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Enzymatic treatment of hemicelluloses and lignin isolated from thermomechanical pulp mill process water
Chemical Engineering Journal 296 (2016) 141–145
Contents lists available at ScienceDirect
Chemical Engineering Journal journal homepage: www.elsevier.com/locate/cej
Enzymatic treatment of hemicelluloses and lignin isolated from thermomechanical pulp mill process water Johan Thuvander a, Petri Oinonen b, Ann-Sofi Jönsson a,⇑ a b
Department of Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden Ecohelix AB, Hägerstensvägen 137, SE-126 48 Hägersten, Sweden
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Mw of thermomechanical pulp mill
process water polymers increased by laccase treatment. Molecular mass of lignin increased from 1 kDa to about 60 kDa. Modest increase of molecular mass of hemicelluloses. High molecular mass polymers purified using diafiltration. Recovery of low molecular mass polymers using nanofiltration.
a r t i c l e
i n f o
Article history: Received 12 January 2016 Received in revised form 4 March 2016 Accepted 19 March 2016 Available online 1 April 2016 Keywords: Galactoglucomannan Thermomechanical pulp Ultrafiltration Nanofiltration Enzymatic treatment
a b s t r a c t Hemicelluloses dissolved in thermomechanical pulp (TMP) mill process streams can be used to manufacture high-value-added products. The molecular mass of the dissolved hemicelluloses is about 10 kDa. In some applications, it would be beneficial with larger molecules. It was therefore investigated whether it is possible to increase the size of hemicelluloses isolated by microfiltration and ultrafiltration from TMP process water using enzymatic treatment with laccase. No significant increase of the size of hemicelluloses was achieved, probably due to only a small percentage of lignin carbohydrate complexes in the process water. The molecular mass of lignin increased however markedly from a peak molecular mass of 1 kDa to about 60 kDa. Diafiltration was used to purify large molecules after the enzymatic treatment. Low-molecular-mass sugar and lignin molecules in the diafiltration permeate were concentrated by nanofiltration. The retention of sugars and lignin was about 98% during nanofiltration to a volume reduction of 80%. Average flux during nanofiltration was 49 l/m2 h. Ó 2016 Elsevier B.V. All rights reserved.
1. Introduction Galactoglucomannan recovered from process streams in thermomechanical pulp (TMP) mills can be used in several highvalue added products, as for example in barrier coatings and food emulsions [1–3]. The molecular size distribution of hemicelluloses is rather broad and differs in various pulp process streams ⇑ Corresponding author. Tel.: +46 46 2228291. E-mail address: [email protected] (A.-S. Jönsson). http://dx.doi.org/10.1016/j.cej.2016.03.087 1385-8947/Ó 2016 Elsevier B.V. All rights reserved.
depending on both wood type [4,5] and pulping process [4,6]. The reported mean size of hemicelluloses recovered from TMP mill process water differs also due to both the cut-off of the ultrafiltration (UF) membrane used to concentrate the hemicelluloses, and the size exclusion chromatography (SEC) system and standards used for calibration when hemicelluloses are analysed. We recently found the peak molecular mass of hemicelluloses recovered from process water in a Swedish TMP mill using a 5 kDa UF membrane to be about 10 kDa [7]. The hemicelluloses were concentrated and purified using a number of stages in a
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J. Thuvander et al. / Chemical Engineering Journal 296 (2016) 141–145
cascade configuration: pre-filtration, microfiltration (MF) of the filtrate and UF of the MF permeate, which is the same method as that used by Persson et al. [8] in an earlier investigation. In some product applications a high molecular mass of the hemicelluloses are desired. Films made of hemicelluloses of higher molecular mass have better mechanical properties [9] and better grease and water vapor properties [2]. One method of increasing the molecular weight of hemicelluloses is by enzymatic treatment with laccase [10]. This method uses covalently bound lignin moieties on the hemicellulose polymers in lignin-carbohydrate complexes (LCC) [11] and link polymers together by the laccase mediated oxidation and coupling of the lignin moieties [12]. Laccase-catalyzed linking of aromatic moieties bound to hemicelluloses has been used to increase the molecular size of hemicelluloses recovered from TMP process water from 15 kDa to 17 kDa [10] and from chemithermomechanical pulp (CTMP) process water from 4 kDa to 12 kDa [6]. The objective of this study was to investigate whether it is possible to use laccase-catalyzed treatment to increase the size of hemicellulose molecules recovered from TMP mill process water in the UF retentate from our previous investigation [7]. The same methodology as that used by Krawczyk et al. [6] was used for enzymatic treatment. Large molecules were purified by diafiltration (DF) using a UF membrane with a nominal cut-off of 10 kDa. The UF permeate, containing small molecules from the DF operation, was concentrated by nanofiltration (NF). 2. Materials and methods 2.1. Process water Process water from a Swedish thermomechanical pulp mill with spruce as primary raw material was used in the investigation. Suspended solids and colloidal extractives were removed by MF with a ceramic 0.1 lm membrane from LiqTech International A/S, Denmark. The MF permeate was concentrated to a volume reduction (VR) of 98% using a UF membrane with a nominal molecular mass cut-off of 5 kDa (UFX5pHt, Alfa Laval Nakskov A/S, Denmark). The performance of the UF membrane during concentration of the MF permeate has been presented previously [7]. 2.2. Experimental procedure 2.2.1. Enzymatic treatment Laccase-catalysed treatment of aromatic moieties was used to increase the molecular mass of hemicelluloses in the UF retentate from the 5 kDa membrane filtration process. The enzymatic treatment was carried out in a glass reactor equipped with an agitator (ReactoMate 30000 CLR, Asynt Ltd., Ely, UK). The solution was heated and maintained at a temperature of 40 °C by circulating temperature-adjusted water around the glass reactor. The reaction was started by adding 20 U/g substrate (1.4 mg/g) of the laccase enzyme (Sigma 51639, Sigma–Aldrich Chemie GmbH, Steinheim, Germany) to the solution. Pure oxygen gas was bubbled through the solution continuously during the total reaction time of 3 h. 2.2.2. Diafiltration The enzymatically treated solution was diluted to four times its initial volume with deionized water. The solution was then concentrated to a VR of 80% using a 2517 spiral-wound UF membrane with a nominal molecular mass cut-off of 10 kDa (UFX10 pHt, Alfa Laval Nakskov A/S, Denmark). The membrane was equipped with a 48-mil spacer (1.2 mm). The temperature was 60 °C, the transmembrane pressure was 2 bar and the cross-flow during both concentration and DF was 1.2 m3/h. The cross-flow was chosen such
that the maximum frictional pressure drop in the spiral-wound element would not exceed 0.6 bar, which is the maximum frictional pressure drop recommended by the membrane manufacturer. 2.2.3. Concentration by nanofiltration The UF permeate was concentrated by NF to a VR of 80% using a 2517 spiral-wound NF membrane with a MgSO4 rejection of P98% (NF99HF, Alfa Laval Nakskov A/S, Denmark). The temperature used was 50 °C, the transmembrane pressure 10 bar and the cross-flow 0.6 m3/h. The cross-flow was chosen so that the maximum frictional pressure drop in the spiral-wound element would not exceed 0.6 bar. 2.3. Analytical methods The total solids content was determined from the weight of the residue after drying samples for 24 h in an oven at 105 °C and cooling to room temperature in a desiccator. The dry sample was then further heated to 575 °C, and this temperature was maintained for 3 h. The ash content was calculated from the weight of the residue after cooling to room temperature in a desiccator. The total lignin content was determined by measuring the light absorption at a wavelength of 280 nm, using a UV-160 spectrophotometer (Shimadzu, Japan) and an absorption coefficient of 17.8 L/(g cm) [13]. The concentration of hemicelluloses was determined by first hydrolysing the polysaccharide to monomeric sugars. The monomeric sugars were then measured using high-performance anion-exchange chromatography coupled with pulsed amperometric detection in an ICS-3000 chromatography system (Dionex Corp., USA) [7]. The molecular mass distribution of hemicelluloses and lignin was determined by SEC using a Waters 600E chromatography system (Waters, USA) equipped with a refractive index (RI) detector (model 2414, Waters) and a UV detector (model 486, Waters). The analytical column was packed with 30 cm Superdex 30 and 30 cm Superdex 200 (GE Healthcare, Sweden). The injection volume was 500 lL. A 125 mM NaOH solution was used as eluent at a flow rate of 1 mL/min. The system was calibrated with polyethylene glycol standards with peak molecular masses of 0.4, 4, 10 and 35 kg/mol (Merck Schuchardt OHG, Germany). Samples were filtered through a 0.2 lm filter (Schleicher & Schuell, Germany) before determination of molecular mass distribution. It should be noted that the SEC column was not calibrated for substances with molecular masses greater than 35 kDa PEG equivalents, and thus sizes above this value are uncertain. 3. Results andiscussion 3.1. Enzymatic treatment Only a slight increase in the size of the hemicelluloses in the TMP process water was seen after the enzymatic treatment, as shown in Fig. 1a. Even though a slightly higher amount of larger molecules were observed in the chromatogram, the peak molecular mass of hemicelluloses was still about 20 kDa after the enzymatic treatment. Lignin on the other hand showed a marked increase of peak molecular mass. The enzymatic treatment increased the peak molecular mass of lignin from 1 kDa to about 60 kDa, as shown in Fig. 1b. These results are in contrast to previous results where the size of hemicelluloses from TMP process water could be increased from 15 kDa to 17 kDa [10] and CTMP process water from 4 kDa to 12 kDa by enzymatic treatment [6]. The difference in results between the present study and the previous studies with TMP mill process water [10] and CTMP mill
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50
100
40
80 2
Flux (l/m h)
Refractive index (mV)
a
60 40
20 10
20 0 0.1
30
0
1
10
100
0
1,000
20
40
Molecular mass (kDa)
80
100
Fig. 2. Flux during diafiltration of the enzymatically treated solution with a 10 kDa membrane. The temperature was 60 °C, the transmembrane pressure 2 bar and the cross-flow 1.2 m3/h.
0.05 0.04 0.03
80
0.02
Concentration (g/l)
UV (Absorbance units)
b
60
Volume reduction (%)
0.01 0.00 0.1
1
10
100
1,000
Molecular mass (kDa) Fig. 1. Molecular mass distribution of (a) hemicelluloses and (b) lignin (measured as UV absorbance at 280 nm) before (—) and after ( ) enzymatic treatment.
Hemicelluloses Lignin
60
40
20
0 0
process water [6] might be surprising. However, there are some major differences in the characteristics of the solutions used in the present study and the previous studies. First, the lignin content of the solution used in the present study was low, with a lignin: hemicellulose ratio of 0.07. In comparison, the lignin:hemicellulose ratio in the previous studies was 0.33 in the study with TMP process water and 0.53 in the study with CTMP process water. Secondly, in the present study the majority of the lignin molecules were found in a considerably lower molecular mass fraction than the hemicelluloses, showing that most of the lignin present in the process water was not covalently bound to hemicelluloses as LCC complexes. In contrast, all lignin in the previous studies [6,10] was found in molecular mass fractions also containing hemicelluloses, indicating that the amount of LCC was higher in these solutions. It is interesting to note the difference between the present and the previous study with TMP mill process water [10]. This shows that the content and characteristics of dissolved components in the process water may be different, even when the same type of raw material and pulping process have been used. Variations in the operation of the pulping process and position of withdrawal of the process water are likely explanations for these differences.
20
40
60
80
100
Volume reduction (%) Fig. 3. Concentration of hemicelluloses and lignin during diafiltration of the enzymatically treated solution.
3.2. Purification of hemicelluloses by diafiltration The composition of the process solution before and after the enzymatic treatment was similar, as can be seen in Table 1. The enzymatically treated solution was treated by DF using a UF membrane with a nominal cut-off of 10 kDa, in order to purify hemicelluloses and separate large and small hemicelluloses. The solution was diluted to four times its initial volume with deionized water and then concentrated to a VR of 80% (corresponding to a volume reduction factor of 5). The flux started at 42 l/m2 h, and decreased to 9 l/m2 h as the VR increased to a VR of 80%, as shown in Fig. 2. The concentration of hemicelluloses increased from 13 g/l before DF to 61 g/l in the retentate at a VR of 80%, and the concentration of lignin increased from 1.2 g/l to 5.8 g/l, as shown in Fig. 3. Hemicelluloses represented 64% of total solids both before and after the enzymatic treatment (see Table 1). Hemicelluloses in the retentate after DF also represent 64% of total solids. The enzymatic
Table 1 Characteristics of the process solution before and after enzymatic treatment, and the retentate and the permeate after DF to a VR of 80%. All measurements were made in triplicate.
Total solids (g/l) Ash (g/l) Lignin (g/l) Hemicellulose (g/l) Arabinan (g/l) Galactan (g/l) Glucan (g/l) Xylan (g/l) Mannan (g/l)
Before enzymatic treatment
After enzymatic treatment
Retentate after DF
Permeate after DF
77.3 ± 0.1 2.1 ± 0.1 3.5 ± 0.2 50.1 ± 1.1 1.2 ± 0.1 6.5 ± 0.1 9.1 ± 0.1 0.7 ± 0.4 32.6 ± 1.1
79.7 ± 0.3 2.1 ± 0.6 4.4 ± 0.2 50.6 ± 1.5 1.3 ± 0.0 6.5 ± 0.1 9.2 ± 0.1 0.8 ± 0.3 32.8 ± 1.8
94.8 ± 0.0 1.0 ± 0.2 5.7 ± 0.2 60.9 ± 3.2 1.5 ± 0.0 8.6 ± 0.1 10.7 ± 0.0 1.0 ± 0.4 39.1 ± 3.5
4.5 ± 0.0 0.5 ± 0.0 0.2 ± 0.0 2.4 ± 0.1 0.1 ± 0.0 0.2 ± 0.0 0.6 ± 0.0 0.0 ± 0.0 1.5 ± 0.1
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J. Thuvander et al. / Chemical Engineering Journal 296 (2016) 141–145
60
2
Flux (l/m h)
80
40
20
0 0
20
40
60
80
100
Volume reduction (%) Fig. 4. Flux during concentration of UF permeate by nanofiltration. The temperature was 50 °C, the transmembrane pressure 10 bar and the cross-flow 0.6 m3/h.
UV (Absorbance units)
a
0.08
0.06
0.04
0.02
0.00 0.1
1.0
10.0
100.0
Molecular mass (kDa)
Refractive index (mV)
b
4. Conclusions
120 100 80 60 40 20 0 0.1
1.0
10.0
shown in Fig 4. The average flux during concentration was 49 l/m2 h. Lignin showed a peak in the SEC chromatogram at 1 kDa. This peak was considerably higher after NF, as can be seen in Fig. 5a. Smaller sugar molecules, <1 kDa, were detected in the NF retentate, whereas the concentration was not sufficiently high to allow detection of these molecules in the DF permeate (Fig. 5b). There is also a hemicellulose peak at 10 kDa, the same molecular mass as in the DF retentate. This is puzzling as the nominal cut-off of the UF membrane used during DF was 10 kDa and molecules of this size should have been retained during DF. The retention of the UFX10 pHt membrane was therefore investigated by UF of a mixture of PEG standards used to calibrate the SEC system. The retention of PEG10 and PEG35 (with peak molecular masses of 10 kDa and 35 kDa) was 81% and 90%, respectively, at a transmembrane pressure of 0.5 bar, and 50% and 53% at 2 bar, revealing that the cut-off of the UFX10 pHt membrane is higher than the nominal cut-off when treating straight polymers, and that the transmembrane pressure has a considerable effect on the retention due to enhanced concentration polarization at higher pressures. The retention of hemicelluloses and lignin was about 98%. The concentration of hemicelluloses increased from 3 g/l to 15 g/l, and the concentration of lignin from 0.3 g/l to 1.3 g/l, in the retentate at VR 83%. The high retention of the NF membrane resulted in a permeate with low concentration of total solids (0.25 g/l), hemicelluloses (0.10 g/l), lignin (0.02 g/l) and ash (0.06 g/l). This permeate can be reused in the pulp mill replacing fresh water.
100.0
Enzymatic treatment of TMP process water with laccase did not have the same effect on the molecular mass of hemicelluloses as in the previous studies, where the size of hemicellulose molecules in process water from a TMP mill [10] and a CTMP mill [6] was increased using the same method. The explanation of the lack of effect in the present study could be that the lignin molecules in the TMP process water were linked to hemicelluloses to a lesser extent. It was also observed that the lignin concentration in the TMP process water is much lower than in the previous. The lowmolecular-mass hemicelluloses in the UF permeate could be recovered and concentrated by NF.
Molecular mass (kDa) Fig. 5. Molecular mass distribution of (a) lignin and (b) hemicellulose in the UF permeate (—) and final NF retentate ( ).
Acknowledgment The Swedish Energy Agency is gratefully acknowledged for financial support.
treatment increased the size of lignin (see Fig. 1b), making the retention of hemicelluloses and lignin almost identical during DF. Hence, no purification of hemicelluloses was achieved by DF of the solution. The initial retention of hemicelluloses and lignin was 85% and 87%, respectively, and 90% for both at the end of DF. This is in sharp contrast to the markedly higher retention of hemicelluloses (97%) than lignin (55%) when the TMP process water was concentrated with a 5 kDa membrane before enzymatic treatment [7]. This confirms that the size of the lignin molecules was increased during enzymatic treatment, while the size of the hemicelluloses was largely unaffected.
3.3. Nanofiltration of ultrafiltration permeate The UF permeate from the DF operation, containing small hemicellulose and lignin molecules, was concentrated by NF. The initial flux was 55 l/m2 h and decreased to 37 l/m2 h at a VR of 83%, as
References [1] S. Willför, K. Sundberg, M. Tenkanen, B. Holmbom, Spruce-derived mannans – a potential raw material for hydrocolloids and novel advanced natural materials, Carbohydr. Polym. 72 (2008) 197–210, http://dx.doi.org/10.1016/ j.carbpol.2007.08.006. [2] V. Kisonen, C. Xu, R. Bollström, J. Hartman, H. Rautkoski, M. Nurmi, J. Hemming, P. Eklund, S. Willför, O-acetyl galactoglucomannan esters for barrier coatings, Cellulose 21 (2014) 4497–4509, http://dx.doi.org/10.1007/s10570-0140428-7. [3] K.S. Mikkonen, C. Xu, C. Berton-Carabin, K. Schroën, Spruce galactoglucomannans in rapeseed oil-in-water emulsions: efficient stabilization performance and structural partitioning, Food Hydrocoll. 52 (2016) 615–624, http://dx.doi.org/10.1016/j.foodhyd.2015.08.009. [4] T. Persson, A.-S. Jönsson, Isolation of hemicelluloses by ultrafiltration of thermomechanical pulp mill process water – influence of operating conditions, Chem. Eng. Res. Des. 88 (2010) 1548–1554, http://dx.doi.org/10.1016/j. cherd.2010.04.002. [5] A. Teleman, M. Nordström, M. Tenkanen, A. Jacobs, O. Dahlman, Isolation and characterization of O-acetylated glucomannans from aspen and birch wood, Carbohydr. Res. 6 (2003) 525–534, http://dx.doi.org/10.1016/S0008-6215(02) 00491-3.
J. Thuvander et al. / Chemical Engineering Journal 296 (2016) 141–145 [6] H. Krawczyk, P. Oinonen, A.-S. Jönsson, Combined ultrafiltration and enzymatic treatment for recovery of high-molecular mass hemicelluloses from chemithermomechanical pulp process water, Chem. Eng. J. 225 (2013) 292– 299, http://dx.doi.org/10.1016/j.cej.2013.03.089. [7] J. Thuvander, A.-S. Jönsson, Extraction of galactoglucomannan from thermomechanical pulp mill process water by microfiltration and ultrafiltration – influence of microfiltration membrane pore size on ultrafiltration performance, Chem. Eng. Res. Des. 105 (2016) 171–176, http:// dx.doi.org/10.1016/j.cherd.2015.12.003. [8] T. Persson, H. Krawczyk, A.-K. Nordin, A.-S. Jönsson, Fractionation of process water in thermomechanical pulp mills, Bioresour. Technol. 101 (2010) 3884– 3892, http://dx.doi.org/10.1016/j.biortech.2009.12.142. [9] A.I. Yaich, U. Edlund, A.-C. Albertsson, Wood hydrolysate barriers: performance controlled via selective recovery, Biomacromolecules 13 (2012) 466–473, http://dx.doi.org/10.1021/bm201518d.
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[10] P. Oinonen, D. Areskogh, G. Henriksson, Enzyme catalyzed cross-linking of spruce galactoglucomannan improves its applicability in barrier films, Carbohydr. Polym. 95 (2013) 690–696, http://dx.doi.org/10.1016/ j.carbpol.2013.03.016. [11] M. Lawoko, G. Henriksson, G. Gellerstedt, Structural differences between the lignin-carbohydrate complexes present in wood and in chemical pulps, Biomacromolecules 6 (2005) 3467–3473, http://dx.doi.org/10.1021/ bm058014q. [12] P. Oinonen, L. Zhang, M. Lawoko, G. Henriksson, On the formation of lignin polysaccharide networks in Norway spruce, Phytochemistry 111 (2015) 177– 184, http://dx.doi.org/10.1016/j.phytochem.2014.10.027. [13] F. Örså, B. Holmbom, J. Thornton, Dissolution and dispersion of spruce wood components into hot water, Wood Sci. Technol. 31 (1997) 279–290, http://dx. doi.org/10.1007/BF00702615.
Contents lists available at ScienceDirect
Chemical Engineering Journal journal homepage: www.elsevier.com/locate/cej
Enzymatic treatment of hemicelluloses and lignin isolated from thermomechanical pulp mill process water Johan Thuvander a, Petri Oinonen b, Ann-Sofi Jönsson a,⇑ a b
Department of Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden Ecohelix AB, Hägerstensvägen 137, SE-126 48 Hägersten, Sweden
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Mw of thermomechanical pulp mill
process water polymers increased by laccase treatment. Molecular mass of lignin increased from 1 kDa to about 60 kDa. Modest increase of molecular mass of hemicelluloses. High molecular mass polymers purified using diafiltration. Recovery of low molecular mass polymers using nanofiltration.
a r t i c l e
i n f o
Article history: Received 12 January 2016 Received in revised form 4 March 2016 Accepted 19 March 2016 Available online 1 April 2016 Keywords: Galactoglucomannan Thermomechanical pulp Ultrafiltration Nanofiltration Enzymatic treatment
a b s t r a c t Hemicelluloses dissolved in thermomechanical pulp (TMP) mill process streams can be used to manufacture high-value-added products. The molecular mass of the dissolved hemicelluloses is about 10 kDa. In some applications, it would be beneficial with larger molecules. It was therefore investigated whether it is possible to increase the size of hemicelluloses isolated by microfiltration and ultrafiltration from TMP process water using enzymatic treatment with laccase. No significant increase of the size of hemicelluloses was achieved, probably due to only a small percentage of lignin carbohydrate complexes in the process water. The molecular mass of lignin increased however markedly from a peak molecular mass of 1 kDa to about 60 kDa. Diafiltration was used to purify large molecules after the enzymatic treatment. Low-molecular-mass sugar and lignin molecules in the diafiltration permeate were concentrated by nanofiltration. The retention of sugars and lignin was about 98% during nanofiltration to a volume reduction of 80%. Average flux during nanofiltration was 49 l/m2 h. Ó 2016 Elsevier B.V. All rights reserved.
1. Introduction Galactoglucomannan recovered from process streams in thermomechanical pulp (TMP) mills can be used in several highvalue added products, as for example in barrier coatings and food emulsions [1–3]. The molecular size distribution of hemicelluloses is rather broad and differs in various pulp process streams ⇑ Corresponding author. Tel.: +46 46 2228291. E-mail address: [email protected] (A.-S. Jönsson). http://dx.doi.org/10.1016/j.cej.2016.03.087 1385-8947/Ó 2016 Elsevier B.V. All rights reserved.
depending on both wood type [4,5] and pulping process [4,6]. The reported mean size of hemicelluloses recovered from TMP mill process water differs also due to both the cut-off of the ultrafiltration (UF) membrane used to concentrate the hemicelluloses, and the size exclusion chromatography (SEC) system and standards used for calibration when hemicelluloses are analysed. We recently found the peak molecular mass of hemicelluloses recovered from process water in a Swedish TMP mill using a 5 kDa UF membrane to be about 10 kDa [7]. The hemicelluloses were concentrated and purified using a number of stages in a
142
J. Thuvander et al. / Chemical Engineering Journal 296 (2016) 141–145
cascade configuration: pre-filtration, microfiltration (MF) of the filtrate and UF of the MF permeate, which is the same method as that used by Persson et al. [8] in an earlier investigation. In some product applications a high molecular mass of the hemicelluloses are desired. Films made of hemicelluloses of higher molecular mass have better mechanical properties [9] and better grease and water vapor properties [2]. One method of increasing the molecular weight of hemicelluloses is by enzymatic treatment with laccase [10]. This method uses covalently bound lignin moieties on the hemicellulose polymers in lignin-carbohydrate complexes (LCC) [11] and link polymers together by the laccase mediated oxidation and coupling of the lignin moieties [12]. Laccase-catalyzed linking of aromatic moieties bound to hemicelluloses has been used to increase the molecular size of hemicelluloses recovered from TMP process water from 15 kDa to 17 kDa [10] and from chemithermomechanical pulp (CTMP) process water from 4 kDa to 12 kDa [6]. The objective of this study was to investigate whether it is possible to use laccase-catalyzed treatment to increase the size of hemicellulose molecules recovered from TMP mill process water in the UF retentate from our previous investigation [7]. The same methodology as that used by Krawczyk et al. [6] was used for enzymatic treatment. Large molecules were purified by diafiltration (DF) using a UF membrane with a nominal cut-off of 10 kDa. The UF permeate, containing small molecules from the DF operation, was concentrated by nanofiltration (NF). 2. Materials and methods 2.1. Process water Process water from a Swedish thermomechanical pulp mill with spruce as primary raw material was used in the investigation. Suspended solids and colloidal extractives were removed by MF with a ceramic 0.1 lm membrane from LiqTech International A/S, Denmark. The MF permeate was concentrated to a volume reduction (VR) of 98% using a UF membrane with a nominal molecular mass cut-off of 5 kDa (UFX5pHt, Alfa Laval Nakskov A/S, Denmark). The performance of the UF membrane during concentration of the MF permeate has been presented previously [7]. 2.2. Experimental procedure 2.2.1. Enzymatic treatment Laccase-catalysed treatment of aromatic moieties was used to increase the molecular mass of hemicelluloses in the UF retentate from the 5 kDa membrane filtration process. The enzymatic treatment was carried out in a glass reactor equipped with an agitator (ReactoMate 30000 CLR, Asynt Ltd., Ely, UK). The solution was heated and maintained at a temperature of 40 °C by circulating temperature-adjusted water around the glass reactor. The reaction was started by adding 20 U/g substrate (1.4 mg/g) of the laccase enzyme (Sigma 51639, Sigma–Aldrich Chemie GmbH, Steinheim, Germany) to the solution. Pure oxygen gas was bubbled through the solution continuously during the total reaction time of 3 h. 2.2.2. Diafiltration The enzymatically treated solution was diluted to four times its initial volume with deionized water. The solution was then concentrated to a VR of 80% using a 2517 spiral-wound UF membrane with a nominal molecular mass cut-off of 10 kDa (UFX10 pHt, Alfa Laval Nakskov A/S, Denmark). The membrane was equipped with a 48-mil spacer (1.2 mm). The temperature was 60 °C, the transmembrane pressure was 2 bar and the cross-flow during both concentration and DF was 1.2 m3/h. The cross-flow was chosen such
that the maximum frictional pressure drop in the spiral-wound element would not exceed 0.6 bar, which is the maximum frictional pressure drop recommended by the membrane manufacturer. 2.2.3. Concentration by nanofiltration The UF permeate was concentrated by NF to a VR of 80% using a 2517 spiral-wound NF membrane with a MgSO4 rejection of P98% (NF99HF, Alfa Laval Nakskov A/S, Denmark). The temperature used was 50 °C, the transmembrane pressure 10 bar and the cross-flow 0.6 m3/h. The cross-flow was chosen so that the maximum frictional pressure drop in the spiral-wound element would not exceed 0.6 bar. 2.3. Analytical methods The total solids content was determined from the weight of the residue after drying samples for 24 h in an oven at 105 °C and cooling to room temperature in a desiccator. The dry sample was then further heated to 575 °C, and this temperature was maintained for 3 h. The ash content was calculated from the weight of the residue after cooling to room temperature in a desiccator. The total lignin content was determined by measuring the light absorption at a wavelength of 280 nm, using a UV-160 spectrophotometer (Shimadzu, Japan) and an absorption coefficient of 17.8 L/(g cm) [13]. The concentration of hemicelluloses was determined by first hydrolysing the polysaccharide to monomeric sugars. The monomeric sugars were then measured using high-performance anion-exchange chromatography coupled with pulsed amperometric detection in an ICS-3000 chromatography system (Dionex Corp., USA) [7]. The molecular mass distribution of hemicelluloses and lignin was determined by SEC using a Waters 600E chromatography system (Waters, USA) equipped with a refractive index (RI) detector (model 2414, Waters) and a UV detector (model 486, Waters). The analytical column was packed with 30 cm Superdex 30 and 30 cm Superdex 200 (GE Healthcare, Sweden). The injection volume was 500 lL. A 125 mM NaOH solution was used as eluent at a flow rate of 1 mL/min. The system was calibrated with polyethylene glycol standards with peak molecular masses of 0.4, 4, 10 and 35 kg/mol (Merck Schuchardt OHG, Germany). Samples were filtered through a 0.2 lm filter (Schleicher & Schuell, Germany) before determination of molecular mass distribution. It should be noted that the SEC column was not calibrated for substances with molecular masses greater than 35 kDa PEG equivalents, and thus sizes above this value are uncertain. 3. Results andiscussion 3.1. Enzymatic treatment Only a slight increase in the size of the hemicelluloses in the TMP process water was seen after the enzymatic treatment, as shown in Fig. 1a. Even though a slightly higher amount of larger molecules were observed in the chromatogram, the peak molecular mass of hemicelluloses was still about 20 kDa after the enzymatic treatment. Lignin on the other hand showed a marked increase of peak molecular mass. The enzymatic treatment increased the peak molecular mass of lignin from 1 kDa to about 60 kDa, as shown in Fig. 1b. These results are in contrast to previous results where the size of hemicelluloses from TMP process water could be increased from 15 kDa to 17 kDa [10] and CTMP process water from 4 kDa to 12 kDa by enzymatic treatment [6]. The difference in results between the present study and the previous studies with TMP mill process water [10] and CTMP mill
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50
100
40
80 2
Flux (l/m h)
Refractive index (mV)
a
60 40
20 10
20 0 0.1
30
0
1
10
100
0
1,000
20
40
Molecular mass (kDa)
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Fig. 2. Flux during diafiltration of the enzymatically treated solution with a 10 kDa membrane. The temperature was 60 °C, the transmembrane pressure 2 bar and the cross-flow 1.2 m3/h.
0.05 0.04 0.03
80
0.02
Concentration (g/l)
UV (Absorbance units)
b
60
Volume reduction (%)
0.01 0.00 0.1
1
10
100
1,000
Molecular mass (kDa) Fig. 1. Molecular mass distribution of (a) hemicelluloses and (b) lignin (measured as UV absorbance at 280 nm) before (—) and after ( ) enzymatic treatment.
Hemicelluloses Lignin
60
40
20
0 0
process water [6] might be surprising. However, there are some major differences in the characteristics of the solutions used in the present study and the previous studies. First, the lignin content of the solution used in the present study was low, with a lignin: hemicellulose ratio of 0.07. In comparison, the lignin:hemicellulose ratio in the previous studies was 0.33 in the study with TMP process water and 0.53 in the study with CTMP process water. Secondly, in the present study the majority of the lignin molecules were found in a considerably lower molecular mass fraction than the hemicelluloses, showing that most of the lignin present in the process water was not covalently bound to hemicelluloses as LCC complexes. In contrast, all lignin in the previous studies [6,10] was found in molecular mass fractions also containing hemicelluloses, indicating that the amount of LCC was higher in these solutions. It is interesting to note the difference between the present and the previous study with TMP mill process water [10]. This shows that the content and characteristics of dissolved components in the process water may be different, even when the same type of raw material and pulping process have been used. Variations in the operation of the pulping process and position of withdrawal of the process water are likely explanations for these differences.
20
40
60
80
100
Volume reduction (%) Fig. 3. Concentration of hemicelluloses and lignin during diafiltration of the enzymatically treated solution.
3.2. Purification of hemicelluloses by diafiltration The composition of the process solution before and after the enzymatic treatment was similar, as can be seen in Table 1. The enzymatically treated solution was treated by DF using a UF membrane with a nominal cut-off of 10 kDa, in order to purify hemicelluloses and separate large and small hemicelluloses. The solution was diluted to four times its initial volume with deionized water and then concentrated to a VR of 80% (corresponding to a volume reduction factor of 5). The flux started at 42 l/m2 h, and decreased to 9 l/m2 h as the VR increased to a VR of 80%, as shown in Fig. 2. The concentration of hemicelluloses increased from 13 g/l before DF to 61 g/l in the retentate at a VR of 80%, and the concentration of lignin increased from 1.2 g/l to 5.8 g/l, as shown in Fig. 3. Hemicelluloses represented 64% of total solids both before and after the enzymatic treatment (see Table 1). Hemicelluloses in the retentate after DF also represent 64% of total solids. The enzymatic
Table 1 Characteristics of the process solution before and after enzymatic treatment, and the retentate and the permeate after DF to a VR of 80%. All measurements were made in triplicate.
Total solids (g/l) Ash (g/l) Lignin (g/l) Hemicellulose (g/l) Arabinan (g/l) Galactan (g/l) Glucan (g/l) Xylan (g/l) Mannan (g/l)
Before enzymatic treatment
After enzymatic treatment
Retentate after DF
Permeate after DF
77.3 ± 0.1 2.1 ± 0.1 3.5 ± 0.2 50.1 ± 1.1 1.2 ± 0.1 6.5 ± 0.1 9.1 ± 0.1 0.7 ± 0.4 32.6 ± 1.1
79.7 ± 0.3 2.1 ± 0.6 4.4 ± 0.2 50.6 ± 1.5 1.3 ± 0.0 6.5 ± 0.1 9.2 ± 0.1 0.8 ± 0.3 32.8 ± 1.8
94.8 ± 0.0 1.0 ± 0.2 5.7 ± 0.2 60.9 ± 3.2 1.5 ± 0.0 8.6 ± 0.1 10.7 ± 0.0 1.0 ± 0.4 39.1 ± 3.5
4.5 ± 0.0 0.5 ± 0.0 0.2 ± 0.0 2.4 ± 0.1 0.1 ± 0.0 0.2 ± 0.0 0.6 ± 0.0 0.0 ± 0.0 1.5 ± 0.1
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2
Flux (l/m h)
80
40
20
0 0
20
40
60
80
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Volume reduction (%) Fig. 4. Flux during concentration of UF permeate by nanofiltration. The temperature was 50 °C, the transmembrane pressure 10 bar and the cross-flow 0.6 m3/h.
UV (Absorbance units)
a
0.08
0.06
0.04
0.02
0.00 0.1
1.0
10.0
100.0
Molecular mass (kDa)
Refractive index (mV)
b
4. Conclusions
120 100 80 60 40 20 0 0.1
1.0
10.0
shown in Fig 4. The average flux during concentration was 49 l/m2 h. Lignin showed a peak in the SEC chromatogram at 1 kDa. This peak was considerably higher after NF, as can be seen in Fig. 5a. Smaller sugar molecules, <1 kDa, were detected in the NF retentate, whereas the concentration was not sufficiently high to allow detection of these molecules in the DF permeate (Fig. 5b). There is also a hemicellulose peak at 10 kDa, the same molecular mass as in the DF retentate. This is puzzling as the nominal cut-off of the UF membrane used during DF was 10 kDa and molecules of this size should have been retained during DF. The retention of the UFX10 pHt membrane was therefore investigated by UF of a mixture of PEG standards used to calibrate the SEC system. The retention of PEG10 and PEG35 (with peak molecular masses of 10 kDa and 35 kDa) was 81% and 90%, respectively, at a transmembrane pressure of 0.5 bar, and 50% and 53% at 2 bar, revealing that the cut-off of the UFX10 pHt membrane is higher than the nominal cut-off when treating straight polymers, and that the transmembrane pressure has a considerable effect on the retention due to enhanced concentration polarization at higher pressures. The retention of hemicelluloses and lignin was about 98%. The concentration of hemicelluloses increased from 3 g/l to 15 g/l, and the concentration of lignin from 0.3 g/l to 1.3 g/l, in the retentate at VR 83%. The high retention of the NF membrane resulted in a permeate with low concentration of total solids (0.25 g/l), hemicelluloses (0.10 g/l), lignin (0.02 g/l) and ash (0.06 g/l). This permeate can be reused in the pulp mill replacing fresh water.
100.0
Enzymatic treatment of TMP process water with laccase did not have the same effect on the molecular mass of hemicelluloses as in the previous studies, where the size of hemicellulose molecules in process water from a TMP mill [10] and a CTMP mill [6] was increased using the same method. The explanation of the lack of effect in the present study could be that the lignin molecules in the TMP process water were linked to hemicelluloses to a lesser extent. It was also observed that the lignin concentration in the TMP process water is much lower than in the previous. The lowmolecular-mass hemicelluloses in the UF permeate could be recovered and concentrated by NF.
Molecular mass (kDa) Fig. 5. Molecular mass distribution of (a) lignin and (b) hemicellulose in the UF permeate (—) and final NF retentate ( ).
Acknowledgment The Swedish Energy Agency is gratefully acknowledged for financial support.
treatment increased the size of lignin (see Fig. 1b), making the retention of hemicelluloses and lignin almost identical during DF. Hence, no purification of hemicelluloses was achieved by DF of the solution. The initial retention of hemicelluloses and lignin was 85% and 87%, respectively, and 90% for both at the end of DF. This is in sharp contrast to the markedly higher retention of hemicelluloses (97%) than lignin (55%) when the TMP process water was concentrated with a 5 kDa membrane before enzymatic treatment [7]. This confirms that the size of the lignin molecules was increased during enzymatic treatment, while the size of the hemicelluloses was largely unaffected.
3.3. Nanofiltration of ultrafiltration permeate The UF permeate from the DF operation, containing small hemicellulose and lignin molecules, was concentrated by NF. The initial flux was 55 l/m2 h and decreased to 37 l/m2 h at a VR of 83%, as
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