BIOPULPING
KURT MESSNER
4.1 Introduction
The main steps in paper making are to separated the fibres from the plant tissue, to .bleach them and to rearrange them to forma paper sheet. The fibres are separated either mechanically by' stone-grinding whole logs or by disc-refining wood chips. Both processes are extremely energy consuming. In mechanical pulping processes the wood tissues break apart mainly at the middle lamella or at the interface of the middle lamella and the wood cell wall. In chemi-thermo-mechanical pulping (CTMP) the refining process is assisted by chemicals and high temperature. As mechanical pulps still contain most of the lignin, their yield is very high (88-96 per cent). They lead to an opaque paper, mainly used for newsprint, books and magazines. Besides the high energy consumption, mechanical pulps suffer from low strength and low brightness (Biermann, 1993).
In chemical pulping processes such as kraft or sulphite pulping, the fibres are separated by dissolution of the middle lamella. Modern sulphur-free processes are being developed. Due to its high strength, unbleached kraft pulp is used for making bags, wrappings and linerboard; after bleaching the pulp could be used to make white papers. Kraft pulp is more resistant to modem totally chlorine-free (TCF) bleaching processes than sulphite pulps, which are weaker. Kraft pulp is used for newsprint, fine paper and tissues. With sulphite pulp, wood resins may pose problems in paper making by leading to sticky deposits. The yield of bleached chemical pulps is about 50 per cent.
Many parameters are used to evaluate the paper making process. The amount of delignification after pulping and bleaching is monitored by the kappa number, an indirect method measuring the amount of permanganate consumed by lignin. The pulp and paper properties are determined by optical parameters such as brightness and cooler; and physical parameters such as tensile index measured on paper strips using a constant force, and tear index, measured by the energy required to propagate an initial tear through several sheets of paper (Biermann, 1993).
The developments in pulp and paper production during the 1990s have been mainly aimed at reducing environmental impact. While no major improvement has been made to reduce the energy input in mechanical pulping, elemental chlorine-free (ECF) and TCF bleaching processes drastically added to the decrease of toxicity and colour of bleach plant effluents from chemical pulping plants. Unfortunately, the new oxygen-, hydrogen peroxide- or ozone-based bleaching technologies reduce the fibre strength and result in lower brightness levels and a higher lignin content of the bleached pulp. One of the strategies to overcome these problems is extended 'cooking' aimed at extracting more lignin during the cooking process.
In its strict sense, biopulping is defined as the pretreatment of wood chips with selectively delignifying white-rot fungi prior to mechanical or chemical pulping. It takes advantage of the diffusible lignin- and hemicellulose-degrading agents excreted by these fungi during incubation of wood chips to cleave the chemical bonds in the wood tissue which must be broken up in mechanical or chemical pulping. This leads to energy reduction and a lignin content after cooking – this is the same objective as the extended cooking process. Ina broader sense, the term biopulping is also used for any biochemical assistance to the pulping process such as the use of non-wood decay fungi for resin degradation or the use of enzyme preparations in pulp and paper pro production, example for bleaching, resin degradation, dewatering etc. For a clearer definition, the prefix `bio' should be used only for whole fungal processes and 'enzymatic' when isolated enzymes are applied.
This chapter describes the biochemical and ultra structural background of fungal activities in biopulping, the technology, and achievements to date; it then discusses - how biopulping could help to solve the problems mentioned above.
4.2 Fungi Used for Biopulping
When wood is decayed by white-rot fungi in nature, it is mostly bleached and may even fall apart into cellulose fibres, strongly resembling pulp. This effect is exerted by some species of fungi and is caused by the selective degradation of lignin and hemicellulose in the middle lamella and also in the wood cell wall. It seems logical to use these fungi for biotechnological processes, either to improve the forage digestibility or to pretreat wood for pulping or other purposes. In fact, wood, delignified by Ganoderma australe and other microorganisms, is traditionally used as cattle feed in Southern Chile and is called palo podrido (Philippi, 1893). There are no records of the use of naturally delignified wood to produce paper, but early attempts were inspired by naturally white-rotted wood (Lawson and Still, 1957; Reis and Libby, 1960; Kawase, 1962). Further biopulping studies were undertaken with Rigidoporus ulmarius at the Forest Products Laboratory, Madison, WI (Kirk et al., 1990). More detailed studies started with the discovery made by Henningsson et al. (1972) describing Phanerochaete chrysosporium as a thermophilic basidiomycete which caused defibration of wood. Mechanical pulp was produced from wood chips pretreated with P. chrysosporium by Ander and Eriksson (1975) at STFI in Stockholm, Sweden. Mutants of this fungus, reduced in cellulose activity, were produced and the first biopulping patent was filed (Eriksson et al., 1976). Adamski et al. (1987) showed that pretreatment of wood chips with the white-rot fungi Phellinus pini and Stereum hirsutum resulted in a decrease of the refining energy in kraft pulping.
A systematic screening for selective lignin degrading fungi was made by Otjen et al. (1987) and Blanchette et al. (1988) who analyzed the relative amount of the structural components of wood blocks after 3 months cultivation. A new screening method, based on Simons stain, proved to be successful to predict energy savings in mechanical pulping (Blanchette et al., 1992a). As a result of about 200 tested fungal strains, P. chrysosporium was found to be the best biopulping fungus for hard wood and Ceriporiopsis subvermispora for hard and soft wood when mechanical pulp was produced (Akhtar et al., 1992a; Blanchette et al., 1992b). A screening programmed to evaluate the best selective white-rot fungi for pretreating wood chips prior to sulphite pulping also identified C. subvermispora as the best choice with Phlebia tremellosa, Phlebia brevispora and Dichomitus squalens ranking next (Messner et al., 1992).
Early strategies of biopulping were aimed at a high degree of delignification of wood chips. Such type of process would lead to a considerable yield loss and additionally to a decrease in paper strength due to the polysaccharide depolymerizing enzymes also excreted by selectively delignifying fungi. A new concept of biopulping was created when it was found that with C. subvermispora and other selectively delignifying fungi, after a relatively short incubation period of 2 weeks and a weight loss of less than 2 per cent with no visible attack on the wood cell walls a considerably lower kappa number, corresponding to a lower lignin content, can be gained "after' sulphite cooking (Messner et al., 1993; Messner and Srebotnik, 1994). The same also applied to refiner mechanical -where high energy savings are reached after incubation times of 2 weeks` Kirk et al., 1993).
Based on morphological studies of wood, arid also on the basic understanding of the penetration of enzymes into wood cell walls, the high effectiveness of biopulping is hard to understand. As postulated earlier (Cowling and Brown, 1969; Stone et al., 1969), molecules of the molecular mass of ligninolytic enzymes (between approx. 45 and 80 kDa) cannot penetrate into the cell walls until rather late stages of decay due to their high molecular weight, resulting in a high hydrodynamic diameter in relation to a low average pore size of the wood cell wall. This was proved by infiltrating wood cell walls with marker proteins and subsequent immunolabelling by Srebotnik et al. (1988) and Daniel et al. (1989). Recently, Blanchette et al. (1997) indicated that after incubation of pine wood with C. subvermispora for 2 weeks – the time when strong biopulping effects are already evident -- only the very small marker protein insulin (molecular mass 5730 kDa) penetrated the secondary wall in a narrow band around the circumference of the lumen. None of the larger proteins were able to penetrate.
It was shown by differential staining and light microscopy (Srebotnik and Messner, 1994) that after 2 weeks, only the cell walls of parenchyma cells, colonized first by hyphae, are delignified. Total delignification of the cell walls and fibre separation appeared only after 6 weeks cultivation, corresponding to 15-20 per cent weight loss. The delignification process mainly started at the lumen surface and progressed towards the middle lamella.
When birch wood chips were incubated with C. subvermispora, until dissolution of the middle lamella and separation of the fibres, they still appeared more or less undegraded in transmission electron microscopy. Infiltrating these fibres with lignin were detected within the wood cell wall by immunoelectron microscopy (Messner and Srebotnik, 1994). Taking into account that hyphal growth and excretion of enzymes takes place in the lumen, one would assume that they would have penetrated into the middle lamella, leading to dissolution of the latter. In this case the infiltrated enzymes would have had to be able to penetrate into the wood cell wall. As this was not the case, it was concluded that sonic kind of highly diffusible, low molecular weight compound must have been produced by the fungus leading to dissolution of the middle lamella (Messner and Srebotnik, 1994).
As similar results were obtained with Dichomitus squalens, another selectively delignifying white-rot fungus, but not with Trametes versicolor, a simultaneous white-rot fungus, it must be assumed that the selectively delignifying white-rot fungi produce a highly diffusible lignin degrading agent of a molecular mass lower than 5000 kDa. This agent obviously diffuses into the cell wall in the early stages of decay, cleaving bonds in the lignin (or hemicellulose) polymers, thereby facilitating an easier chemical delignification and a decreased energy input in mechanical pulping. Retrospectively, it was fortuitous to select fungi excreting these so far unidentified agents leading to the biopulping effect before delignification at only 2 per cent weight loss.
Due to this new understanding of the biopulping mechanism, the strategy has changed from a long-term process with a high degree of delignification to a relatively short-term process with almost no loss of substrate. This is essential progress as high yield is desirable both in mechanical and chemical pulping. The solubilized lignin received after cooking is used for internal heat and energy generation in chemical pulp mills.
Another approach to biopulping is the use of ascomycetes such as Ophiostorna piliferum. This fungus rapidly colonizes the wood chips but does not alter the wood cell wall. Its main effect is the degradation of extractives after colonizing the resin canals and rupturing the ray parenchyma cells and the distruction of pit membranes (Blanchette et al., 1992c). It is assumed that by the latter activity a more even distribution of the cooking chemicals is brought about, resulting in a more uniform pulp production (Wall, unpublished results).
4.3 Lignolytic Enzyme Systems
The consumption of wood or other highly lignified plant tissues by microorganisms requires the excretion of a lignolytic enzyme system. White-rot fungi can be considered to be the most efficient organisms in this respect. So far, four types of lignin degrading enzymes have been isolated from white-rot fungi.
Lactase: A copper-containing oxidase of a molecular mass between 60 and 80 kDa. It catalyzes four one-electron oxidations of mostly phenolic compounds. Free phenoxy radicals are formed as intermediates. Various compounds such as ABTS (Bourbonnais and Paice, 1992), HBT (Call and Mücke, 1997), or 3-HAA (3-hydroxyanthranilate) (Eggert et al., 1995, 1996) can act as co substrates or mediators. While ABTS and HBT are not produced by fungi, 3-HAA was isolated from the white-rot fungus Pycnoporus cinnabarinus. It was shown that these low molecular weight compounds also enable the fungus to oxidize non-phenolic lignin and to act at a distance from the fungal hypha.
Manganese peroxidase: Like lignin peroxidase, this was discovered in P. chrysosporium and characterized by Kuwahara et al. (1984). It is commonly produced by white-rot fungi. Its molecular weight is similar to that of lignin peroxidase, it contains protoporphyrin IX and catalyzes one-electron oxidations of phenolic and non-phenolic compounds. The primary substrate is Mn(II) which is oxidized to Mn(III) and stabilized by forming complexes with organic acids. The Mn(III)-complex must be diffusible in wood and can depolymerize lignin (Wariishi et al., 1991). A new mechanism involving lipid peroxidation, leading to oxidation of the recalcitrant non-phenolic structures in lignin was found by Moen and Hammel (1994) and Bar et al. (1994). Recent results pointed to the participation of this mechanism in the depolymerization of lignin by C. subvermispora (Srebotnik et al., 1994; Jensen et al., 1996).
Lignin peroxidase has a molar mass of 38 to 43 kD, also contains protoporphyrin IX as a prosthetic group and catalyzes one-electron oxidations of phenolic and non-phenolic compounds generating phenoxy radicals and cation radicals. In the presence of veratryl alcohol, an organic acid, H202 and oxygen, lignin peroxidase was also found to be able to oxidize Mn(II) (Popp et al., 1990). The enzyme was first detected in the culture fluid of P. chrysosporium by Tien and Kirk (1983) and Glenn and Gold (1985) but it is excreted by only a few white-rot fungi.
Other peroxidases: Several other manganese independent peroxidases, also different from lignin peroxidase, have been described in white-rot fungi (Heinzkill and Messner, 1997).
Principally, all lignin degrading enzymes appear to be able to act at a distance from the hypha in the depth of the wood cell wall either via manganese complexes or via other low molecular weight compounds, also called mediators. Other compounds able to penetrate because of their low molecular weight and to oxidize wood components were discovered recently (Enoki et al., 1997; Goodell et al., 1997). The understanding of the underlying mechanism for biopulping is closely related to the discovery of the chemical and biochemical reactions of such low molecular weight compounds.
4.4 The Biopulping Process
Wood is the natural substrate for white-rot fungi; consequently wood chips offer suitable conditions for biopulping fungi but a non-optimized process would be rather slow. It must be speeded up by optimizing parameters such as nutrient supply, quantity and type of inoculum, moisture content of wood chips, and aeration. As all parameters except aeration have to be set at the beginning, scaling-up is of even greater importance for this type of process than for liquid fermentations.
Development of the biopulping process has reached the pilot scale as far as the use of white-rot fungi for mechanical pulping and sulphite pulping is concerned, and has already been tested on a commercial scale with the ascomycete Ophiostoma piliferum for kraft pulping.
4.4.1 Inoculation and Nutrient Supply
Like many other basisiomycetes, C. subvermispora differs from O. piliferum insofar as it only produces clamydospores integrated in the fungal mycelium, but has no conidiospores. From this point of view it might be easier to produce large amounts of fungal inoculum from 0. piliferum. With C. subvermispora the mycelium must be fragmented to increase the number of inoculation points. Nevertheless, clamydospores are resting spores and probably will also guarantee a stable inoculum after drying. Inocula of O. piliferum have already been produced in large scale and this fungus is on the market under the trade name Cartapip 97 and is used to decrease the content of extractives on wood chips. Both types of inocula are produced in liquid fermentation at the same time, but solid substrate fermentation may also be a method for inoculum production, especially for Ceriporiopsis (Majcherczyk et al., 1996).
In a wood chip pile, conditions such as humidity, available nutrients and temperature are highly favorable to fungal growth and many fungi are able to colonize the chips. One of the most ubiquitous fungi is Trichoderina. Some of its species excrete a broad range of compounds, controlling the growth of basidiomycetes (Horvath et al., 1994). In fact, this fungus is commercially used for plant and wood protection (Freitag et al., 1991). As Ceriporiopsis is unable to compete with the indigenous microorganisms on wood, the chips have to be decontaminated prior to inoculation. This can be done either by chemicals such as sodium bisulphate (Akhtar et al., 1995a, b) or by a short atmospheric steaming. A steaming period of only 15 seconds was sufficient to give Ceriporiopsis the competitiveness needed for an even colonization of the wood chips and to cause the desired biopulping effect (Akhtar et al., 1997). O. piliferum is reported to be competitive against the natural wood chip organisms and can be inoculated on contaminated chips. Chen and Schmidt (1996) reported a method to grow P. chrysosporium on unsterilized wood chips. According to Pearce et al. (1995), some wood decay fungi identified after screening more than 200 strains were found to be able to colonize unsterilized wood chips; this led to high energy savings in mechanical pulping.
The amount of inoculum needed for an evenly distributed and dense growth of C. subverispora or other basidiomycetes on the chip surfaces and a dense colonization of the interior of the chips was found to be strongly dependent on the amount of nutrients available to the fungus. When the fungal inoculum was applied to the wood chips suspended in unsterilized corn steep liquor the amount of fungal inoculum could be reduced to 0.25 g/ton (dw/dw) of wood (Akhtar et al., 1996). Corn steep liquor is a cheap, semi-solid by-product of corn milling, and contains mainly protein, lactic acid and sugars. It is used as a feed supplement and also for other fungal fermentations. Experiments with lactose and other sources of organic nitrogen showed comparable results (Akhtar et al., 1997).
4.4.2 Fermentation
Considering the amount of wood chips to be treated in a pulp and paper mill, fermentation processes using rotating fermentors would not be feasible. The only acceptable technology will be a static-bed fermentation type as for example in chip silos or even on wood chip piles. Solid-state fermentations are much harder to control than liquid fermentations. They are considered to be gas—liquid—solid mixtures in which an aqueous phase is intimately associated with solid surfaces and is in contact with a gas phase continuous with the external gas environment (Mudget, 1986).
The moisture content of commercial wood chips varies, but is mostly lower than the optimum range for white-rot fungi which also differs between species. Usually, a broad range around 100 per cent (dew) is acceptable. The only method of controlling process parameters such as oxygen—carbon dioxide exchange or heat transfer is via the gas phase. In experiments carried out in 20-litre laboratory fermentors, aeration rates below 0.01 litre/litre/minute were found to have a negative effect on biopulping with C. subvermispora (Heimel, 1993). Similar thresholds have been reported for P. chrysosporium (Akhtar et al., 1997) and forced aeration was also found to be needed in solid-state fermentations of aspen wood with Phlebia tremellosa (Reid, 1989). In Ceriporiopsis fermentations the temperature built up from room temperature to 31-33°C within 4 days and remained constant to day 14 (Heimel, 1993). Taking into consideration that in wood chip piles temperatures of 45-50°C are usually reached, and even may increase to the incineration point, this is a surprising result. It can be explained by the fact that in biopulping, after steaming, the wood chips are colonized by C. subvermispora as a monoculture, while in wood chip piles thermophilic organisms may succeed mesophilic microorganisms. Obviously C. subvermispora is able to stabilize the temperature at its optimum. The difference in the temperatures reached at aeration rates of 0.001, 0.01: and 0.1 volume/volume/minute (vvm) was only 2°C (Heimel, 1993). It demonstrates that the temperature development in biopulping can hardly be controlled by the aeration rate due to the slow heat transfer from the interior of the wood chip to t e gas phase of the void volume. It appears that only the oxygen—carbon dioxide exchange can be improved by aeration.
According to biomass determinations based on the ergosterol content of the mycelium of C. subvermispora, it was reported that after a period of vigorous growth of 6 days the fungal growth largely decreased until day 14 although the optimum temperature of 33°C was reached in the solid substrate fermentation (Messner et al., 1997). An ergosterol content of 0.7 per cent correlated to a fungal biomass of 5 mg/g wood (dw) (Koller, 1996) (Figure 4.1).
It should be further investigated whether this model based on experiments in 20-litre laboratory fermentors is also applicable to larger volumes. From the experiments it can be concluded that either aerated chip silos or aerated chip piles will be
Figure 4.1 Development of biomass of Ceriporiopsis subvermispora on spruce wood chips supplemented with 2 per cent corn steep liquor/2 per cent glucose; 2 per cent corn steep liquor; and water, calculated from the ergosterol content of the fungal mycelium
needed to create optimum conditions for C. subvermispora or other biopulping Scale-up of the biopulping process is under way at the Forest Products Laboratory, Madison, Wisconsin, and probably also at some pulp and paper companies.
4.5 Biomechanical Pulping
Refiner mechanical pulp (RMP) is produced by disintegrating chips between rotating metal discs at atmospheric pressure. Refining is carried out in two stages: the first is aimed at fibre separation at the middle lamella after prior softening of the chips by steam while the second step alters the fibre surface for improved fibre bonding in the final paper. Power requirements are 1600-18000 kWh/ton. In thermo mechanical pulping (TMP) the refiners are at 110-130°C and elevated pressure in the first stage to promote fibre liberation at the S, cell wall layer, leading to improved fibre bonding compared with RMP. Energy requirements are 1900-2900 kWh/ton, over two-thirds of which is used in the primary pressurized refining step (Biermann, 1993).
It can be concluded from the high values of electrical energy demand that a softening of wood chips by fungal pretreatment can result in great benefits, especially in mechanical pulping. Consequently, most efforts have been invested into this type of application of fungal pretreatment. Fungi which do not lead to a modification of the structure of the wood cell wall, such as 0. pilifreum, may reduce the resin content but do not bring about energy savings (Fischer et al., 1994). Most of the work on biomechanical pulping was done by the Biopulping Consortium at the Forest Products Laboratory, Madison, Wisconsin, and was summarized by Kirk et al. (1993) and Akhtar et al. (1996, 1997). After fungal screening (Otjen et al., 1987; Blanchette et al., 1992a,b) research focused on C. subvermispora and P. chrysosporium on aspen and loblolly pine chips. A US patent was issued on the use of C. subvermispora for biomechanical pulping (Blanchette et al., 1991). Typical results obtained with two selected strains are shown in Table 4.1 (Akhtar et al., 1997).
These results demonstrate that by selecting efficient fungal strains and optimizing the process, energy savings of up to almost 40 per cent can be achieved in RMP even after 2 weeks incubation in laboratory tests. Low strength is one of the weaknesses of mechanical compared with chemical pulp. Pretreatment of wood chips with biopulping fungi clearly increases the strength properties. The tear index of hand sheets made from pretreated aspen chips increased from 1.01 to 3.62 (mN n2g-1) and from 2.18 to 3.36 with loblolly pine (Akhtar et al., 1992b). Similar
Table 4.1 Energy savings and tear index improvement over control during biomechanical pulping of fresh loblolly pine and aspen chips with strains of C. subvermispora
Wood
Strain
Incubation time (Weeks)
Energy (%)
Tear Index (%)
Loblolly pine
Aspen
CZ-3
SS-3
Fp-90031
SS-3
FP-90031
2
2
4
2
4
23
34
42
38
40
19
46
131
0
67
improvements were achieved for Norway spruce and birch, but not for eucaly wood after fungal treatment (Setliff et al., 1990). It can be concluded that a scaled-up industrial process will lead to great benefits for the pulping industry.
Due to chromophore production during fungal pretreatment the brightness adversely affected and decreased compared with control pulp. Unbleached a biopulp reached a brightness level of 51.8 per cent (Elrepho) compared with 62.: cent in the control, and 3 per cent H2O2 bleached biopulp reached 76.0 per compared with 80.0 per cent in the control. Nevertheless, in a two-step bleaching, 78 per cent was reached with the biopulp (Sykes, 1993), demonstrating that bleat should not create a major problem after biopulping.
Another positive effect of biopulping was identified when waste water from first refiner passes of aspen chips, treated with either P. chrysosporiurn or C. vermispora, was analyzed for biological oxygen demand (BOD), chemical ox demand (COD) and Microtox toxicity. The toxicity of the waste water decreased from 17 to 4 (100/EC50, where EC50 is the median effective concentration), due the consumption of extractives by the fungus. BOD (g/kg pulp) decreased from, 36 but COD (g/kg pulp) increased from 74 to 100, due to lignin fragments released as a result of fungal pretreatment (Sykes, 1994).
Wood resins cause a number of serious problems in pulp and paper production by creating sticky deposits. Cleaving the ether bonds of triglycerides either by mercial lipases used on pulp (Fischer and Messner, 1992; Fischer et al., 1993) lipases produced by fungi during chip colonization (Wendler et al., 1991; Farrell al., 1994) was found to reduce the pitch problem. When the capacity to decrease pitch content of loblolly pine chips by C. subvermispora during biopulping was pared with that of the commercial pitch control fungus O. piliferum both fungi the same result – a decrease of approximately 30 per cent – after 4 weeks bation. After 2 weeks C. subvermispora had already degraded 24 per cent pared with 15 per cent for O. piliferum, correlating with a 53 per cent decrease triglycerides (Fischer et al., 1996). These results indicate an additional bone biopulping, namely pitch reduction. While no effect of pretreatment with 0. forum on paper strength was found by Fischer et al. (1994), an increase in strength parameters was detected by Fordo Kohler et al. (1996), after pretreatment with tapip 97, a commercial product from 0. piliferum.
4.6 Biochemical Pulping
Various chemical methods exist to break down the chemical structure of lignir render it soluble in water. Common methods are the sulphite process and the sulphate or alkaline process, which is most extensively used.
4.6.1 Bio-sulphite Pulping
Sulphite processes use mixtures of sulphurous acid and/or its alkali salt~ example, K+, Ca2+, Mg2+) to solubilize lignin through the formation of sulphonate, functionalities and cleavage of lignin bonds. Only a few paper mills are still calcium sulphite. In Europe magnesium-based cooking liquors are widely applied for example in the magnefite process, enabling an efficient chemical and e recovery.
Biopulping in connection with magnefite pulping was investigated using birch and spruce wood chips and five selected strains of fungi (Messner et al., 1992). The chips were sterilized, supplemented with either synthetic or complex media such as corn steep liquor and inoculated with a suspension of blended mycelium. After 2 and 4 weeks of incubation in a 20-litre static bed laboratory bioreactor aerated with humidified air at 0.01 vvm the chips were cooked. The kappa number of unbleached control pulp was approximately 24 and reached 6.3 after laboratory bleaching in a two-step sequence (EOP–P, where EOP represents alkaline oxygen/ peroxide treatment and P a further peroxide bleaching stage). The effect of fungal pretreatment on the pulp properties was monitored by brightness, tear index and tensile index.
Table 4.2 shows that C. subvermispora was the best fungus for magnefite biopulping. On spruce chips 30 per cent kappa reduction was gained with this fungus after 2 weeks incubation time (Messner and Srebotnik, 1994). Contrary to C. subvermispora, P. chrysosporium showed a great selectivity for the wood species as no effect was detected after 2 weeks incubation on spruce chips. Except for P. chrysosporium, all fungi tested, known to be selective lignin degraders, showed good results on birch chips. The paper strength properties of handsheets prepared from birch chips, incubated for 4 weeks and measured as tear index and tensile index after 10 and 20 minutes beating time, were reduced by about 10 per cent (Messner and Srebotnik, 1994). These results indicate that after pretreatment of wood chips with selective lignin degrading basidiomycetes, such as C. subvermispora, about 30 per cent more lignin can be solubilized in magnefite cooking at a rather low impact on the physical properties of the pulp.
When wood chips supplemented with corn steep liquor or other complex media are incubated with C. subvermispora, the chip colour changes to brown after a few days. Unfortunately these chromophoric compounds are not destroyed during cooking, leading to a brightness drop from 62 per cent ISO-brightness to 50 per cent. When this pulp was bleached in an EOP–P bleaching sequence, 4 per cent ISO was still lost compared with the unbleached control of 70 per cent ISO-brightness (Messner et al., 1997). With wood chips supplemented with a synthetic medium the brightness loss was lower and resulted in 1.5 per cent ISO after cooking and 0.8 per cent after bleaching at similar kappa reductions.
Besides optimizing the media or screening for fungal stains that do not decrease the brightness, an approach to avoid the problem of brightness loss is to reduce the cooking time as additional chromophores are created during longer cooking times. Table 4.3 shows that despite a high kappa reduction after magnefite cooking for 315 minutes, no brightness gain was achieved after bleaching due to a more intensive
Table 4.2 Percentage decrease of kappa number of biopulped birch chips after magnefite cooking
Fungus
2 weeks
4 weeks
Ceriporiopsis subvermispora
33
48
Phlebia tremollosa
40
Phlebia brevispora
35
Dichomitus squalens
33
Phanerochaete chrysosporium
12
20
Table 4.3 Kappa reduction and brightness loss/gain of spruce wood chips, supplemented with a synthetic medium, pretreated for 2 weeks with Ceriporiopsis subvermispora and subsequent magnefite cooking at various times and EOP-P bleaching
Cooking time
Kappa
reduction
(%)
Brightness (%ISO)
loss/gain
unbleached
Brightness (%ISO)
loss/gain
bleached
315 min
-37.7
-1.5
-0.8
240 min
0
+2.3
+3.5
chromophore production at the low kappa values. By reducing the cooking time by 75 minutes, the same kappa level (24) as with the untreated control chips is reached after fungal pretreatment caused by the enzymatic modification of the lignin. Fewer chromophores were produced during cooking, leading to a brightness increase of 3.5 per cent ISO (brightness level 75) after a two-step EOP-P laboratory bleaching sequence. By decreasing the cooking time the capacity of the digester could be increased, leading to a higher productivity, still leading to a small brightness increase. Further work is needed to evaluate the reactivity of fungal pretreated magnefite pulp to various bleaching chemicals and sequences. An Austrian patent has been granted on bio-sulphite pulping (Messner et al., 1995).
4.6.2 Sodium- and Calcium-based Sulphite Pulping
The effect of pretreatment of pine chips (Finds taeda) with two strains of Ceriporiopsis subvermispora (CZ-3 and L-14807 SS-3) for 2 weeks on kappa number, Pulp yield, chemical consumption, brightness and colour, as well its on the effluent parameters has been reported (Scott et tit., 1995a, 1996; Akhtar et al., 1997). After sodium bisulphate pulping 27 per cent kappa reduction was reached with both strains at a control value of k 31.2. Calcium-acid sulphite pulped wood chips pretreated with the strain CZ-3 showed 49 per cent kappa reduction compared with 21 per cent kappa reduction for strain SS-3. After sodium sulphite pulping the pulp yield was decreased by 1.7 while after calcium-acid sulphite pulping the yield was comparable with the control. Furthermore, it was found that the consumption of pulping liquor did not increase after calcium-acid sulphite cooking.
Similar bleaching results, as reported above for magnefite pulp, were obtained for calcium-acid sulphite pulp. Due to the fungus induced chromophores the brightness of the unbleached pulp decreased from 54 to 49 per cent brightness. Nevertheless, after 4 per cent hydrogen peroxide bleaching as well as after I per cent FAS (formamidine sulphinic acid) bleaching, the same brightness level (SO per cent) was reached with biopulp. The biopulp appeared to be brighter as a result of a lower amount of the yellow component of the reflected light.
As water discharges from pulp and paper mills have been subjected to dramatic regulatory measures and will still have to be decreased in the future, the effect of biopulping on water parameters was assayed (Scott et al., 1995b). While no change was measured in the BOD and COD content of the liquor effluent compared with the control, toxicity was decreased from 17.4 to 7.2 toxicity units, probably due to fungal degradation of extractives, as was also the case with biomechanical pulping.
4.6.3 Conclusions on Bio-sulphite Pulping
The results obtained so far show that sulphite pulping may profit from pretreatment of wood chips with selectively delignifying fungi by a reduced cooking time and a decrease in kappa of the bleached pulp at the same or slightly increased brightness. However, the main objective of chemical pulping is to decrease the amount of bleach chemicals. Chlorine or chlorine dioxide are substituted by oxygen–hydrogen peroxide- or ozone-bleaching sequences because of their adverse environmental effects, but the new totally chlorine-free bleaching sequences are less specific for lignin and lead to pulps with lower strength properties. An increased extraction of lignin during cooking resulting in a lower kappa number, as caused by fungal pretreatment, would be an ideal prerequisite for reducing the amount of bleaching chemicals, resulting in a lower environmental impact and/or a pulp of higher quality. At present the production of chromophores during the modification of the cell wall components by fungi reduces the positive effects of bio-sulphite pulping. More research is needed to overcome this drawback.
Another advantage of fungal pretreatment for sulphite pulping comes from the associated decrease in resin and other extractives thereby making wood species with higher resin content more acceptable for sulphite pulping.
Interesting results were obtained when C. subvermispora was used in biobleaching of sulphite pulps for dissolving pulp production (Christov et al., 1996). In contrast to all other experiments described in this chapter, pulp instead of wood chips was inoculated with C. subvermispora and incubated for 10 days. Fungal pretreatment of pulp very effectively increased the bleachability of the pulp leading to kappa numbers of around 1.0 compared with 6.7 in the control after an ECF bleaching sequence. Contrary to the results obtained on wood, no adverse effect was observed on brightness. The fungal prebleached pulp reached a brightness level of approximately 80 per cent ISO compared with 56 per cent for the untreated control, but some cellulose degradation occurred. The long incubation time of the pulp and the cellulose loss will probably exclude a technical application for this method, but it is interesting to see that the brightness loss observed with all other biopulping experiments did not occur.
4.6.4 Bio-kraft Pulping
Kraft pulping gives a high strength pulp and is useful for any wood species including high resin wood types. Most of the chemical pulp produced worldwide is kraft pulp. A disadvantage is the difficulty with which the pulp is bleached compared with sulphite pulp (Biermann, 1993). From this point of view a pretreatment with selectively delignifying white-rot fungi to decrease the lignin content after cooking and to render the pulp more accessible for bleaching chemicals would be the ideal combination of methods. Surprisingly, less work has been done on bio-kraft pulping than on bio-sulphite or biomechanical pulping, and has delivered rather inconclusive results. Experiments using P. chrysosporium (Oriaran et al., 1990, 1991; Labor-sky et al., 1991) on glucose supplemented aspen and red oak chips for 20 and 30 days, respectively, led to 3 and 9 per cent kappa reduction compared with untreated chips. Brightness of the handsheets prepared from unbleached pulp decreased dramatically by 54 and 62 per cent. Comparable with that described for bio-sulphite pulping, pulp with a similar kappa number could be produced at a cooking time reduced by one-third. The pulp produced responded better to refining and had higher tensile and burst indices. As a result of a large screening programme on white-rot fungi from South Africa (Bosnian et al., 1993; Wolfaardt et al., 1993) large scale bio-kraft pulping experiments were performed with S. hirsutum, Pycnoporus sanguineus and T. versicolor following a 9-week treatment. The kappa number decreased by up to 17 per cent but was accompanied by a yield loss and an increase of alkali consumption (Wolfaardt et al., 1996).
From the results reported on bio-kraft pulping it can be concluded that the effect of pretreatment with white-rot fungi on kappa reduction is lower than in biosulphite pulping and shows a much higher tendency for colour reduction of the resulting unbleached pulp.
Another approach to biopulping is to use non-wood decay fungi such as the ascomycetes sapstain fungus 0. piliferum. Similar to the white-rot fungi it penetrates the wood via wood rays and the pit pores of the cell walls and is also able to rupture the pit membranes. While the white-rot fungi are able to modify the wood cell walls after colonization, no attack on lignified cell walls takes place with 0. piliferum due to its lack of lignolytic enzymes. An important factor in chemical pulping is a uniform liquor penetration of the wood chips. As the liquor penetrates into the lumina of the fibres via pit pores, a prior fungal disruption or dissolution should improve penetration, leading to improved pulping results.
A 2-week treatment of northern Pine softwood chips with 0. piliferum resulted in a kappa reduction of 9 per cent. Yield, viscosity and strength properties remained constant and the demand of bleach chemicals in the first chlorination step was reduced by 9 per cent at a brightness level of 91.9 per cent (Wall et al., 1994, 1996). On aspen chips treated for 3 weeks, up to 29 per cent kappa reduction was measured. When the bio-kraft pulping effect of 0. piliferum was compared with that of the white-rot fungus Phlebia tremellosa, 5.8 per cent kappa reduction was measured compared with 14.3 per cent (Rocheleau et al., submitted for publication). No brightness loss was created by either of the fungi. The results show that, as with white-rot fungi, improved pulping results can be obtained by increasing penetration of the cooking liquor. Confirmation of this assumption comes from the observation that 0. pilifrum has no influence on the result in mechanical pulping (Fischer et al., 1994).
4.6.5 Bio-organosolv Pulping
Ferran et al. (1996) investigated the use of white-rot decay as a pretreatment for organosolv delignification of Eucalyptus grandis wood and found a threefold ~ P
increase of the delignification rate after 1 month incubation with T. versicolor. Longer incubation times as well as pretreatment with P. chrysosporium did not lead to any further improvements in delignification.
4.7 Biopulping of Non-wood Plants
About 10 per cent of the paper produced worldwide is made from non-wood plants such as cotton, straw, canes, grasses and hemp, and paper making from such sources is increasing. The fibres are mostly cooked with sodium hydroxide at a lower temperature and shorter time than wood pulp due to lower lignin content. Straw pulp is similar to hardwood pulp. Fibres may be used for fine papers but also along with secondary fibres in a mixture containing approximately 25-50 per cent non-wood fibres for corrugating medium. The disadvantages of straw for pulping are its high silica content and low drainage rates (Biermann, 1993).
Reed grass (Phalaris arundinacea) was treated by Hatakka et al. (1996), with selectively delignifying fungi (P. radiata, P. tremellosa, Pleurotus ostreatus and C. subvermispora) for 7 and 14 days and cooked for 15 min in NaOH solution with anthraquinone. The highest loss in lignin after 2 weeks cultivation was caused by C. subvermispora, leading to kappa 19.7 compared with 21.6 for the control. The fines content was reduced from 9.8 to 7.7 per cent. The viscosity was negatively affected, decreasing by 17 per cent. Consequently, the handsheet properties of a pine/grass fine paper also decreased slightly.
Solid substrate fermentation studies on wheat straw, including 14C-labelled lignin, with different selectively delignifying white-rot fungi strongly suggested lignin degradation by manganese peroxidase mediated by Mn(III) (Martinez, 1997).
Atmospheric refining of jute bast was studied after pretreatment with C. subvermispora by Sabharwal et al. (1995). The energy consumption in refining was reduced by 33 per cent for fungal treated jute and burst, tensile and tear strengths were enhanced by 39, 22 and 33 per cent, respectively. Similar results were achieved when kenaf bast was biopulped. Similar to that described for the various kinds of biopulping of wood, the brightness increase after a single stage alkaline peroxide bleaching was much less for biopulp, reaching a brightness level of only 57 per cent compared with 70 per cent of the control (Sabharwal et al., 1996).
4.8 Concluding Remarks
The pretreatment of wood chips or non-wood plants with C. subvermispora as well as with other selectively delignifying white-rot fungi was found to be beneficial for all kinds of pulp production. Nevertheless, the greatest advantages seem to be related to mechanical pulping. High energy savings, an increase in paper strength, reduced resin content and reduced toxicity of the effluent all point to a great future for biopulping. The benefits for chemical pulping are not so clear at the moment. Although substantially lower kappa values are reached after sulphite cooking, it seems to be critical to overcome the brightness loss in bleaching. More research is needed to study the chemistry of chromophore production, induced by the fungal enzyme system, as well as the effect of different bleaching sequences on biopulp. An interesting approach to the problem could be the high bleaching effect of the same fungus when growing on unbleached pulp instead of wood chips and this seems worth further investigation. The chemical reactions taking place in kraft cooking are even less favourable for wood biopulped with basidiomycetes and increase the trend of brightness loss. However, incubation of wood chips with ascomycetes exerting no enzymatic changes of the wood cell wall lead to some benefits in kraft cooking due to a better penetration of the wood chips by the cooking liquor. The importance of biopulping for straw and other non-wood plants will increase with the growing importance of these raw materials.
The technical development of biopulping has made good progress. With O. piliferum, a fully developed industrial process commercially known as CARTAPIP for inoculum production and for inoculation is available. No additional adjustments of the wood chip pile are needed. For C. subvermispora, the fungus with potentially greater benefits, the process has been scaled-up at the Forest Products Laboratory in Madison, Wisconsin, to pilot plant level and results are comparable with results achieved in the laboratory (Akhtar, personal communication). It must be decided whether an aerated wood chip pile will be sufficient or whether a silo-type reactor as already used in Scandinavian countries for chip storage will be needed.
The argument of a long incubation time of 2 weeks is sometimes used against biopulping. If it is considered that wood chips are already stored in piles for at least 2 weeks prior to use and that ample space is available at chip yards, biopulping can be regarded as a more controlled procedure, comparable with the present situation.
Theoretically, a pretreatment process for wood chips based purely on enzymes or other biological low molecular weight catalysts can be imagined, but is not feasible at this time. Although research into the decay mechanisms in the wood cell wall has received an important impetus by focusing on low molecular weight compounds, a full understanding of the biochemical mechanisms of biopulping will still take some time.
