BioMatNet Item: AIR3-CT94-2065 - Use of New Technical Enzyme and Its Biomimetric System to Improve the Properties of Poplar High Yield Pulp

AIR3-CT94-2065
Use of New Technical Enzyme and Its Biomimetric System to Improve the Properties of Poplar High Yield Pulp

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AIR Cluster VII - Forestry and Forest Products : Biological Conversion : Biotechnology : Drying/Pretreatment : Fibre : Paper/Pulp : Plant Genetics : Process Engineering : Pulping : Wood (Lignocellulose)

	Proposal No:	AIR3-CT94-2065
	Date Prepared:	September 1999, May 1999
	Source:	Final technical report November 1998 Final Summary Report

Final technical report November 1998

Introduction

Enzymes, that are more efficient and environmentally friendly compared to chemicals, are becoming of more interest to the pulp and paper industry. The first application of enzymes was in 1959 for the fibrillation of paper pulps with cellulases. The first industrial utilisation occurred in the 1980's, when xylanases were introduced for improving refining or beating of chemical pulps. These enzymes were also tested for the pre-bleaching of chemical pulps. The discovery of laccases and manganese peroxidases that oxidise lignin led, in the mid 1990's, to their testing in the bleaching of chemical pulps.

One of the disadvantages of enzymes is their lack of stability under industrial conditions (higher temperatures, pH. oxidising agents, some metallic ion salts). Hence, research was needed to modify them in order to use them for industrial applications. Xylanases, laccases and manganese peroxidases have been widely studied for the bleaching of chemical pulps. Applied to the unbleached pulps, they could facilitatethe further lignin removal by the conventional oxidising bleaching chemicals such as chlorine dioxide, ozone and hydrogen peroxide. Some chemical pulp mills are using enzymes in their bleaching sequence to facilitate lignin extraction and save some bleaching chemical, as a result of which about 10 % of the chemical pulp is manufactured with an enzymatic stage.

The high-yield pulps represent about 20 % of the world pulp production. Several processes are available to manufacture mechanical pulps that vary in equipment used pulp yield. These fall into two main classes:

pulp processes based on mechanical treatment, that may or may not be associated with thermal treatment of the wood chips, including the stoneground wood processes (SGW, PGW) and the refiner thermomechanical processes (TMP)
pulp processes based on mechanical treatment associated with a thermal and chemical treatment of the wood chips, including chemi-thermomechanical (CTMP) and chemi-mechanical (CMP) processes

The former processes generate pulp with higher yield, lower pulp strengths but higher optical properties. They are used in the manufacture of newsprint and lightweight coated papers. The latter processes produce a pulp with higher quality but lower yield. These pulps are added to the fibrous mixtures used for printing, writing papers, board and sanitary papers.

In the 1990's, a new CMP/CTMP process was developed, based on the impregnation of the wood chips with alkaline peroxide before the primary and secondary refining stages. High quality pulp is obtained. This pulp can be added to printing papers, replacing some hardwood chemical pulps. The main advantages of this process are a better use of hardwood resource. Some energy savings of primary and secondary refiners and a total treatment and recycling of the liquid effluents. Mills have been installed in North America for the production of hardwood alkaline peroxide mechanical pulp (APP) with zero effluent discharge to the environment.

The use of enzymes in high-yield pulping processes is rather new. Processes involving a biochemical treatment of wood chips have been developed using the white-rot fungi for wood chip treatment before primary and secondary refining. With such a treatment, carried out over 2 to 4 weeks, a biomechanical pulp can be produced, with a pulp quality comparable to CTMP with important energy savings. This process was adapted to industrial applications. For a good fungus development, a steam treatment is used to eliminate microorganisms that can attack the applied fungus. Experiments on large scale were carried out and the results obtained at laboratory scale were confirmed.

Although the interest of enzymatic treatment to pulp and paper industries is well established, only a few studies have concerned the introduction of such a treatment in high-yield pulping processes. As the lignin is still present into the fibre wall, it was of high interest to test specific diffusing ligninolytic oxidative enzymes on the last generation of high-yield pulps. Manganese peroxidases, a peroxidase from ligninolytic fungi, can oxidise lignin structures by the way of manganese III ion complexes. Since manganese III ion act as a mild oxidising agent, when chelated with organic acids, it is assumed to be a primary lignin oxidising agent in fungal biodegradation of wood in nature. Hence, their application to high-yield pulps rich in lignin might be of benefit. However, at the start of the project, the productivity of manganese peroxidase was low, hampering the technical development of such biotreatments for the industry.

This production had to be improved in order to generate sufficient amounts of enzymes for use in pulping processes. As Phanerochaete chrysosporium is the most efficient fungus for the production of manganese peroxidases and lignin peroxidases, the optimisation of culture conditions and the selection of hypersecretory strains were necessary.

Objectives

The objectives of this project were as follows.

Comparison of different poplar clones for then-non-mechanical and alkaline peroxide mechanical pulping in order to determine the effect of wood structure and genetic modification on the pulp quality and on the process parameters.
Selection of manganese peroxidase hypersecretory strains from Phanerochaete chrysosporium
Optimisation of culture conditions (medium composition. inducers, oxygenation, stirring) of the selected strain for the enzyme production.
Scale-up of the manganese peroxidase production in pilot plant bioreactor (100 1) and industrial fermenter (2 M³).
Production of manganese peroxidases in sufficient quantities for pilot plant and industrial scale pulp treatment.
Development of a manganese peroxidase treatment of a poplar alkaline peroxide mechanical pulp in order to improve pulp quality.
Understanding of the manganese peroxidase action on the fibre component and on the fibre wall structure by the way of analytical, chemical and microscopic methods.

Activities

To meet the main objective - to improve the quality of an industrial poplar high-yield pulp through the introduction of an enzymatic treatment based on the new ligninolytic oxidative enzymes (the manganese peroxidases) - the alkaline peroxide-based high-yield pulping process based on hardwood resources was selected with the collaboration of an European industrial producer of such commercial pulp. The manganese peroxidases were studied, adapted and optimised. Their production had to be improved and scaled-up.

Four different commercial poplar clones (I214, Lux, Raspalje and Spiado) were compared for the manufacture of alkaline-peroxide mechanical pulp. The behaviour of these poplar clones differed, depending on the nature of the wood structure, chemical composition and fibre morphology. Raspalje poplar clone had the lowest lignin content, the highest cellulose content with a slightly brighter wood colour. This clone was thus the most interesting in terms of alkaline-peroxide mechanical pulping and thermomechanical pulping. The fibres were longer, more flexible and developed higher interfibre bonding potential.

Some lignin structure modifications occurred during chemical and mechanical treatments of the process. The non-condensed part of the lignin structure was more labile than the condensed part. The observation of the fibre structure with transmission electron microscopy revealed that the Raspalje poplar clone presented higher mechanical properties because of the presence of G layer in the secondary layer of the fibre wall. This layer, rich in polysaccharides, facilitated fibre separation (lower cohesion forces with other fibre wall layers) and fibrillation.

The manganese peroxidases were optimised from an hypersecretory strain of Phanerochaete chrysosporium: I-1512 strain. The culture conditions and media composition were optimised to produce the highest activity of manganese peroxidases recorder in 2.5 1 fermenter, at ore than 3500 units/l. These conditions were transferred to a 12 1 reactor and then to 100 l fermenterduring scale-up. The geometry of the reactor was kept constant and the design of the fungus carrier maintained. The result of this scale-up was an activity of 7200 units/l, reached in 150 hours.

The manganese peroxidase treatment was adapted to the pulp manufacture industrial process. The enzymatic treatment was carried out on the secondary refined poplar pulp in order to increase the enzyme accessibility. All the treatment conditions were optimised, as a result the treatment time was reduced from 8 hours to 1 hour, The pulp concentration was increased from 0. 5 % to 10 % and the temperature raised to 40 degrees C The peroxide charge was reduced and the peroxide introduction mode simplified The buffer was changed for a commercial cheaper one: lactate. Under these conditions, the manganese peroxidase treatment induced energy savings during beating (7 to 20 %) and enhanced the pulp properties. This improvement was the result of lignin modification and internal fibrillation of the microfibrils. These results were confirmed at pilot plant scale by treating 100 kg of o.d. poplar alkaline-peroxide pulp. Some printing paper was produced with success on an experimental paper machine.

Conclusions

The comparison of four different poplar clones for the manufacture of thermomechanical and alkaline peroxide mechanical pulps have enabled the effects thatwood structure and genetic difference had on pulp quality. Among the four poplar clones, Raspalje gave the best results with TMP and CMP. This can be explained by the lower lignin content, the higher cellulose content and by the presence of tension wood. The tension wood induced a new layer in the secondary wall that facilitated fibre separation.

The production of manganese peroxidases was enhanced by the selection of an hypersecretory strain of Phanerochaete chrysosporium (I- 1512). With this new strain, MnP activity was improved in 2.5 1 fermentors by optimizing the medium composition and the culture conditions. The scale-up to 12 1 and 100 l bioreactors was achieved by maintaining the same geometry and the same culture conditions. The MnP activity was increased: from 3600 U/l in the 2.5 1 fermentor to 7200 U/l in the 100 l bioreactor, the highest ever obtained for the production of manganese peroxidases.

Unfortunately, the scale-up in 2m³ bioreactor did not succeed, due to problems of reactor geometry, oxygen supply and presence of bacteria. However, the 100 litre bioreactor enabled sufficient enzyme to be produced for pilot plant pulping experiments. The enzymatic treatment of poplar alkaline peroxide mechanical pulp was adapted to industrial conditions. All the parameters of the manganese peroxidase treatment were optimized with this pulp. The presence of manganese ions, complexes and oxide decreased the pulp brightness. An acidic washing stage and a post-bleaching stage to recover or increase the brightness was found necessary. In these conditions, the pulp quality was improved and the beating behaviour greatly modified. Energy savings could be anticipated following MnP treatment. Printing papers were manufactured with this MnP-treated pulp without any problems.

Final Summary Report

Summary
The objective of the AIR3-CT94-2065 project was to improve the quality of an industrial poplar high-yield pulp by introducing an enzymatic treatment based on the new-ligninolytic oxidation enzymes, the manganese peroxydases.

The alkaline peroxide-based high-yield pulping process was the last generation of the chemimechanical pulping process developed to a better use of hardwood resources and to enhance the pulp properties. A process based on this technology was applied at industrial scale in many countries and was the only one able to manufacture commercial pulps with zero liquid effluent. Due to these reasons, this process was selected with the collaboration of an European industrial producer of such a commercial pulp.

The manganese peroxidases were oxidative enzymes capable to oxidise lignin in lignocellulosic raw materials but its action might be studied, adapted and optimised. Their production had to be improved and scaled-up.

Four different commercial poplar clones: I 214, Lux, Raspalje and Spiado were compared for the manufacture of alkaline-peroxide mechanical pulp. The behaviour of these poplar clones differed. depending on the nature of the wood structure, chemical composition and fibre morphology. Raspalje poplar clone presented the lowest lignin content. the highest cellulose content with a slightly brighter wood colour. This clone was also the most interesting in terms of alkaline-peroxide mechanical pulping and thermomechanical pulping. The fibres were longer. more flexible and developed higher interfibre bonding potential. Some lignin structure modifications occurred during chemical and mechanical treatments of the process. The non-condensed part of the lignin structure was more sensible than the condensed part. The observation of the fibre structure with transmission electron microscopy revealed that the Raspalje poplar clone presented higher mechanical properties because of the presence of G layer in the secondary layer of the fibre wall. This layer, rich in polysaccharides, facilitated the fibre separation (lower cohesion forces with other fibre wall layers) and the fibrillation. Therefore more interfibre bonds were created during paper manufacture.

The manganese peroxidases was optimised from an hypersecretory strain of Phanerochaete chrysosphorium: I-1512 strain. The culture conditions. medium composition were optimised. In this conditions, it was possible to produce the highest activity of manganese peroxidases never published in the world in 2.5 I fermentor: more than 3500 unit/s. These conditions were transposed to 12 I reactor and then to 100 I fermentor for scale-up. The geometry of the reactor was kept constant and the design of the fungus carrier maintained. The result of this scale-up was an increase in the manganese peroxidase production: an activity of 7200 units/l was reached in 150 hours.

Finally, the manganese peroxidase treatment was adapted to the pulp manufacture industrial process. The enzymatic treatment was carried out on the secondary-refined poplar pulp in order to increase the enzyme accessibility. All the treatment conditions were optimised:

The treatment time was reduced from 8 hours to 1 hour,
The pulp consistency was increased from 0.5 % to 10 % and the temperature to 40°C
The peroxide charge was reduced and the peroxide introduction mode simplified
The buffer was changed for a commercial cheaper one: lactate

In these conditions. the manganese peroxidase treatment induced energy savings during beating (7 to 20%) and enhanced the pulp properties. This improvement was the result of lignin modification and internal fibrillation of the microfibrils. These results were confirmed at pilot plant scale by treating 100 kg of o.d. poplar alkaline-peroxide pulp. Some printing paper was produced with success on an experimental papermachine.

EXECUTIVE SUMMARY

Scientific Objectives
This project was devoted to the improvement of the quality of poplar high-yield pulps, especially chemimechanical pulps manufactured with alkaline peroxide impregnation, by an enzymatic treatment based on manganese peroxidases These objectives will be reached in:

comparing different poplar clones for TMP and CMP pulping based on the process of the industrial partner
providing engineering basis for a biobleaching process of the poplar CMP to improve the paper strengths to reduce the colour reversion with light exposure and to decrease the chemical charges if possible
using non conventional enzymatic treatment based on manganese peroxidases and Mn III chelates and improving the enzyme action with the biomimetic manganic complexes - improving the manganese peroxidases production at laboratory and at industrial scales - and testing this bioprocess at pilot plant and industrial scales if interesting results are obtained.

WORK PERFORMED DURING FOUR YEARS

Wood characterisation of poplar clones
Four commercial poplar clones were selected and studied I 214, Lux, Raspalje and Spiado The characterisation of the wood structure and composition, by chemical analysis, transmission electron microscopy revealed that Raspalje clone was different from the others This poplar clone possessed lower lignin content, higher cellulose content with the same wood extractives (table 1) General examination of the wood structure indicated that Raspalje seemed to have more vessels, thinner and more compact fibre walls More tension wood was observed indicated by the presence of the G-layer after the S2-layer Two kinds of parenchyma cells

radial parenchyma characterised by an unusual "isotropic layer", localised within the secondary wall and certainly highly lignified
Axial parenchyma with no isotropic layer

Immunochemical localisation of lignins according to their chemical nature (guaïacyl and guaïacyl-syringyl units) revealed that GS lignin was the most abundant in all tissues The nature of lignin into the cell corners and middle lamella was different This could influence the behaviour during enzymatic treatment No qualitative lignin distribution was observed between the poplar clones.

Poplar clone	I 214	Raspalje	Lux	Spiado
DCM extractives, %	0.73	0.70	0.47	0.55
Lignin, %	24.7	21.0	22.6	23.6
Cellulose, %	58.4	60.9	60.2	59.6
Basic density g/cm³	0.284	0.325	0.357	0.346
Yellow component	15.86	15.92	16.25	16.87
Metallic ion content, ppm:
Iron	22	10	53.7	36.7
Manganese	2.3	8.5	3.5	4.5
Copper	< 2	< 2	< 2	< 2

Table 1 : Wood characteristics of the four poplar clones

Manufacture of TMP/CMP poplar pulps at pilot plant scale
Thermomechanical (TMP) and alkaline-peroxide mechanical pulps were manufactured at pilot plant scale (capacity: 40 kg/h) to simulate the behaviour of the wood in industrial pulping units. The TMPs brought information about the wood behaviour during mechanical pulping based on thermal treatment, primary and secondary refining. Differences in pulp quality and energy consumption were observed between the four clones: Raspalje clone presented the best compromise between the pulp quality and the energy consumption (table 2). Its bleachability with hydrogen peroxide permitted to obtain 80 % ISO brightness.

Poplar clone	I 214	Raspalje	Lux	Spiado
Defibering energy, kWh/t	1130	1090	1160	1045
Bulk, cm³/g	2.57	2.36	2.74	2.83
Breaking lenght, m	1880	2440	1820	1310
Stretch %	0.95	1.21	0.97	0.76
Burst index, kPam²/g	0.90	1.18	0.91	0.71
Tear index, mNm²/g	2.10	3.10	2.00	2.30
Brightness, % ISO	76.6	79.1	77.8	77.1
Opacity, %	90.0	88.3	85.8	89.8
Absorption coefficient cm²/g	5.7	4.4	3.8	5.7
Scattering coefficient cm²/g	600	605	485	585
Mean fibre length, mm	0.61	0.71	0.75	0.70

Table 2. Properties of the peroxide bleached TMPs at 100 ml CSF for the four poplar clones.

Even with a lower fibre length, the Raspalje TMP had higher mechanical properties indicating that the fibres were more flexible and developed more hydrogen bonds. certainly due to the presence of the G-layer. The transmission electron microscopy showed a large proportion of woody elements with traces of fractures between primary wall and S2 layer. After defibering and refining. Raspalje TMP presented more delamination of fibres and fibrillation. mainly in the G-layer. Besides. the lignin structure was more condensed into the Raspalje TMP fibres than in other poplar clone TMPs.

The simulation of the alkaline-peroxide mechanical pulping process was carried out at pilot plant scale. To perfectly simulate the industrial process. some difficulties were encountered, explaining the lower pulp quality of these pulps (table 3). The obtained results confirmed the previous ones: the Raspalje clone was much more suitable for high-yield pulping than the others.

Poplar clone	I 214	Raspalje	LUX	Spiado
Breaking length, m	2250	2515	1750	2200
Stretch, %	0.93	1.16	1.05	1.05
Burst index, kPam²/g	0.84	0.96	0.68	1.01
Tear index, mNm²/g	1.95	2.80	1.60	1.90
Brightness, % ISO	73.3	72.2	73.2	71.3
Opacity, %	8.4	89.1	81.8	84.8
Absorption coef., cm²/g	8.3	9.5	9.5	12.0
Scattering coef., cm²/g	495	490	500	535
Mean fibre length, mm	0.58	0.65	0.53	0.56

Table 3: Properties of the alkaline-peroxide mechanical pulps at 100 ml CSF for the four poplar clones

Production of manganese peroxidases at laboratory scale and scale-up
Phanerochaete chrysosporium was chosen for the production of manganese peroxidases (MnP). The strains presenting the best MnP were isolated and compared to the parental strain BKM-F-1767. A very interesting strain for MnP production was found: I-1512. The culture conditions of this strain were studied and optimised in immobilised cell bioreactors in which the MnP production was the most effective. With 2.5 l bioreactor, a maximum MnP activity of more than 3500 units/l was obtained in 80 hours with a lignin peroxidase (LiP) activity 6 fold lower. This activity was the highest activity never obtained at laboratory scale in the world.

The oxygen transfer and the stirring mode and rate were important factors for the MnP production. The carrier on which the fungus developed, affected the enzyme production and activity. Some inducers could improve the MnP production. The fungus growth and the enzyme production depended strongly on the mode of oxygenation, on the medium composition and on the mode of operation. A relation between average final pellet size and MnP activity as found. Immobilisation was the most efficient process for the MnP production with Phanerochaete chrysosporium.

The scale-up of the MnP production was carried out first in 12 l bioreactor and then in 100 l bioreactor. The geometry of the 2.5 l fermentor was directly transposed to l 21 bioreactor and then to 100 l bioreactor. An enhancement of the manganese peroxidase activity and production was observed during this scale-up (table 4). The LiP activity was still at the same ratio. Aeration and oxygenation seemed to be the main parameters to control during scale-up.

Bioreactor volume	2.51	12l	l001
Glycerol, g/l/h	0.038	0.039	0.044
Ammonium, 10^-3 g/l/h	0.5	0.91	1.05
MnP activity, U/l	3600	5880	7200
Specific activity, U MnP/mg	185.8	56	120
LiP activity, U/l	600	2520	2100
Specific activity, U LiP/mg	31	25	35

Table 4: Comparison of the results obtained with bioreactors of different sizes. Scale-up of MnP production.

The only difference observed during the scale-up was the delay of the MnP production. Into the 100 l bioreactor the MnP activity appeared after 150 hours of incubation, compared to 80 hours for the 2.5 l fermentor.