m L-1, time of 0-5 h, pH of 3.6-6.5, and temperature of 26-46 oC. Once the reaction was completed, PHL was filtered on a Whatman nylon membrane (0.45µm pore size). 2.4 Flocculation experiments Singular or dual polymer system was carried out. For a singular polymer system, the specified amount of the Poly-DADMAC solution was added into the PHL sample in 30 mL vial. Then, the vial was shaken for 10 min at 150 rpm, and settled for 24 h. Samples from the solution were filtered on a Whatman nylon membrane (0.45µm pore size). For a dual polymer system, the desired charge of Poly-DADMAC was added in the PHL, which was stirred for 10 min at 150 rpm; subsequently, CPAM was added to the system, again stirring for 10 min at 150 rpm. The subsequent procedures were the same as above. 2.5 Lignin isolation from the original and laccase treated PHL The lignin present in the original PHL was initially adsorbed onto activated carbon (CR325W-Ultra, CARBON RESOURCES Comp.) by following the method in the literature (Shen et al. 2013). The weight ratio of PHL to activated carbon was 30:1. 6
The mixture was shaken at 250 rpm and room temperature for 5 h. Then, the mixture was filtered on Whatman filter paper (Q8). The adsorbed lignin, together with the activated carbon, was dried at room temperature for 48 h, the desorption of lignin from activated carbon was conducted using methanol (methanol: activated carbon= 30:1) at 300 rpm and room temperature for 5 h with three replicates. The lignin sample was obtained by filtration and evaporation, then dried in vacuum oven at 50 o
C for 48 h.
2.6 Acetylation and gel permeation chromatography The lignin samples were acetyl-brominated in a mixture of acetyl bromide and acetic acid (8:92) and kept for 72 h (Iiyama and Wallis, 1988). Once the bromination was completed, the solvent was evaporated in a rotary evaporator. Then, the acetylated lignin was dissolved in THF and filtered through a 0.22 µm filter for GPC analysis, then subjected to the molecular weight analysis of lignin based on the GPC method (Agilent 1260). 2.7. Lignin, hemicelluloses, acetic acid and furfural analysis The lignin content of the PHL was determined based on the UV-vis spectrometric method at 205 nm by following Tappi UM250 (Huo et al., 2013). The concentration of hemicelluloses in the samples was determined using an ion chromatography (IC) unit equipped with a CarboPacTMPA1 column (Dionex-300, Dionex Corporation, Canada) and a pulsed amperometric detector (PAD). Acid hydrolysis of samples was carried out with sulfuric acid at 121oC in an oil bath for 1h (Neslab Instruments, Inc., Portsmouth, NH, USA) as described elsewhere (Zhang et 7
al., 2013). A Varian 300 NMR-spectrometer was employed for acetic acid and furfural concentrations determination as described (Yang et al.2013b), with D2O to water ratio of 1:4.
3 Results and discussion 3.1 Proposed process for lignin removal from PHL by laccase-induced polymerization The hemicelluloses and part of lignin can be removed from hardwood chips in the prehydrolysis stage prior to Kraft pulping in the production of dissolving pulp (Sixta 2006). The 5- carbon hemicelluloses are potential raw material for producing value-added products, such as furfural or xylitol. Lignin in the PHL, on the other hand, needs to be removed prior to the value-added utilization of others in the PHL, because the presence of lignin has a number of drawbacks (Duarte et al., 2010). The main constituents in the PHL include lignin, xylose, acetic acid and furfural (Yang et al. 2013b). As discussed before, the lignin removal is essential prior to the value-added utilization of others, such as acetic acid, hemicelluloses. It was reported that the acid precipitation method could only remove 22% PHL lignin when acidified to pH 2, followed by PEO flocculation (Shi et al., 2011). One potential method to improve the removal of PHL lignin is to polymerize the PHL lignin, for example by the laccase-induced polymerization. Fig. 1 shows a proposed process diagram of the prehydrolysis kraft-based dissolving pulp production process, with the add-on regarding the PHL utilization. 8
The present concept of the laccase-induced polymerization for enhancing the lignin removal from the PHL, followed by filtration or flocculation, is included. As can be seen, the PHL is first treated with laccase, then filtration or polymer flocculation will then follow. Due to the increased molecular weight of lignin as a result of laccaseinduced polymerization, the filtration/polymer flocculation becomes more effective, resulting in an enhanced lignin removal. The removed lignin may be used potentially as a polymer or bio-fuel (Mattinen et al., 2011). The lignin-depleted PHL, which mainly contains hemicelluloses and acetic acid, can be further recovered/ utilized for value-added products. 3.2 Optimization of the laccase- induced polymerization pH was studied for optimizing the laccase treatement. As can be seen (Fig. 2 (A)), a wide pH range (3.6-5) was suitable for lignin removal, which may be beneficial for the laccase treatment. This is in agreement with literature results (Ibarra et al., 2006) that the optimal pH was 3-4.5 in a totally chlorine free bleaching of eucalyptus pulp using laccase-mediator system. The optimal temperature for the laccase treatment for the purpose of removing lignin from PHL is about 36 oC, as shown in Fig 2 (B). Under the conditions of 36 oC, laccase concentration of 2 U mL-1, 2 h and pH as is, about 45% lignin removal can be achieved. The optimal temperature for laccase was lower than literature report (Gouveia et al. 2013), showing that temperature of 60 oC led to the extensive lignin polymerization during laccase- assisted polymerizing of kraft lignin from black liquor. Shown in Fig. 2 (C) is the reaction time effect. It can be found that a 3 h would 9
be sufficient for the laccase treatment. As shown in Fig. 2 (D), a laccase of 2-3 U mL-1 is the optimum to reach the maximum lignin removal (50-58%) from the PHL under the conditions studied. 3.3 Lignin molecule weight The lignin molecular weight of original and laccase treated PHL was listed in Table 2. It can be seen that the molecular weight (Mw) of laccase treated lignin increased about 160% from the original one, supporting the conclusion of lignin grafting/ coupling reactions induced by laccase. The lignin polymerization can then facilitate lignin removal through filtration or flocculation. Previous studies showed lignin molecular weight increase when using laccase to polymerize commercial lignosulfonates (original Mw range 7000-25000) for polymer utilization (Areskogh et al. 2010b), and eucalyptus globulus kraft lignin (original molecular weight of 4200) (Gouveia et al. 2012). 3.4 Flocculation/ coagulation process of laccase treated PHL The interaction of polymers with the original lignin present in the PHL was studied previously (Saeed et al., 2011; Shi et al., 2011), these polymers included PEO, PAC, Poly-DADMAC, chitosan, their combinations with acidification or activated carbon adsorption were also studied. The effect of using polymers to further improve the lignin removal on the laccase-treated PHL was thus, further investigated in this study. 3.4.1 Poly-DADMAC treatment The complexes of Poly-DADMAC and lignocelluloses material can be induced 10
by charge interaction and hydrogen bond (Saeed et al., 2011). The lignin removals from the PHL treated by Poly-DADMAC were presented in Fig. 3, the original PHL without laccase treated was included as the control. As can be seen from Fig. 3, the lignin removal of original PHL was much lower compared with the laccase treated PHL. At a Poly-DADMAC charge of 100 ppm, about 23.6% of lignin removal was obtained, while under otherwise the same conditions, about 57% lignin removal was achieved for the laccase treated PHL. This can be attributed to the lignin polymerization by laccase treatment, since the patch flocculation could be more effective by increasing the molecular weight of lignin (Saeed et al., 2011). With increasing the Poly-DADMAC charge, the lignin removal of the laccase treated PHL reached the maximum at 250 ppm Poly-DADMAC. Lignin-PolyDADMAC complexes were formed (Duarte et al., 2010) and precipitated when adding Poly-DADMAC to the PHL, and the effect was more pronounced with the increased charge of Poly-DADMAC until 250 ppm, beyond which the excessive PolyDADMAC has limited effect on the lignin complexes formation due to completion of charge neutralization. Similar results were reported by Saeed et al (2011). In their research on the effect of chitosan dosage on the lignin removal degree from the original PHL, it was found that with the chitosan dosage increasing, the lignin removal reached the maximum at high molecular weight of chitosan at 1 mg/g (Saeed et al., 2011). Duarte et al. (2010) reported that 36% of lignin was removed from dilution sample of sugar maple hydrolyzate using 47 ppm Poly-DADMAC. 11
3.4.2 Dual polymer treatment A dual polymer system has been recognized as an efficient coagulation/ flocculation process for removing lignin in the PHL of the kraft-based dissolving pulp production (Shi et al., 2011). The mechanism is that: 1) firstly lignocelluloses material formed complexes with Poly-DADMAC, which is highly charged, but with a low molecular weight; 2) subsequently, the lignin-Poly-DADMAC complexes can be bridged by CPAM, which has high molecular weight but low charge. As shown in Fig. 4, the lignin removal from the original PHL was much lower than the laccase treated PHL under otherwise the same conditions. For the laccase treated PHL, the lignin removal has negligible effect when increasing the dual polymer charges in the given range. The lignin removal of 58% was achieved at 35 ppm for both Poly-DADMAC and CPAM. Compared with the Poly-DADMAC polymer system, the dual polymer system has higher lignin removal at lower polymer charges. In addition, much more compact sediment was formed after dual polymer system treatment, facilitating the subsequent separation process. Similar results were reported by Yasarla and Ramarao (2012), they employed polymers (PEI, PolyDADMAC, CPAM) to flocculate colloidal material in lignocellulosic hydrolyzates, and found that the particles flocculated with PEI were larger, and had more compact structures than those from Poly-DADMAC. Shi et al. (2012) studied the PAC/PEO dual polymer system to remove lignin from the original PHL, and 37.5% lignin removal was obtained. 3.5 Characterization of treated PHL 12
It is important that the concentrations of hemicellulosic sugars and acetic acid, which are to be further utilized in the downstream processing, would be negligibly affected during the laccase polymerization process. The chemical compositions of the original and the treated PHL are listed in Table 1 and 3, respectively. As can be seen, xylan/ xylose was the major component, which represents about 74% of the total sugars in the original PHL. The laccase treatment of PHL had negligible loss of monosaccharide: the total monosaccharide sugar in the original PHL was 9.87g/L, while it was 9.56g/L and 10.2g/L after laccase treatment at 2 and 3 U/mL-1 laccase concentrations, respectively. On the other hand, there was some loss of monosaccharide sugars after the post- flocculation treatment, and the total monosaccharide sugar concentration was 8.92- 9.47g/L using the poly-DADMAC, and 8.99- 9.18 g/L using the dual polymers of poly- DADMAC/ CPAM. The oligosaccharide sugars changed from 52.32 g/L to 49.19- 51.05 g/L after the laccase treatment, and 44.0-44.2g/L after the post- flocculation using poly- DADMAC, and 43.29-45.29 g/L after the post- flocculation using poly- DADMAC/ CPAM dual polymers. The total sugar loss was in the range of 12-15%. One possible explanation is the oligo-sugar was removed through charge neutralized with cationic polymer, i.e. Poly-DADMAC and CPAM, because oligo-sugar is rich in hydroxyl groups. It was reported that about 7% of xylose loss occurred when applying 300 ppm PEO, to flocculate the original lignin in the PHL (Shi et al., 2011). Additionally, Saeed et al., (2011) reported that 30% of total sugars were lost when using 500 ppm high molecular weight chitosan in their study to remove the lignin present in the original 13
PHL.
4 Conclusions The laccase treatment of prehydrolysis liquor (PHL) of the kraft-based dissolving pulp production process can increase the lignin molecular weight, which then facilitates its removal in the subsequent filtration/flocculation process. The optimum laccase-induced polymerization conditions were: Laccase conc=2 U mL-1, t=3 h, T=36 oC, pH 3.6 (the original pH). The application of flocculation concept in a subsequent step can further enhance the lignin removal. The concentrations of hemicelluloses and acetic acid in the PHL were only marginally affected during the laccase treatment, although the hemicelluloses removal was 12-15% when combining the use of polymers in the subsequent flocculation step.
Acknowledgements This project was funded by an NSERC CRD grant, an Atlantic Innovation Fund, Canada Research Chairs programs of the Government of Canada. The authors are also grateful for the financial support from the National Science Foundation of China (Grant No. 31270627, 31370580) and Natural Science Foundation of Shandong Province (Grant No. ZR2010CM065, ZR2011CM011).
References 1. Areskogh, D., Li, J., Gellerstedt, G., and Henriksson, G., 2010a. Structural 14
modification of commercial lignosulphonates through laccase catalysis and ozonolysis. Ind. Crops Prod. 32, 458-466. 2. Areskogh, D., Li, J., Gellerstedt, G., and Henriksson, G., 2010b. Investigation of the Molecular Weight Increase of Commercial Lignosulfonates by Laccase Catalysis. Biomacromolecules 11, 904-910. 3. Duarte, G.V., Ramarao, B.V., and Amidon, T.E., 2010. Polymer induced flocculation and separation of particulates from extracts of lignocellulosic materials. Bioresour. Technol. 101, 8526-8534. 4. Gouveia, S., Fernández-Costas, C., Sanromán, M.A., and Moldes, D., 2012. Enzymatic polymerisation and effect of fractionation of dissolved lignin from Eucalyptus globulus Kraft liquor. Bioresour. Technol. 121, 131-138. 5. Gouveia, S., Fernández-Costas, C., Sanromán, M.A., and Moldes, D., 2013. Polymerisation of Kraft lignin from black liquors by laccase from Myceliophthora thermophila: Effect of operational conditions and black liquor origin. Bioresour. Technol. 131, 288-294. 6. Hage, R.E., Chrusciel, L., Desharnais, L., and Brosse, N., 2010. Effect of autohydrolysis of Miscanthus x giganteus on lignin structure and organosolv delignification. Bioresour. Technol. 101, 9321-9329. 7. Huo, D., Fang, G. G., Yang, Q., Han, S. M., Deng, Y. J., Shen, K. Z., and Lin, Y., 2013. Enhancement of eucalypt chips’ enzymolysis efficiency by a combination method of alkali impregnation and refining pretreatment. Bioresour. Technol. 150: 73-78. 15
8. Hu, R., Lin, L., Liu, T., and Liu, S., 2010. Dilute sulfuric acid hydrolysis of sugar maple wood extract at atmospheric pressure. Bioresour. Technol. 101: 35863594. 9. Huttermann, A., Mai, C., and Kharazipour, A., 2001. Modification of lignin for the production of new compounded materials. Appl. Microbiol. Biotechnol. 55, 387-394. 10. Ibarra, D., Romero, J., Martínez, M.J., Martínez, A.T., Camarero, S., 2006. Exploring the enzymatic parameters for optimal delignification of eucalypt pulp by laccase-mediator. Enzyme Microb. Technol. 39, 1319-1327. 11. Iiyama, K. and Wallis, A., 1988. An improved acetyl bromide procedure for determining lignin in woods and wood pulps. Wood Sci. Technol. 22: 271-280. 12. Jurado, M., Prieto, A., Martínez-Alcalá, Á., Martínez, Á.T., Martínez, M.J., 2009. Laccase detoxification of steam-exploded wheat straw for second generation bioethanol. Bioresour. Technol. 100, 6378-6384. 13. Kolb, M., Sieber, V., Amann, M., Faulstich, M., Schieder, D., 2012. Removal of monomer delignification products by laccase from Trametes versicolor. Bioresour. Technol. 104, 298-304. 14. Lee, H. J., Lim, W. S., Lee, J. W., 2013. Improvement of ethanol fermentation from lignocellulosic hydrolysates by the removal of inhibitors. J. Ind. Eng. Chem. 19: 2010-2015. 15. Liu, H., Hu, H., Jahan, M.S., and Ni, Y., 2013. Furfural formation from the prehydrolysis liquor of a hardwood kraft-based dissolving pulp production 16
process. Bioresour. Technol. 131, 315-320. 16. Ludwig, D., Amann, M., Hirth, T., Rupp, S., Zibek, S., 2013. Development and optimization of single and combined detoxification processes to improve the fermentability of lignocellulose hydrolyzates. Bioresour. Technol. 133, 455461. 17. Mansfield, S.D., 2002. Laccase impregnation during mechanical pulp processing improved refining efficiency and sheet strength. Appita J. 55, 49-53. 18. Mattinen, M. L., Maijala, P., Nousiainen, P., Smeds, A., Kontro, J., Sipilä, J., Tamminen, T., Willför, S., and Viikari, L., 2011. Oxidation of lignans and lignin model compounds by laccase in aqueous solvent systems. J. Mol. Catal. B: Enzym. 72, 122-129. 19. Salehian, P., Karimi, K., Zilouei, H., and Jeihanipour, A., 2013. Improvement of biogas production from pine wood by alkali pretreatment. Fuel 106, 484-489. 20. Shen, J., Kaur, I., Baktash, M.M., He, Z., and Ni, Y., 2013. A combined process of activated carbon adsorption, ion exchange resin treatment and membrane concentration for recovery of dissolved organics in pre-hydrolysis liquor of the kraft-based dissolving pulp production process. Bioresour. Technol. 127, 5965. 21. Shi, H., Fatehi, P., Xiao, H., and Ni, Y., 2011. A combined acidification/PEO flocculation process to improve the lignin removal from the pre-hydrolysis liquor of kraft-based dissolving pulp production process. Bioresour. Technol. 102, 5177-5182. 17
22. Shi, H., Fatehi, P., Xiao, H., and Ni, Y., 2012. Optimizing the Poly Ethylene Oxide Flocculation Process for Isolating Lignin of Prehydrolysis Liquor of a KraftBased Dissolving Pulp Production Process. Ind. Eng. Chem. Res. 51: 53305335. 23. Saeed, A., Fatehi, P., and Ni, Y., 2011. Chitosan as a flocculant for pre-hydrolysis liquor of kraft-based dissolving pulp production process. Carbohydr. Polym. 86, 1630-1636. 24. Schorr, D., Diouf, P. N., and Stevanovic, T., 2014. Evaluation of industrial lignins for biocomposites production. Ind. Crops Prod. 52: 65-73. 25. Sixta, H., 2006. Hand Book of Pulp, first ed., vol. 1. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. 26. Wei, L., Shrestha, A.. Tu, M., and Adhikari S., 2011. Effects of surfactant on biochemical and hydrothermal conversion of softwood hemicellulose to ethanol and furan derivatives. Process Biochem. 46: 1785-1792. 27. Yang, G., Jahan, M.S., and Ni, Y., 2013a. Structural Characterization of Prehydrolysis Liquor Lignin and Its Comparison with Other Technical Lignins. Curr. Org. Chem. 17: 1589-1595. 28. Yang, G., Sarwar Jahan, M., Ahsan, L., Zheng, L., and Ni, Y., 2013b. Recovery of acetic acid from pre-hydrolysis liquor of hardwood kraft-based dissolving pulp production process by reactive extraction with triisooctylamine. Bioresour. Technol. 138, 253-258. 29. Yasarla, L.R., Ramarao, B.V., 2012. Dynamics of Flocculation of Lignocellulosic 18
Hydrolyzates by Polymers. Ind. Eng. Chem. Res. 51, 6847-6861. 30. Yu, Y., Zeng, Y., Zuo, J., Ma, F., Yang, X., Zhang, X., and Wang, Y., 2013. Improving the conversion of biomass in catalytic fast pyrolysis via white- rot fungal pretreatment. Bioresour. Technol. 134, 198-203. 31. Zhang, D. S., Yang, Q., Zhu, J. Y., and Pan, X. J., 2013. Sulfite (SPORL) pretreatment of switchgrass for enzymatic saccharification. Bioresour. Technol. 129: 127-134.
19
Fig. 1 Process diagram for lignin removal by laccase-induced polymerization in a kraft- based dissolving pulp production process
20
Fig. 2 Lignin removals from the PHL by laccase-induced polymerization and filtration (A) effect of pH, (B) effect of temperature, (C) effect of time, (D) effect of laccase concentration
21
Fig. 3 Lignin removal from PHL (with/without laccase treatment) as a function of Poly-DADMAC charge (Laccase treatment conditions: : Laccase concentration 2 U mL-1, time 3 h, temperature 36 oC, pH of the original PHL (3.63))
22
Fig. 4 Lignin removal from PHL (with/without laccase treatment) in the dual-polymer system of Poly-DADMAC/CPAM (Laccase treatment conditions: Laccase concentration 2 U mL-1, time 3 h, temperature 36 oC, pH of the original PHL (3.63))
23
Table 1 Composition of the original PHL Total Rhammose
Arabinose
Galactose
Glucose
Xylose
Mannose
HAcb
Lignin
11.3
12.2
sugars Mono-
0.70
1.58
0.85
0.95
5.36
0.43
Oligo-
0.63
NDa
1.62
4.45
40.7
4.92
61.9
Note: NDa - Not detected. HAcb - Acetic acid.
24
Table 2 Molecular weight of lignin before and after laccase polymerization Original PHL
Laccase treated
Mw/Mn
1.72
1.99
Mn (Da)
564
802
Mw (Da)
972
1593
Note: laccase treated condition: laccase concentration 2 UmL-1, temperature 36 o C, time 3 h, pH of the original PHL (3.63).
25
Table 3 Effect of laccase polymerization followed by either filtration, or polyDADMAC or dual polymer treatment on the concentrations of hemicelluloses, and acetic acid (g L-1) Poly-
Dual polymer
DADMAC
Poly-DADMAC/CPAM
Laccase L2
L3
250
300
35/35
45/45
Mono-
0.60
0.70
0.61
0.61
0.61
0.61
Oligo-
0.63
0.58
0.55
0.51
0.40
0.51
Mono-
1.46
1.54
1.37
1.39
1.39
1.38
Oligo-
ND
a
ND
ND
ND
ND
ND
Mono-
0.90
1.02
0.84
0.85
0.86
0.83
Oligo-
1.29
1.30
1.24
1.39
1.32
1.51
Mono-
1.06
1.14
0.87
1.03
1.00
0.99
Oligo-
4.04
4.30
3.69
3.80
3.57
3.50
Mono-
5.14
5.36
4.84
5.20
4.87
4.74
Oligo-
39.0
39.77
34.6
33.9
34.3
35.3
Rhammose
Arabinose
Galactose
Glucose
Xylose
Mannose
26
Mono-
0.40
0.44
0.39
0.39
0.45
0.44
Oligo-
4.23
5.10
4.12
4.40
3.70
4.47
Total sugars
58.8
61.3
53.1
53.5
52.5
54.3
HAcb
10.6
11.1
6.45
6.47
7.07
6.78
Note: L2, L3, represent laccase concentrations of 2 and 3 U mL-1, respectively, other conditions such as time 3h, temperature 36 oC, pH of the original PHL (3.63).
27
1.
A new process enhances lignin removal from the prehydrolysis liquor was proposed.
2.
Laccase treatment was incorporated into the new process.
3.
Laccase treatment can polymerize low Mw lignin.
4.
The concentration of hemicelluloses and acetic acid were largely unaffected.
28
S0960-8524(14)00664-6 http://dx.doi.org/10.1016/j.biortech.2014.05.005 BITE 13413
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
21 March 2014 30 April 2014 2 May 2014
Please cite this article as: Wang, Q., Sarwar Jahan, M., Liu, S., Miao, Q., Ni, Y., Lignin removal enhancement from prehydrolysis liquor of kraft-based dissolving pulp production by laccase- induced polymerization, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.05.005
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.
Lignin removal enhancement from prehydrolysis liquor of kraftbased dissolving pulp production by laccase- induced polymerization Qiang Wanga,b*, M. Sarwar Jahanb,c, Shanshan Liu a,b, Qingxian Miao b,d, Yonghao Nib a
Key Laboratory of Pulp & Paper Science and Technology (Qilu University of Technology), Ministry of
Education, Jinan, Shandong, P. R. China 250353 b
Limerick Pulp and Paper Centre, University of New Brunswick, Fredericton, New Brunswick, Canada
E3B 5A3 c
Pulp and Paper Research Division, BCSIR Laboratories, Dhaka, Dr. Qudrat-i-Khuda Road, Dhaka
1205, Bangladesh d
College of Material Engineering, Fujian Agriculture and Forestry University, Fuzhou, P. R. China
350002
Abstract: Lignin removal is essential for value-added utilization of hemicelluloses and acetic acid present in the prehydrolysis liquor (PHL) of a kraft- based hardwood dissolving pulp production. In this paper, a novel process concept, consisting of laccase-induced lignin polymerization, followed by filtration/ flocculation, was developed to enhance the lignin removal. The results showed that the lignin removal increased from 11% to 46-61% at laccase concentration of 1-4 U mL-1. The GPC results showed that the molecular weight of the lignin from the laccase treated PHL was increased by 160% in comparison with the original one. The subsequent flocculation using singular Poly-DADMAC system or dual polymer system of Poly1
DADMAC/CPAM can further remove 10-15% lignin. The concentrations of hemicelluloses and acetic acid were negligibly affected during the laccase treatment, while flocculation caused 12-15% of total sugar loss. Additionally, the process incorporates this new concept into the kraft-based dissolving pulp production process was proposed. Keywords: Prehydrolysis liquor; Lignin removal; Laccase; Polymerization; Flocculation; Biorefinery. 1. Introduction Lignocellulose material, wood in particular, has the greatly potential to produce bio-fuel and bio-materials (Salehian et al., 2013). For this reason, the Forest biorefinery has gained much interest in the recent years (Yu et al., 2013). The kraft-based dissolving pulp production process fits well into the forest biorefinery concept (Shen et al., 2013). Prehydrolysis is an important step in the kraftbased dissolving pulp production process to remove hemicelluloses, which are not desired in the final product. In the prehydrolysis process, a part of lignin is also dissolved, along with hemicelluloses (Hage et al., 2010). The presence of lignin in the prehydrolysis liquor (PHL) is detrimental for its value-added utilization of hemicelluloses. For example, xylose/xylan fermentation for ethanol or xylitol production can be negatively affected (Lee, et al., 2013). The prehydrolysis process also releases acetic acid from the bound acetyl groups of hemicelluloses (Wei, et al., 2011), in particular if a mixture of hardwood was used as the raw material. The separation and concentration of acetic acid from the PHL is crucial in implementing 2
the biorefinery concept; however, the presence of lignin may hamper the separation and concentration of acetic acid from the PHL (Yang et al., 2013b). In the process of converting xylose/xylan in the PHL to furfural, the presence of lignin can cause side reactions (Liu et al., 2013). Therefore, lignin should be removed first in order for successful exploitation of hemicelluloses and acetic acid in the PHL. Once separated from the PHL, lignin itself can be raw material for many valueadded products (Schorr et al., 2014), e.g. phenols, biofuel, and plastics, which can generate additional revenues to the kraft-based dissolving pulp mills. A number of methods have been proposed for the lignin removal from PHL in the literature: (1) by acidification (Shi et al., 2011), although lowering its pH is an effective method for lignin precipitation/separation from the black liquor of kraft pulping process, only 3.8% of lignin can be removed from PHL; (2) by adding polymers such as polyethylene oxide (PEO) (Shi et al., 2011); (3) using adsorbent (Shen et al., 2013), e.g., lime mud and activated carbon, to remove lignin via adsorption; (4) nanofiltration or microfiltration (Hu et al., 2010). However, lignin in PHL (pH 3.6) is quite different from that in the black liquor: for example, the molecular weight and structure are much different (Yang et al., 2013a). Activated carbon performed well on the lignin removal (Shen et al. 2013); however, it still has practical challenges. Therefore, development of more effective/ efficient methods for removing/ separating lignin from the PHL is still desirable. The bio- and/ or enzyme technology has received much attention in the pulp and paper industry (Huttermann et al., 2001), because of (i) high selectivity, (ii) mild 3
reaction conditions, (iii) no formation of undesired by-product. For example, the aerobic/ anaerobic processes have been widely used in mill effluent treatment, xylanase for pulp bleaching, enzymatic deinking/ pitch control (Areskogh et al., 2010a). Laccase, a copper-containing oxidase, was of interest for detoxification and polymerization in lignocellulosic material utilization. Jurado et al (2009) reported the detoxification wheat straw from steam explosion process by laccase, and found that the toxic phenolic compounds were polymerized in the process, hence improving the enzymatic efficiency of the cellulose. Kolb et al (2012) using HS-SPME/GC-MS to monitor the removal of phenolic monomers during laccase treatment, and reported that they can be removed in the process. Ludwig et al (2013) using laccase and laccase-anion exchanger to improve the fermentability of cooked wheat straw from the ethanol- based organosolv process, and successfully enhanced the enzymatic efficiency. Gouveia et al (2012; 2013) employed laccase to polymerize different lignin fractionation isolated from black liquor through successive organic solvent extraction, the results showed that the initial lignin with higher molecular weight lead to the pronounce molecular weight increase. The objective of present study is to enhance the lignin removal from PHL by the laccase- treatment, followed by flocculation. The hypothesis was that laccase can induce polymerization of lignin present in the PHL, which can then facilitate the lignin removal in the subsequent step. The laccase treatment was optimized by varying pH, temperature, time and laccase concentration. The molecular weight of 4
laccase-treated lignin was determined based on the gel permeation chromatography (GPC) technique. The addition of coagulant (Poly-DADMAC) and/or flocculant (CPAM) on the lignin removal after the laccase treatment was also investigated. The effect on other dissolved organics, like hemicelluloses, acetic acid in the PHL were characterized.
2. Materials and Methods 2.1 Raw materials, Enzyme, and chemicals The prehydrolysis liquor (PHL) was collected from a commercial plant in Eastern Canada that produces dissolving pulp based on the kraft technology and uses a mixture of maple (70% wt.), poplar (20% wt.) and birch (10% wt.) as raw materials. The large particles of PHL were filtered using Whatman qualitative filter paper. The compositions of the original PHL are listed in Table 1, and pH is 3.63. The laccase sample (based on Trametes versicolor) and 2,2’-azino-bis (3ethylbenzthiazoline-6-sulfonate) (ABTS) were purchased from Sigma-Aldrich. Poly-diallyl dimethyl ammonium chloride (Poly-DADMAC) with a molecular weight of 400-500 kDa was purchased from Aldrich. Cationic polyacrylamide (CPAM) with a molecular weight of 1 MDa was received from BASF. Prior to use, solutions of 4 g L-1 and 1 g L-1 for Poly-DADMAC and CPAM were prepared, respectively. 2.2 Determination of laccase activity The laccase activity was determined spectrophotometrically at 420 nm (ε=36000 M-1 cm-1), as described by Mansfield (2002), at pH 4.5 and 20 oC with ABTS 0.5 mM, 5
as the substrate. One activity unit was defined as the amount of enzyme that oxidized 1µmol of ABTS per min. The activities were expressed in U mL-1. 2.3 Laccase treatments 60 mL PHL was added into 250 mL Erlenmeyer flask. Then, a required amount of 30 U mL-1 laccase solution was added to the flask, the reaction was carried out in shaker in a water bath. The treatment conditions were: Laccase concentration of 0-4 U m L-1, time of 0-5 h, pH of 3.6-6.5, and temperature of 26-46 oC. Once the reaction was completed, PHL was filtered on a Whatman nylon membrane (0.45µm pore size). 2.4 Flocculation experiments Singular or dual polymer system was carried out. For a singular polymer system, the specified amount of the Poly-DADMAC solution was added into the PHL sample in 30 mL vial. Then, the vial was shaken for 10 min at 150 rpm, and settled for 24 h. Samples from the solution were filtered on a Whatman nylon membrane (0.45µm pore size). For a dual polymer system, the desired charge of Poly-DADMAC was added in the PHL, which was stirred for 10 min at 150 rpm; subsequently, CPAM was added to the system, again stirring for 10 min at 150 rpm. The subsequent procedures were the same as above. 2.5 Lignin isolation from the original and laccase treated PHL The lignin present in the original PHL was initially adsorbed onto activated carbon (CR325W-Ultra, CARBON RESOURCES Comp.) by following the method in the literature (Shen et al. 2013). The weight ratio of PHL to activated carbon was 30:1. 6
The mixture was shaken at 250 rpm and room temperature for 5 h. Then, the mixture was filtered on Whatman filter paper (Q8). The adsorbed lignin, together with the activated carbon, was dried at room temperature for 48 h, the desorption of lignin from activated carbon was conducted using methanol (methanol: activated carbon= 30:1) at 300 rpm and room temperature for 5 h with three replicates. The lignin sample was obtained by filtration and evaporation, then dried in vacuum oven at 50 o
C for 48 h.
2.6 Acetylation and gel permeation chromatography The lignin samples were acetyl-brominated in a mixture of acetyl bromide and acetic acid (8:92) and kept for 72 h (Iiyama and Wallis, 1988). Once the bromination was completed, the solvent was evaporated in a rotary evaporator. Then, the acetylated lignin was dissolved in THF and filtered through a 0.22 µm filter for GPC analysis, then subjected to the molecular weight analysis of lignin based on the GPC method (Agilent 1260). 2.7. Lignin, hemicelluloses, acetic acid and furfural analysis The lignin content of the PHL was determined based on the UV-vis spectrometric method at 205 nm by following Tappi UM250 (Huo et al., 2013). The concentration of hemicelluloses in the samples was determined using an ion chromatography (IC) unit equipped with a CarboPacTMPA1 column (Dionex-300, Dionex Corporation, Canada) and a pulsed amperometric detector (PAD). Acid hydrolysis of samples was carried out with sulfuric acid at 121oC in an oil bath for 1h (Neslab Instruments, Inc., Portsmouth, NH, USA) as described elsewhere (Zhang et 7
al., 2013). A Varian 300 NMR-spectrometer was employed for acetic acid and furfural concentrations determination as described (Yang et al.2013b), with D2O to water ratio of 1:4.
3 Results and discussion 3.1 Proposed process for lignin removal from PHL by laccase-induced polymerization The hemicelluloses and part of lignin can be removed from hardwood chips in the prehydrolysis stage prior to Kraft pulping in the production of dissolving pulp (Sixta 2006). The 5- carbon hemicelluloses are potential raw material for producing value-added products, such as furfural or xylitol. Lignin in the PHL, on the other hand, needs to be removed prior to the value-added utilization of others in the PHL, because the presence of lignin has a number of drawbacks (Duarte et al., 2010). The main constituents in the PHL include lignin, xylose, acetic acid and furfural (Yang et al. 2013b). As discussed before, the lignin removal is essential prior to the value-added utilization of others, such as acetic acid, hemicelluloses. It was reported that the acid precipitation method could only remove 22% PHL lignin when acidified to pH 2, followed by PEO flocculation (Shi et al., 2011). One potential method to improve the removal of PHL lignin is to polymerize the PHL lignin, for example by the laccase-induced polymerization. Fig. 1 shows a proposed process diagram of the prehydrolysis kraft-based dissolving pulp production process, with the add-on regarding the PHL utilization. 8
The present concept of the laccase-induced polymerization for enhancing the lignin removal from the PHL, followed by filtration or flocculation, is included. As can be seen, the PHL is first treated with laccase, then filtration or polymer flocculation will then follow. Due to the increased molecular weight of lignin as a result of laccaseinduced polymerization, the filtration/polymer flocculation becomes more effective, resulting in an enhanced lignin removal. The removed lignin may be used potentially as a polymer or bio-fuel (Mattinen et al., 2011). The lignin-depleted PHL, which mainly contains hemicelluloses and acetic acid, can be further recovered/ utilized for value-added products. 3.2 Optimization of the laccase- induced polymerization pH was studied for optimizing the laccase treatement. As can be seen (Fig. 2 (A)), a wide pH range (3.6-5) was suitable for lignin removal, which may be beneficial for the laccase treatment. This is in agreement with literature results (Ibarra et al., 2006) that the optimal pH was 3-4.5 in a totally chlorine free bleaching of eucalyptus pulp using laccase-mediator system. The optimal temperature for the laccase treatment for the purpose of removing lignin from PHL is about 36 oC, as shown in Fig 2 (B). Under the conditions of 36 oC, laccase concentration of 2 U mL-1, 2 h and pH as is, about 45% lignin removal can be achieved. The optimal temperature for laccase was lower than literature report (Gouveia et al. 2013), showing that temperature of 60 oC led to the extensive lignin polymerization during laccase- assisted polymerizing of kraft lignin from black liquor. Shown in Fig. 2 (C) is the reaction time effect. It can be found that a 3 h would 9
be sufficient for the laccase treatment. As shown in Fig. 2 (D), a laccase of 2-3 U mL-1 is the optimum to reach the maximum lignin removal (50-58%) from the PHL under the conditions studied. 3.3 Lignin molecule weight The lignin molecular weight of original and laccase treated PHL was listed in Table 2. It can be seen that the molecular weight (Mw) of laccase treated lignin increased about 160% from the original one, supporting the conclusion of lignin grafting/ coupling reactions induced by laccase. The lignin polymerization can then facilitate lignin removal through filtration or flocculation. Previous studies showed lignin molecular weight increase when using laccase to polymerize commercial lignosulfonates (original Mw range 7000-25000) for polymer utilization (Areskogh et al. 2010b), and eucalyptus globulus kraft lignin (original molecular weight of 4200) (Gouveia et al. 2012). 3.4 Flocculation/ coagulation process of laccase treated PHL The interaction of polymers with the original lignin present in the PHL was studied previously (Saeed et al., 2011; Shi et al., 2011), these polymers included PEO, PAC, Poly-DADMAC, chitosan, their combinations with acidification or activated carbon adsorption were also studied. The effect of using polymers to further improve the lignin removal on the laccase-treated PHL was thus, further investigated in this study. 3.4.1 Poly-DADMAC treatment The complexes of Poly-DADMAC and lignocelluloses material can be induced 10
by charge interaction and hydrogen bond (Saeed et al., 2011). The lignin removals from the PHL treated by Poly-DADMAC were presented in Fig. 3, the original PHL without laccase treated was included as the control. As can be seen from Fig. 3, the lignin removal of original PHL was much lower compared with the laccase treated PHL. At a Poly-DADMAC charge of 100 ppm, about 23.6% of lignin removal was obtained, while under otherwise the same conditions, about 57% lignin removal was achieved for the laccase treated PHL. This can be attributed to the lignin polymerization by laccase treatment, since the patch flocculation could be more effective by increasing the molecular weight of lignin (Saeed et al., 2011). With increasing the Poly-DADMAC charge, the lignin removal of the laccase treated PHL reached the maximum at 250 ppm Poly-DADMAC. Lignin-PolyDADMAC complexes were formed (Duarte et al., 2010) and precipitated when adding Poly-DADMAC to the PHL, and the effect was more pronounced with the increased charge of Poly-DADMAC until 250 ppm, beyond which the excessive PolyDADMAC has limited effect on the lignin complexes formation due to completion of charge neutralization. Similar results were reported by Saeed et al (2011). In their research on the effect of chitosan dosage on the lignin removal degree from the original PHL, it was found that with the chitosan dosage increasing, the lignin removal reached the maximum at high molecular weight of chitosan at 1 mg/g (Saeed et al., 2011). Duarte et al. (2010) reported that 36% of lignin was removed from dilution sample of sugar maple hydrolyzate using 47 ppm Poly-DADMAC. 11
3.4.2 Dual polymer treatment A dual polymer system has been recognized as an efficient coagulation/ flocculation process for removing lignin in the PHL of the kraft-based dissolving pulp production (Shi et al., 2011). The mechanism is that: 1) firstly lignocelluloses material formed complexes with Poly-DADMAC, which is highly charged, but with a low molecular weight; 2) subsequently, the lignin-Poly-DADMAC complexes can be bridged by CPAM, which has high molecular weight but low charge. As shown in Fig. 4, the lignin removal from the original PHL was much lower than the laccase treated PHL under otherwise the same conditions. For the laccase treated PHL, the lignin removal has negligible effect when increasing the dual polymer charges in the given range. The lignin removal of 58% was achieved at 35 ppm for both Poly-DADMAC and CPAM. Compared with the Poly-DADMAC polymer system, the dual polymer system has higher lignin removal at lower polymer charges. In addition, much more compact sediment was formed after dual polymer system treatment, facilitating the subsequent separation process. Similar results were reported by Yasarla and Ramarao (2012), they employed polymers (PEI, PolyDADMAC, CPAM) to flocculate colloidal material in lignocellulosic hydrolyzates, and found that the particles flocculated with PEI were larger, and had more compact structures than those from Poly-DADMAC. Shi et al. (2012) studied the PAC/PEO dual polymer system to remove lignin from the original PHL, and 37.5% lignin removal was obtained. 3.5 Characterization of treated PHL 12
It is important that the concentrations of hemicellulosic sugars and acetic acid, which are to be further utilized in the downstream processing, would be negligibly affected during the laccase polymerization process. The chemical compositions of the original and the treated PHL are listed in Table 1 and 3, respectively. As can be seen, xylan/ xylose was the major component, which represents about 74% of the total sugars in the original PHL. The laccase treatment of PHL had negligible loss of monosaccharide: the total monosaccharide sugar in the original PHL was 9.87g/L, while it was 9.56g/L and 10.2g/L after laccase treatment at 2 and 3 U/mL-1 laccase concentrations, respectively. On the other hand, there was some loss of monosaccharide sugars after the post- flocculation treatment, and the total monosaccharide sugar concentration was 8.92- 9.47g/L using the poly-DADMAC, and 8.99- 9.18 g/L using the dual polymers of poly- DADMAC/ CPAM. The oligosaccharide sugars changed from 52.32 g/L to 49.19- 51.05 g/L after the laccase treatment, and 44.0-44.2g/L after the post- flocculation using poly- DADMAC, and 43.29-45.29 g/L after the post- flocculation using poly- DADMAC/ CPAM dual polymers. The total sugar loss was in the range of 12-15%. One possible explanation is the oligo-sugar was removed through charge neutralized with cationic polymer, i.e. Poly-DADMAC and CPAM, because oligo-sugar is rich in hydroxyl groups. It was reported that about 7% of xylose loss occurred when applying 300 ppm PEO, to flocculate the original lignin in the PHL (Shi et al., 2011). Additionally, Saeed et al., (2011) reported that 30% of total sugars were lost when using 500 ppm high molecular weight chitosan in their study to remove the lignin present in the original 13
PHL.
4 Conclusions The laccase treatment of prehydrolysis liquor (PHL) of the kraft-based dissolving pulp production process can increase the lignin molecular weight, which then facilitates its removal in the subsequent filtration/flocculation process. The optimum laccase-induced polymerization conditions were: Laccase conc=2 U mL-1, t=3 h, T=36 oC, pH 3.6 (the original pH). The application of flocculation concept in a subsequent step can further enhance the lignin removal. The concentrations of hemicelluloses and acetic acid in the PHL were only marginally affected during the laccase treatment, although the hemicelluloses removal was 12-15% when combining the use of polymers in the subsequent flocculation step.
Acknowledgements This project was funded by an NSERC CRD grant, an Atlantic Innovation Fund, Canada Research Chairs programs of the Government of Canada. The authors are also grateful for the financial support from the National Science Foundation of China (Grant No. 31270627, 31370580) and Natural Science Foundation of Shandong Province (Grant No. ZR2010CM065, ZR2011CM011).
References 1. Areskogh, D., Li, J., Gellerstedt, G., and Henriksson, G., 2010a. Structural 14
modification of commercial lignosulphonates through laccase catalysis and ozonolysis. Ind. Crops Prod. 32, 458-466. 2. Areskogh, D., Li, J., Gellerstedt, G., and Henriksson, G., 2010b. Investigation of the Molecular Weight Increase of Commercial Lignosulfonates by Laccase Catalysis. Biomacromolecules 11, 904-910. 3. Duarte, G.V., Ramarao, B.V., and Amidon, T.E., 2010. Polymer induced flocculation and separation of particulates from extracts of lignocellulosic materials. Bioresour. Technol. 101, 8526-8534. 4. Gouveia, S., Fernández-Costas, C., Sanromán, M.A., and Moldes, D., 2012. Enzymatic polymerisation and effect of fractionation of dissolved lignin from Eucalyptus globulus Kraft liquor. Bioresour. Technol. 121, 131-138. 5. Gouveia, S., Fernández-Costas, C., Sanromán, M.A., and Moldes, D., 2013. Polymerisation of Kraft lignin from black liquors by laccase from Myceliophthora thermophila: Effect of operational conditions and black liquor origin. Bioresour. Technol. 131, 288-294. 6. Hage, R.E., Chrusciel, L., Desharnais, L., and Brosse, N., 2010. Effect of autohydrolysis of Miscanthus x giganteus on lignin structure and organosolv delignification. Bioresour. Technol. 101, 9321-9329. 7. Huo, D., Fang, G. G., Yang, Q., Han, S. M., Deng, Y. J., Shen, K. Z., and Lin, Y., 2013. Enhancement of eucalypt chips’ enzymolysis efficiency by a combination method of alkali impregnation and refining pretreatment. Bioresour. Technol. 150: 73-78. 15
8. Hu, R., Lin, L., Liu, T., and Liu, S., 2010. Dilute sulfuric acid hydrolysis of sugar maple wood extract at atmospheric pressure. Bioresour. Technol. 101: 35863594. 9. Huttermann, A., Mai, C., and Kharazipour, A., 2001. Modification of lignin for the production of new compounded materials. Appl. Microbiol. Biotechnol. 55, 387-394. 10. Ibarra, D., Romero, J., Martínez, M.J., Martínez, A.T., Camarero, S., 2006. Exploring the enzymatic parameters for optimal delignification of eucalypt pulp by laccase-mediator. Enzyme Microb. Technol. 39, 1319-1327. 11. Iiyama, K. and Wallis, A., 1988. An improved acetyl bromide procedure for determining lignin in woods and wood pulps. Wood Sci. Technol. 22: 271-280. 12. Jurado, M., Prieto, A., Martínez-Alcalá, Á., Martínez, Á.T., Martínez, M.J., 2009. Laccase detoxification of steam-exploded wheat straw for second generation bioethanol. Bioresour. Technol. 100, 6378-6384. 13. Kolb, M., Sieber, V., Amann, M., Faulstich, M., Schieder, D., 2012. Removal of monomer delignification products by laccase from Trametes versicolor. Bioresour. Technol. 104, 298-304. 14. Lee, H. J., Lim, W. S., Lee, J. W., 2013. Improvement of ethanol fermentation from lignocellulosic hydrolysates by the removal of inhibitors. J. Ind. Eng. Chem. 19: 2010-2015. 15. Liu, H., Hu, H., Jahan, M.S., and Ni, Y., 2013. Furfural formation from the prehydrolysis liquor of a hardwood kraft-based dissolving pulp production 16
process. Bioresour. Technol. 131, 315-320. 16. Ludwig, D., Amann, M., Hirth, T., Rupp, S., Zibek, S., 2013. Development and optimization of single and combined detoxification processes to improve the fermentability of lignocellulose hydrolyzates. Bioresour. Technol. 133, 455461. 17. Mansfield, S.D., 2002. Laccase impregnation during mechanical pulp processing improved refining efficiency and sheet strength. Appita J. 55, 49-53. 18. Mattinen, M. L., Maijala, P., Nousiainen, P., Smeds, A., Kontro, J., Sipilä, J., Tamminen, T., Willför, S., and Viikari, L., 2011. Oxidation of lignans and lignin model compounds by laccase in aqueous solvent systems. J. Mol. Catal. B: Enzym. 72, 122-129. 19. Salehian, P., Karimi, K., Zilouei, H., and Jeihanipour, A., 2013. Improvement of biogas production from pine wood by alkali pretreatment. Fuel 106, 484-489. 20. Shen, J., Kaur, I., Baktash, M.M., He, Z., and Ni, Y., 2013. A combined process of activated carbon adsorption, ion exchange resin treatment and membrane concentration for recovery of dissolved organics in pre-hydrolysis liquor of the kraft-based dissolving pulp production process. Bioresour. Technol. 127, 5965. 21. Shi, H., Fatehi, P., Xiao, H., and Ni, Y., 2011. A combined acidification/PEO flocculation process to improve the lignin removal from the pre-hydrolysis liquor of kraft-based dissolving pulp production process. Bioresour. Technol. 102, 5177-5182. 17
22. Shi, H., Fatehi, P., Xiao, H., and Ni, Y., 2012. Optimizing the Poly Ethylene Oxide Flocculation Process for Isolating Lignin of Prehydrolysis Liquor of a KraftBased Dissolving Pulp Production Process. Ind. Eng. Chem. Res. 51: 53305335. 23. Saeed, A., Fatehi, P., and Ni, Y., 2011. Chitosan as a flocculant for pre-hydrolysis liquor of kraft-based dissolving pulp production process. Carbohydr. Polym. 86, 1630-1636. 24. Schorr, D., Diouf, P. N., and Stevanovic, T., 2014. Evaluation of industrial lignins for biocomposites production. Ind. Crops Prod. 52: 65-73. 25. Sixta, H., 2006. Hand Book of Pulp, first ed., vol. 1. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. 26. Wei, L., Shrestha, A.. Tu, M., and Adhikari S., 2011. Effects of surfactant on biochemical and hydrothermal conversion of softwood hemicellulose to ethanol and furan derivatives. Process Biochem. 46: 1785-1792. 27. Yang, G., Jahan, M.S., and Ni, Y., 2013a. Structural Characterization of Prehydrolysis Liquor Lignin and Its Comparison with Other Technical Lignins. Curr. Org. Chem. 17: 1589-1595. 28. Yang, G., Sarwar Jahan, M., Ahsan, L., Zheng, L., and Ni, Y., 2013b. Recovery of acetic acid from pre-hydrolysis liquor of hardwood kraft-based dissolving pulp production process by reactive extraction with triisooctylamine. Bioresour. Technol. 138, 253-258. 29. Yasarla, L.R., Ramarao, B.V., 2012. Dynamics of Flocculation of Lignocellulosic 18
Hydrolyzates by Polymers. Ind. Eng. Chem. Res. 51, 6847-6861. 30. Yu, Y., Zeng, Y., Zuo, J., Ma, F., Yang, X., Zhang, X., and Wang, Y., 2013. Improving the conversion of biomass in catalytic fast pyrolysis via white- rot fungal pretreatment. Bioresour. Technol. 134, 198-203. 31. Zhang, D. S., Yang, Q., Zhu, J. Y., and Pan, X. J., 2013. Sulfite (SPORL) pretreatment of switchgrass for enzymatic saccharification. Bioresour. Technol. 129: 127-134.
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Fig. 1 Process diagram for lignin removal by laccase-induced polymerization in a kraft- based dissolving pulp production process
20
Fig. 2 Lignin removals from the PHL by laccase-induced polymerization and filtration (A) effect of pH, (B) effect of temperature, (C) effect of time, (D) effect of laccase concentration
21
Fig. 3 Lignin removal from PHL (with/without laccase treatment) as a function of Poly-DADMAC charge (Laccase treatment conditions: : Laccase concentration 2 U mL-1, time 3 h, temperature 36 oC, pH of the original PHL (3.63))
22
Fig. 4 Lignin removal from PHL (with/without laccase treatment) in the dual-polymer system of Poly-DADMAC/CPAM (Laccase treatment conditions: Laccase concentration 2 U mL-1, time 3 h, temperature 36 oC, pH of the original PHL (3.63))
23
Table 1 Composition of the original PHL Total Rhammose
Arabinose
Galactose
Glucose
Xylose
Mannose
HAcb
Lignin
11.3
12.2
sugars Mono-
0.70
1.58
0.85
0.95
5.36
0.43
Oligo-
0.63
NDa
1.62
4.45
40.7
4.92
61.9
Note: NDa - Not detected. HAcb - Acetic acid.
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Table 2 Molecular weight of lignin before and after laccase polymerization Original PHL
Laccase treated
Mw/Mn
1.72
1.99
Mn (Da)
564
802
Mw (Da)
972
1593
Note: laccase treated condition: laccase concentration 2 UmL-1, temperature 36 o C, time 3 h, pH of the original PHL (3.63).
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Table 3 Effect of laccase polymerization followed by either filtration, or polyDADMAC or dual polymer treatment on the concentrations of hemicelluloses, and acetic acid (g L-1) Poly-
Dual polymer
DADMAC
Poly-DADMAC/CPAM
Laccase L2
L3
250
300
35/35
45/45
Mono-
0.60
0.70
0.61
0.61
0.61
0.61
Oligo-
0.63
0.58
0.55
0.51
0.40
0.51
Mono-
1.46
1.54
1.37
1.39
1.39
1.38
Oligo-
ND
a
ND
ND
ND
ND
ND
Mono-
0.90
1.02
0.84
0.85
0.86
0.83
Oligo-
1.29
1.30
1.24
1.39
1.32
1.51
Mono-
1.06
1.14
0.87
1.03
1.00
0.99
Oligo-
4.04
4.30
3.69
3.80
3.57
3.50
Mono-
5.14
5.36
4.84
5.20
4.87
4.74
Oligo-
39.0
39.77
34.6
33.9
34.3
35.3
Rhammose
Arabinose
Galactose
Glucose
Xylose
Mannose
26
Mono-
0.40
0.44
0.39
0.39
0.45
0.44
Oligo-
4.23
5.10
4.12
4.40
3.70
4.47
Total sugars
58.8
61.3
53.1
53.5
52.5
54.3
HAcb
10.6
11.1
6.45
6.47
7.07
6.78
Note: L2, L3, represent laccase concentrations of 2 and 3 U mL-1, respectively, other conditions such as time 3h, temperature 36 oC, pH of the original PHL (3.63).
27
1.
A new process enhances lignin removal from the prehydrolysis liquor was proposed.
2.
Laccase treatment was incorporated into the new process.
3.
Laccase treatment can polymerize low Mw lignin.
4.
The concentration of hemicelluloses and acetic acid were largely unaffected.
28
