Enzyme and Microbial Technology 29 (2001) 160 –165
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Energy saving with fungal enzymatic treatment of industrial poplar alkaline peroxide pulps J.C. Sigoillota,*, M. Petit-Conilb, I. Herpoe¨la, J.P. Joseleauc, K. Ruelc, B. Kurekd, C. de Choudensb, M. Asthera a
Unite´ INRA de Biotechnologie des Champignons Filamenteux, IFR-BAIM, Universite´s de Provence et de la Me´diterrane´e, ESIL, 163 Avenue de Luminy, Case Postale 925, 13288 Marseille cedex 09, France b Centre Technique du Papier, domaine universitaire, B.P. 7110, 38020 Grenoble Cedex, France c Centre de Recherches sur les Macromole´cules Ve´ge´tales, domaine universitaire de Saint-Martin-d’He`res-Gie`res, 601 Rue de la Chimie, B.P. 53, 38041 Grenoble Cedex 9, France d Laboratoire de Physicochimie et Biotechnologie des Polyme`res, Institut National de la Recherche Agronomique, Moulin de la Housse, BP 1039, 51687 Reims cedex 2, France Received 10 August 2000; received in revised form 29 March 2001; accepted 23 April 2001
Abstract An alkaline peroxide industrial pulp from poplar was treated with a manganese peroxidase (MnP) from the hypersecretory strain of Phanerochaete chrysosporium I-1512 after a second stage of refining. The enzymatic treatment caused an improvement in the pulp quality by inducing an enzymatic refining onto the fibers. Transmission electron microscopy showed that the enzymatic refining was characterized by internal and external fibrillation of the fibers. Chemical modifications of lignin caused by MnP treatment facilitated the post-refining stage before papersheet manufacture. All together the enzymatic treatment resulted in energy savings of 25% during beating. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Manganese peroxidase; Phanerochaete chrysosporium I-1512; Pulp and paper; Enzymatic refining; Poplar; Alkaline peroxyde
1. Introduction Mechanical pulps represent about 20% of the world pulp production. The mechanical pulping processes generate high yield pulps rich in lignin but with relatively low strength properties. Moreover, the manufacture of mechanical pulps are energy consuming. Considerable research has been devoted to improve mechanical pulp properties and to reduce the production cost of the process. In recent years, the use of white-rot fungi for treating wood chips before the refining stages to facilitate fiber separation and to reduce the refiner energy consumption has been developed [1–3]. Several reports have shown the effectiveness of biomechanical pulping process but the fungal treatments needed incubation times too long for an industrial scale application. In contrast, enzymatic treatment which take only few hours are more compatible with mill processes. The effects of en* Corresponding author. Tel.: ⫹33-4-91-82-86-06; fax: ⫹33-4-91-8286-01. E-mail address:
[email protected] (J.C. Sigoillot).
zymes on mechanical pulps depend largely on their penetration into the pulp. Ruel et al. [4] have shown that the action of ligninolytic enzymes such as manganese peroxidase (MnP) and laccase on high yield pulp fibers was more efficient after secondary pulp refining when the fiber structure was deeply opened. The use of isolated enzymes in high-yield pulping processes was rather new. However some studies have already revealed the interest of the introduction of such a treatment in high-yield pulping processes. Sigoillot et al. [5] have shown that treatment of wheat straw chemimechanical (CMP) pulp with MnP enhanced the mechanical properties of the pulp by a selective action of MnP on the lignin. A patent has been filed by Farell [6] for the use of lignin peroxidases to improve the optical properties and strength of mechanicals pulps. Recently, Pere et al. [7] demonstrated that a primary refined mechanical pulp treated with cellobiohydrolase gave energy savings of between 10 to 40% in the secondary refining stage without modifications of the fiber length and paper strength. Consequently, alternative technology results in net savings in production costs.
0141-0229/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 1 ) 0 0 3 6 8 - 4
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In this work, the treatment of a commercial poplar alkaline peroxide pulp with a manganese peroxidase from the hypersecretory strain Phanerochaete chrysosporium I-1512 was investigated to evaluate the potential of MnP treatment in increasing pulp strength and in energy savings during beating.
2. Material and methods 2.1. Fungal strain and enzyme production Phanerochaete chrysosporium I-1512 (CNCM, Institut Pasteur, Paris, France) was used in this study. The crude manganese peroxidase extract was produced using a new bioreactor design in which an airlift column (100 L working volume) was combined with biofilm immobilization, as previously described [8]. After 6 –7 days of incubation, the culture supernatant was harvested and concentrated 10 fold by ultrafiltration using a 10000 Da membrane (Millipore S.A., Molsheim, France). 2.2. Lignin and manganese peroxidase assays Lignin peroxidase activity was determined spectrophotometrically at 30°C by the method of Tien and Kirk [9]. One unit of enzyme activity is equivalent to 1 mol of product formed per min. Manganese (II) dependent peroxidase activity was determined spectrophotometrically at 30°C by the method of Paszczynski et al. [10]. One unit of enzyme activity is equivalent to 1 mol of substrate oxidized per min. 2.2.1. Pulp origin Poplar alkaline peroxide mechanical pulp was industrial undried pulp produced by Sicem Saga S.p.A (Italy). The wood was reduced into matches in a refiner equipped with specially designed plates before impregnation with alkaline peroxide. After chemical reaction, the wood matches were primary and secondary refined. The pulp had a freeness of 290 ml CSF.
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the pulp before and after MnP treatment. The drainage capability (Freeness) is expressed in ml of water drain off from a suspension of 2 g of pulp (Dry weight) according to TAPPI standards (ml CSF). It depends on the surface state and swelling of fibers and represent an useful index of the importance of the mechanical treatment applied to the pulp. The beating behavior was evaluated by comparing energy consumption versus drainage index for treated and untreated pulps. In mechanical pulping, energy consumption is an important economic factor to be considered. The PFI mill is a standard laboratory apparatus for delaminating fiber cell wall and liberating microfibrills from the cell wall. These microfibrills are essential elements for developing strengths necessary to form paper sheet by interfiber bonding. 2.4. Pulp quality evaluation Optical and physical measurements were performed according to international standards (TAPPI or ISO standards). Ten handsheets were made for each pulp sample and results are expressed as the mean of individual measurements. Characteristics of fibers were determined by image analysis with PQM 1000 (Sunds defibrator) and CyberMetrics analyser [11,12]. 2.5. Transmission electron microscopy Small samples of pulps were fixed in a freshly prepared mixture of 0.2% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer pH 7.0. Samples, dehydrated through a series of increasing ethanol concentrations up to 70% (v/v), were embedded in London Resin White (LRW) (hard mixture) and polymerized over 24 h at 50°C. Chemical staining of polysaccharides was done on thin sections by the periodic acid-thiocarbohydrazide-silver proteinate (PATAg method of Thiery, modified as in Burlat et al. [13]. 2.6. Scanning electron microcopy Samples of pulps were fixed and subjected to drying by the critical point method in liquid carbon dioxide.
2.3. Pulp treatment 3. Results The screened refined poplar CMP pulp, suspended to 5 or 10% consistency in 100 mM lactate as buffer containing 1 mM MnSO4, was treated with 25 U MnP/g of pulp during 1 h. Hydrogen peroxide solution (0,1 mM) was introduced into the suspension at the beginning of the treatment, just before enzyme addition. The enzymatic treatment was followed by an acidic wash (20 min with H2SO4 at 2% pulp consistency), a chelating treatment (0,4% DTPA, 25 min, 60°C, 4% pulp consistency) and a peroxide stage (1% H2O2, 1,5% NaOH, 4% Na2SiO3, 2 h, 60°C, 16% pulp consistency). The pulp was then beaten in a PFI mill to develop the paper properties and to determine the refining behavior of
The screened refined poplar CMP was treated with MnP at 5 or 10% pulp consistency. Characteristics of the MnP treated pulp were compared with the untreated reference pulp. The pulp properties are summarized in Table 1. MnP treatment improved the physical properties of the pulp, especially the tensile strength which is the most important parameter for the runability on paper machine and the quality of the paper produced. However a decrease in the tear index was observed. This can be explained by internal fibrillation of the fibers (as shown in Fig. 2D) that fragilize internal structure and give the fiber more sensitive to shear.
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Table 1 Physical properties and mean dimensions of poplar alkaline peroxide fibers after MnP treatment
Physical properties Freeness (ml) Bulk (cm3/g) Tensile index (Nm/g) Tear index (mNm2/g) Brightness (% ISO) Opacity (%) Scattering coefficient (cm2/g) Fiber ultrastructure Fiber length (mm) Fiber width (m) Coarseness (mg/m) Bonding potential RBA index (%) Shive content (%)
Untreated pulp reference
MnP treated pulp 5% consistency
MnP treated pulp 10% consistency
80 1.34 62.9 3.31 74.4 66.7 235
60 1.27 68.5 2.81 72.4 67.0 200
50 1.19 68.2 2.61 78.6 61.2 165
0.89 26.6 0.153 30.2
0.86 28.8 0.141 48.0
0.93 34.1 0.099 38.8
0.02
0.05
0.02
The evolution of the mechanical properties was similar at 5 and 10% consistency. However, treatment of the higher pulp consistency gave a better brightness of the pulp. Although high consistency experiments are difficult to perform at lab scale, they are closer from industrial conditions. The morphology of the fibers was unmodified by MnP treatment. An important effect of the MnP treatment was the modification of the beating behavior. As indicated in Table 1, the freeness of the MnP treated pulp decreased faster than the reference pulp. This result revealed that the MnP treatment could result in energy savings during beating. To confirm the better beating ability of the MnP-treated pulp, the energy requirement during beating versus freeness was plotted for the MnP-treated pulp and compared to a nontreated reference pulp (Fig. 1). The MnP-treated poplar CMP was more rapidly beaten than the reference pulp. Important energy saving was obtained in the PFI mill: about 25% of the beating energy could be saved with a MnP treatment of the pulp at a freeness level of 90 ml CSF. Modification of the fiber wall and of the fiber behavior after enzyme treatment were evaluated by the analysis of the fiber characteristics. The wet fiber flexibility index indicated that the MnP-treated fibers were transformed during beating (Fig. 3A). The flexibility variation was more important for MnP-treated fibers at comparable freeness indexes. This modification was associated with an increase in the relative bonded area index (RBA index) which increased faster during beating for the MnP-treated fibers. Moreover, in order to determine the fibrillation or the cutting phenomena which were involved during the mechanical treatment, fiber dimensions during beating of reference and MnP-treated poplar CMP pulp were analyzed (Fig. 3B). The refining treatment had a direct effect on the fiber characteristics. As the refining intensity increased, the fiber length somewhat decreased. The fibers treated by the manganese peroxidases
Fig. 1. Evolution of the energy consumption during post-refining in PFI mill with freeness for the poplar alkaline peroxide pulp in presence (F) or in absence (䡬) of MnP treatment.
were longer and cutting was less important. Moreover, refining had a direct effect on the fiber coarseness. Thus, the fiber wall peeling-off phenomenon was predominant, indicating a certain fibrillation. These increases in fibrillation by MnP treatment was revealed by scanning electron microscopy examination. Numerous fibrils of the MnP-treated pulp were clearly individualized indicating a delamination of the fiber walls (Fig. 2). As shown in Fig. 2C and 2D, in the most accessible and opened structures the effect of the enzymes in the fiber walls was an internal fibrillation which resulted in the individualization of microfibrills. Following the enzymatic treatment, the microfibrills were devoid of protecting lignin and hemicelluloses, as indicated by their low stability under the electron beam.
4. Discussion For paper making, pulp fibers must be refined or beaten in specific equipment by delaminating cell wall on metallic discs equipped with bars and grooves in aqueous medium. Beating induces two important phenomena: fiber cutting or fibrillation. To develop high paper strengths, fibrillation must be the predominant effect of beating. To achieve this, application of energy is a result. MnP treatment of poplar CMP induced some beating behavior modifications: Y mean fiber length was less reduced than the reference pulp (Fig. 3B), revealing that the cutting phenomenon was reduced or delayed; Y energy saving (Fig. 1): it was observed a decrease in energy consumption of about 25% for MnP treated pulp to reach a drainage capability of 100 ml;
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Fig. 2. A & B, Scanning Electron Microscopy. After MnP treatment (B), the surface of fibers show more cellulose microfibrils with an outward orientation (arrow-heads) than before enzymatic treatment (A). C & D, Transmission Electron Microscopy. Transverse section of pulp fibers after staining by PATAg technique. C: control. D: after MnP treatment, a seperation of cellulose microfibrils is obvious. The loosening is now more important in the inner part of the wall which, because of its loosening, was more easily penetrated by the enzyme.
Y modification of beaten status of the pulp: the MnP treatment decreased the drainage capability compared to the control (320 ml versus 370 ml), indicating increased fiber hydratation; Y MnP treatment enhanced internal and external fibrillation phenomenon, as revealed by SEM and TEM examination of fiber surface and wall (Fig. 2) Y MnP treatment seemed to have a weak effect on wet fiber flexibility and bonding potential (Fig. 3A and 3B). All these results allow us to conclude that MnP treatment had an “enzymatic refining effect” on the polplar CMP fibers. It promotes liberation of numerous and fine fibrils compared to the reference pulp (Fig. B). The results presented in this study showed that the beating of poplar alkaline peroxide pulps treated with MnP was easier than that of the reference pulp. At the same energy level, lower freeness levels could be reached compared to
the control. Examination in transmission electron microscopy revealed that the treatment with MnP increased internal and external fibrillations. Similar results have already been observed on mechanical pulps treated by MnP [4,5, 14]. A rather extensive fibrillation could indeed be observed in transmission electron microscopy. The action of manganese peroxidase of inducing a significant removal of the inter-microfibril material individualizes the cellulose microfibrils. Although the mechanism of action of MnP involves an oxidative degradation of lignin, a part of the hemicellulose may have also been removed because of their attachment to lignin molecules via lignin-carbohydrate covalent bonds [15,16]. It is interesting to note that the action of the enzyme concerned all of the S2 layer of the fiber walls. The homogeneous action of the enzyme corresponds to the regular distribution of lignin throughout the S2 layer. This agrees with the presence in this anatomic zone of the fiber wall of significant amounts of non-condensed -O-4 aryl
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Fig. 3.: Effect of beating (expressed as freeness level) on fibers characteristics: A, flexibility in presence (f) or in absence (䡺) of MnP treatment and bonding potential index (RBA index) in presence (F) or in absence (䡬) of MnP treatment. B, Mean fiber length in presence (F) or in absence (E) of MnP treatment.
alkyl subunits [13] which are also highly sensitive to manganese peroxidase degradation [17]. The observation of an extended microfibril separation, in the secondary wall and at the periphery of the fiber surface, suggests that, in the secondary refined pulp, the opening of the structure allowed a good penetration of MnP and chelated Mn3⫹ into the fiber wall, as shown by Ruel et al. [4]. The fibers treated with MnP were more fibrillated and could then develop more interactions through hydrogen bonding. Consequently, the MnP action could be assimilated to enzymatic refining increasing inter-fiber bonding through fibrillation. Pulp consistency seem to be an important parameter, and better results were obtained at 10% consistency. In the latter conditions, the freeness level lower than with the 5% pulp consistency at 3000 rounds is probably correlated with a more important fibrillation due to the enzymatic treatment. An other consequence of the higher pulp consistency was to enhance the brightness. Due to this improvement, the opacity was reduced because of a modification of the scattering behavior of the fibers. In itself the refining effect of the enzymes onto the fibers resulted in 25% energy saving during beating. Analysis of the behavior of fibers during the mechanical treatment showed that the enzymatic treatment had a slight protective effect on the fibers in facilitating fibrillation without important cutting. This behavior could explain the improvement of the tensile and burst strengths and the limited reduction in tear index. For the same energy consumption, a decrease of the freeness level of MnP treated pulp was associated with an increase in pulp strengths. Moreover, the more fibrillated and more flexible fibers induced a decrease of the bulk by increasing inter-
fiber contacts. This resulted in an increased of the density of the paper. However, a decrease of the tear index revealed some intrinsic degradation of the fiber due to the increase in internal fibrillation. The enzymatic treatment conditions were defined in order to ensure compatibility with industrial processes. Indeed, the short time needed by the treatment (one hour), the simplified peroxide introduction (at the beginning of the treatment) and the low temperature (40°C) used are suitable for industrial application. Another interest of MnP treatment is that is consists of a one-stage treatment carried out in a stirred reactor, and consequently requires limited investment.
5. Conclusion The pulp quality and the beating behavior of a commercial poplar high-yield pulp were improved by a treatment with manganese peroxidase. The enzymatic treatment induced an enzymatic refining which facilitated fibrillation. The fibrillation enhancement was in agreement with pulp properties development. Tensile strength was improved while tear strength decreased slightly due to fiber structure damage. As a result of the development of fibrillation, the energy consumption was reduced during beating. Energy savings of about 25% were achieved in the post-refining stage before papersheet manufacture in beating conditions similar to those used in the pulp industry. Consequently, a more cost-effective manufacturing process could be envis-
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aged by the introduction of an enzymatic stage in paper manufacture from alkaline poplar pulp.
Acknowledgments This work was carried out in the framework of European Union Research Program AIR3-CT94 –2065 (POPLAR) and GIS-EBL (Re´gion PACA and Conseil Ge´ne´ral 13). Isabelle Herpoe¨l is grateful to the Conseil Re´gional Provence-Alpes-Coˆte d’Azur, the Institut National de la Recherche Agronomique and Cellurhoˆne for a PhD scholarship.
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