FIELD OF INVENTION The present invention relates to a method and means for treatment of wood chips.

BACKGROUND TO INVENTION It is known that any fibrous raw material, such as wood, straw, bamboo, bagasse, sisal, flax and cotton can be defibered and used in paper manufacture.

Separation of the fibres of such materials is called pulping, regardless of the extent of purification involved in the process.

Apart from mechanical pulping, two chemical pulping processes are known.

These are the alkaline pulping method (Kraft or Soda-AQ process) and the acid pulping method respectively.

Chemical pulping has been the preferred method because chemical pulps have good strength properties and can also have high brightness values with bleaching. In order to obtain higher pulp yields a great amount of research and development was done in recent years on the pretreatment steps of the chemical pulping process. One of these developments is where the wood chips are, for example, impregnated with aqueous solutions containing sodium hydroxide and/or sulphite liquor and then cooked at temperatures of between 120°C and 180°C (US 4 486 267).

An acid pretreatment process with potassium monopersulfate as part of the chemical pulping process is also known (US 4 404 061).

In an Australian Patent specification (AU 158,917) an alkaline process for the production of paper making pulp from tannin containing raw materials is described. According to this process the tannin content of the materials is diminished prior to alkaline digestion. Thereby a black liquor substantially free of undesirable complex organic compounds is secured. This black liquor may be

readily evaporated and burned to destroy organic waste matter and permit recovery of alkali present.

As mentioned above another process involves the pretreatment of wood chips with an alkali followed by a chemical pulping treatment with peroxymonosulphate at a relative low temperature and pressure (US 5 433 825).

Escalating demands in Kraft pulp qualities have focussed attention on a continuous advancement of cooking technology. The so-called Lo-solidsX pulping resulted in the EAPC process (enhanced alkali profile cooking), which is especially designed for softwood pulping when high pulp strengths are required.

The alternating cooking stages, particularly the higher effective alkali (EA) concentration at the end of the cooking, give higher pulp strengths and improved bleachability.

A further process involves the pretreatment of wood chips by inoculating the wood chips with an inoculum of fungi and a nutrient adjuvant. The wood chips are then introduced into a bioreactor and incubated and then pulped. The addition of the nutrient adjuvant dramatically reduces the amount of fungal inoculant needed.

This process also includes a step where the wood chips are steamed to surface sterilize it prior to bio-pulping (US 5 851 351). Some of the objectives of these pretreatment processes are: (a) to obtain a higher pulp yield ; (b) to produce stronger fibres; (c) to extract lignin more efficiently; and (d) to lower the Kappa number of the pulp.

It is very difficult with these processes to achieve all the objectives simultaneously. Usually one or two objectives are achieved, but not without compromising one for the other.

Some of the major disadvantages of these pretreatment processes are that

(a) it is difficult to treat wood chips uniformly with the substance/chemical that is used; (b) it is difficult to penetrate the interior mass of the wood chips; (c) in the case of the pretreatment of wood chips by inoculating the wood chips with an inoculum of fungi and a nutrient adjuvant, the sterilization of the wood chips prior to the fungal pretreatment occurs only on the surface of the wood chips, because no real penetration occurs with the steaming process as described therein; (d) deposit formation in pulp mills occurs.

For several years, much research has been carried out to overcome the disadvantages and inherent constraints associated with chemical pulping.

However, few of these attempts have produced acceptable modifications to the alkaline pulping process. Current aims to improve alkaline pulping, besides, optimizing pulp quality and quality control, are listed below : \'accelerate the delignification rate, \'increase the delignification selectivity, increase pulp yields, improve pulp properties, and eliminate or reduce air pollution in Kraft pulping.

Increasing environmental pressures have made it necessary to investigate methods for reducing the amount of energy, sulphur, and chlorine-containing compounds used during pulping and bleaching. Environmental protection is one of the most important incentives for the development of sulphur-free chemical processes as alternatives to Kraft pulping. Moreover, concerns about the discharge of chlorinated organic compounds (AOX) from the bleach plant, has led to a decrease in the amount of chlorine used. The amount of AOX discharged from the bleach plant is dependent upon the amount of chlorine used, and the consumption of elemental chlorine during bleaching is a function of the residual lignin content, or Kappa number, of pulp. Environmental pressures have thus led

to the design of many technologies aimed at lowering the lignin content of the pulp entering the bleach plant. Lowering the Kappa number of the pulp by increasing the efficiency of the pulping process has led to a reduction in chemicals necessary for pulp bleaching and a concomitant reduction in polluants discharged from the bleach plant.

The residual pulp lignin content can be reduced by extended delignification during pulping, although at the expense of both pulp yield and pulp strength properties.

An important process modification is alkaline pulping with catalytic additives to increase pulp yield during extended delignification. Of these methods, the most spectacular and widely investigated field of additive pulping is alkaline pulping with anthraquinone (AQ) or its derivatives, e. g. Soda-AQ pulping. The benefits of alkaline pulping AQ generally include increased delignification rates, selectivity, as well as reduced alkali charges and improved pulp properties and yields. More specifically, emphasis has been placed on AQ additives in soda pulping to improve the non-sulphur process with regards to pulping rates, and pulp properties and yields. Recently, the introduction of biotechnology, or bio-alkaline pulping, has been investigated to alleviate some of the problems associated with conventional Kraft pulping.

It is also known that a variety of fibrous raw materials, such as wood or annual plants can be used in paper manufacture. Wood is a complex material and consists of a variety of macromolecular compounds of which cellulose, hemicellulose and lignin are the most abundant.

Separation of the fibers of such materials is called pulping, regardless of the extent of purification involved in the process. Alkaline pulping is one chemical pulping method that is used by industry and fiber separation can be achieved by either using sulphur containing compounds (the Kraft process) or without the use of sulphur containing compounds (the Soda-AQ process). The general objective of alkaline pulping is to separate the wood fibers, which consist mainly of cellulose, to a desired level of pureness from the complex matrix in which they are embedded in the native wood chips so that the pulp yield can be maximized.

To achieve this objective the lignin must be removed. Lignin is undesirable

because it protects the cellulose polysaccharides in complexes known as lignocellulose. During fiber separation the reaction sites for the delignification medium will become more accessible due to a partial dissolution of the hemicelluloses.

One of the problems related to alkaline pulping is that the required high pulp yield, without compromising fiber strengths, is very difficult to obtain without the use of expensive and high volumes of chemicals. Therefore bio-pulping has been introduced to assist with the pulping process and several attempts to create bio- pulping systems have been reported.

One of the first bio-pulping processes that was disclosed is where wood chips are inoculated with fungal cultures that are capable of forming lignin-decomposing enzymes without effecting the cellulose. The Pycnoporous sanguineus fungus, or mutants thereof is one of the suggested fungus (US 3 962 033).

In another bio-pulping process wood chips are first treated with sulfite salt, and then incubated under conditions favoring the propagation of white-rot fungus and then it is mechanically pulped. (US 5 460 697).

Another bio-pulping process involves the use of the white-rot fungus Phanerochaete chrysosporium (also known as Sporotrichum pulverulentum), together with a nutrient adjuvant (US 5 750 0005) and in yet another bio-pulping process the fungus specimen Ceriporiopsis subvermispora is used (US 5 055 159).

The major disadvantages of these processes are that: (a) sterilization of the wood chips is not adequate. Conventional sterilization occurs by means of a surface steaming process and does not penetrate the wood chips, leaving unwanted fungi behind; (b) none of the suggested cultures were evaluated on Southern Hemisphere occurring wood species like Pinus patula, Eucalyptus grandis and Accacia meamsii ;

(c) screening processes to evaluate suitable cultures are only applicable for the identification of mono-cultures and no screening process for the identification of co-cultures or a consortium of cultures has yet been disclosed; (d) nutrient adjuvants are needed; (e) it only applies to bio-pulping prior to mechanical pulping processes; and (f) only one culture is used or suggested and in particular to break down only the lignin, effectively ignoring xylanolytic cultures, which are also important in bio-pulping processes.

It is an object of the invention to suggest a method and means for improving wood chip treatment.

SUMMARY OF INVENTION According to the invention, a method of treating wood chips, includes sterilization of wood chips under pressure and heat to obtain sterilized wood chips.

The wood chips may be water-soaked prior to sterilizing.

The wood chips may be water-soaked up to a water content of about 60%.

The wood chips may be steam-sterilized.

The wood chips may be sterilized at a temperature between 110°C and 180°C.

The wood chips may be sterilized at a temperature of about 170°C.

The sterilized wood chips may be cooled down to room temperature prior to further processing.

The sterilized wood chips may be washed with water.

The wood chips may be supplemented with nutrients after sterilization.

The nutrients may be selected from the group consisting of liquid malt extract medium, liquid glucose-nitrogen medium, and liquid molasses medium.

The sterilized wood chips may be subjected to a hot-water extraction to obtain extractives, and relatively extractive-free wood chips.

The wood chips may be selected from the group consisting of Eucalyptus grandis, Acacia mearnsii and Pinus patula.

The relatively extractive-free wood chips may be chemically treated.

The relatively extractive-free wood chips may be pulped.

Also according to the invention, a method of treating wood chips, includes the steps of sterilization of wood chips to obtain sterilized wood chips, and of inoculating the sterilized wood chips with selected fungal test cultures, which constitute a consortium of co-cultures.

The sterilization may be done under pressure and heat.

The method may include the steps of inoculating sterilized wood chips and subjecting the inoculated wood chips to a sulphur free alkaline pulping process (Soda-AQ pulping).

The sterilized wood chips may be cooled to room temperature prior to inoculation with the selected fungal test cultures.

The test cultures may be sub-cultured on malt extract.

The wood chips may be water-soaked prior to sterilization.

The wood chips may be water-soaked up to a water content of about 60 %.

The wood chips may be steam sterilized.

The wood chips may be sterilized at a temperature to between 110°C and 180°C.

The wood chips may be sterilized at a temperature of about 170°C.

The wood chips may be supplemented with nutrients prior to inoculation.

The nutrients may be selected from the group consisting of liquid malt extract medium, liquid glucose medium and liquid molasses medium.

The sterilized wood chips may be subjected to a hot water extraction to obtain extractives and relatively extractive-free wood chips.

The method may include the further step of subjecting the wood chips to an alkaline pulping process.

The wood chips may be inoculated with ligninolytic Pycnoporous sanguineus and xylanolytic Aspergillus flavipes co-cultures in a suitable bioreactor.

The wood chips may be selected from the group consisting of Eucalyptus grandis, Acacia mearnsii and Pinus patula.

The extracted wood chips may be chemically treated.

The extracted wood chips may be pulped.

The wood chips may be inoculated with ligninolytic Pycnoporous sanguineus and xylanolytic Aspergillus flavipes co-cultures in a suitable bioreactor prior to sulphur free alkaline pulping process (Soda-AQ pulping).

The wood chips may be treated by a bio-pulping method prior to conventional alkaline pulping processes.

The bio-pulping may occur by means of a consortium of co-cultures.

The bio-pulping may occur by means of synergism between ligninolytic and xylanolyticco-cultures.

A hot water extraction process of the wood chips may take place prior to the bio- pulping process.

The present invention thus provides a pretreatment method for extractive-rich wood chips which includes hot water extraction prior to chemical pulping of both

hardwood and softwood or other suitable lignocellulosic residues, which will assist in improving the efficiency of a subsequent chemical pulping processes in the following respects: (a) in the case of Pinus patula, Eucalyptus grandis and Acacia meamsii wood chips, to reduce the quantity of active alkali required in conventional chemical pulping ; (b) in the case of Pinus patula, Eucalyptus grandis and Acacia mearnsii wood chips, to produce unbleached chemical pulp with a lower residual lignin content (lower Kappa number); and (c) in the case of Acacia meamsii, to obtain higher pulp yield after alkaline delignification.

The invention also provides a pretreatment process for wood chips to create sterile conditions before fungal inoculation of wood chips for bio-pulping, e. g. with an inoculum of fungi and a nutrient adjuvant occurs.

The hot water extraction of wood chips prior to the fungal inoculation serves two purposes: it firstly provides penetrating sterile conditions of the material to be fungal inoculated ; and secondly it will remove substantial amounts of extractives contained in the wood chips which normally retard fungal growth after inoculation.

A screening can take place to identify synergism of co-cultures.

Successful bio-pulping results in terms of pulp yield increases, Kappa number reduction and lower active alkali consumption are obtained on Eucalyptus grandis, Pinus patula and Acacia meamsii wood chips. Treatment of wood chips with synergistic co-cultures also enhances their fiber strength development, resulting in stronger paper and introduces better bleaching performance.

BRIEF DESCRIPTION OF DRAWINGS The invention will now be described by way of example with reference to the accompanying schematic drawings.

In the drawings there is shown in:

FIGURE 1 a schematic presentation indicating all the steps of the process of wood chip treatment in accordance with the invention; FIGURE 2 a schematic flow diagram of the steps of the process of wood chip treatment in accordance with the invention indicating the pulping process and also showing the integration of bio-pulping into conventional chemical pulping ; FIGURE 3 a pilot plant bioreactor in accordance with the invention for allowing a solid substrate fermentation of wood chips on a laboratory scale ; and FIGURE 4 an L-shape cross-streaking inoculation technique of two fungi.

DETAILED DESCRIPTION OF THE INVENTION The wood chip extraction procedure as set out in Figure 1 involves the following : (a) Filling of any suitable enclosed container with wood chips of hardwood, softwood or any other suitable lignocellulosic residues.

(b) Addition of a suitable substance to the container.

(c) PLC controlled heating between 156 °C and 172 °C of the substance in the container.

(d) Degassing of the container for at least 12 minutes and at a temperature of at least 156 °C.

(e) Final extraction of the substance under a certain pressure, for at least 3 hours and at a temperature of at least 156 °C.

(f) The temperature in the described steps are not to exceed 172 °C at any time.

The extracted wood chips can then be inoculated according to conventional methods. Thereafter the wood chips can then be introduced into a bioreactor and incubated, also according to conventional methods.

Finally the extracted wood chips are delignified according to conventional chemical methods like the Kraft, or Soda-AQ methods.

The method according to the present invention is therefore concerned with the value that is added prior to conventional bio-pulping and chemical pulping processes.

A substantial time period is to be allocated after step (f) above and before conventional chemical pulping resumes during which selective fungal growth can be stimulated. This stimulation can occur in any suitable enclosed container.

It is envisaged that in a preferred arrangement the suitable substance used in the present invention can be ordinary tap water. The substance can, however, also be water with a suitable admixture of any suitable chemical to provide for better pretreatment results.

The suitable enclosed container used in the present invention can be a digester.

In a preferred arrangement the described pretreatment process of wood chips, where the inoculating of wood chips with an inoculum of fungi and a nutrient adjuvant occurs, is used after the hot water extraction invention.

In a preferred arrangement the water extraction of wood chips will result in a reduced deposit formation in pulp mills.

The method of hot water extraction can further be utilized as a pierce sterilization technique before fungal inoculation takes place as indicated in Figure 2. This combined effect of hot water extraction and conventional bio-pulping thereafter will result in further benefits with regards to: (a) lowering the active alkali demand; (b) lowering the residual lignin content; and (c) increasing the pulp yield.

In a preferred arrangement the thickness of wood chips with a high extractive content has a synergistic effect on reject formation during chemical pulping. It is

therefore recommended that pulp makers adjust their chippers to produce thinner chips, when pulping wood with a high extractive content.

The hot water extraction can further be repeated as many times as needed to sterilise the wood chips satisfactory.

The inventive method will be illustrated by way of the following examples: EXAMPLE 1 A laboratory pulping schdule that was applied in the following preferred embodiment.

Screened wood chips of the 4-8 mm thickness range were treated in tap water for three hours, applying a standard laboratory Kraft pulping schedule with regards to temperature and pressure. The applied schedule included the following steps: (a) Filling of digester (15-liter capacity) with 1500 g of screened wood chips; (b) Addition of 10 liter water; (c) PLC controlled heating; (d) Degassing for 12 minutes at 156°C ; (e) Final extraction at 172°C for 2 hours; (f) A temperature of 172°C was not exceeded.

(g) After 3 hours hot water treatment, the chips were removed, separated from the extracted liquid, washed with water and where then delignified, using the Kraft and Soda-AQ-methods ; (h) The pre-pulping extraction steps were carried out for Acacia mearnsii, Eucalyptus grandis, Eucalyptus macarthurii and Pinus patula chips.

Hot water extraction in the case of Acacia mearnsii before Kraft pulping resulted in:

(a) 127.4 % increase in residual active alkali (RAA).

(b) 11.28 % reduction in residual lignin content (Kappa number).

(c) 0.92 % increase in pulp yield after alkaline Kraft delignification.

Hot water extraction in the case of Acacia mearnsii before Soda-AQ pulping resulted in: (a) 111.85% increase in residual active alkali (RAA).

(b) 15.93 % reduction in residual lignin content (Kappa number).

(c) 4.54 % increase in pulp yield after alkaline Kraft delignification.

In the case of Eucalyptus grandis, a hardwood species with a lower extractive content, before Kraft pulping it resulted in: (a) 46.36% increase in residual active alkali (RAA).

(b) 11.01 % reduction in residual lignin content (Kappa number).

(c)-0.24 % increase in pulp yield after alkaline Kraft delignification.

In the case of Eucalyptus grandis, a hardwood species with a lower extractive content, before Soda-AQ pulping it resulted in: (a) 76.93% increase in residual active alkali (RAA).

(b) 11.30 % reduction in residual lignin content (Kappa number).

(c) 0.23 % increase in pulp yield after alkaline Soda/AQ delignification.

In the case of Pinus patula, a softwood species with a lower extractive content, before Kraft pulping it resulted in: (a) 46.59% increase in residual active alkali (RAA).

(b) 13.04 % reduction in residual lignin content (Kappa number).

(c) 3.88 % increase in pulp yield after alkaline Soda/AQ delignification.

In the case of Pinus patula, a softwood species with a lower extractive content, before Soda-AQ pulping it resulted in: (a) 55.76% increase in residual active alkali (RAA).

(b) 10.97 % reduction in residual lignin content (Kappa number).

(c) 7.39% increase in pulp yield after alkaline Soda/AQ delignification.

In the case of Eucalyptus macarthurii, a hardwood species with a high extractive content, it resulted for the 2-4 mm chip thickness fraction in: (a) 2.40% increase in residual active alkali (RAA).

(b) 3.30 % reduction in residual lignin content (Kappa number).

(c) 6.20% increase in pulp yield after alkaline Soda/AQ delignification.

EXAMPLE 2 In yet a further example the beneficial effect of pressurized hot water extraction of both softwood and hardwood chips has been evaluated. The beneficial effect of hot water extraction becomes increasingly better for wood species with higher wood extractive contents.

A number of extractive rich wood species as well as other suitable lignocellulosic residues contain hot water extractable organic substances which suppress selective fungal growth upon inoculation. Removal of such deterrent organic substances by hot water extraction will result in reduced active alkali consumption during chemical delignification and lower amounts of lignin left in the unbleached pulp fiber.

FURTHER EXPERIMENTS A number of further experiments were conducted. These were briefly as follows : Wood Raw Material

Three wood species were evaluated, viz., Eucalyptus grandis, Acacia mearnsii and Pinus patula, and were received as industrially prepared wood chips. Wood chip samples were screened for thickness with a Wennberg chip screen. Accept- sized wood chips were further screened for material which were considered to be detrimental to alkaline pulping, e. g., chips that contained resinous and knotty material, and were therefore removed by hand. It was thought that even small amounts of such material could influence pulping properties in view of the small volume of chips used in the microbomb digesters, and were therefore removed.

Following screening of wood chips, the moisture content of the accept-sized wood chips was adjusted to approximately 55 % by soaking in water for 24 hours.

Wood chips were then placed in plastic bags and stored at-4°C until inoculated with selected fungal test cultures.

Fungal Pre-treatment of wood chips Fungal material for inoculation Six unknown fungal cultures and one c-culture combi-nation were selected and compared against seven well established fungal monocultures.

Bioreactors Suitable bioreactors were made by the conversion of 5 litre steel containers with removable lids. The conversion included the installation of an elevated stainless steel mesh at the bottom of the container, which served as a support for the wood chips. Wood chips, when placed inside the bioreactors, were thus elevated above the bottom of steel containers. This was done to prevent wood chips in the bottom of the container being submerged in liquid nutrients and water. Moreover, it was felt that the elevation, and thus the void space in the bottom of the bioreactor, would improve natural circulation of air in the bioreactor. A further conversion included the placement of an air-exchange flange in the removable lid of the steel container. Holes, 25 mm in diameter, were cut in the removable lids, and cylindrical steel flanges, 15 mm in height, were knitted to the holes. Cotton wool stoppers could thus be replaced inside the cylindrical flanges, which served

as biological filters during air exchange, thus preventing contamination of wood chips during the incubation period.

Sterilization Approximately 1 800g wet chips, or a. 800g oven dry, were then placed inside each bioreactor and the removable lids sealed airtight. Mouths of flanges were plugged with cotton wool stoppers, and covered with aluminium foil caps.

Bioreactors containing wood chips were placed in an autoclave and steam- sterilized at 121°C for 2 hours. Bioreactors were removed from the autoclave at the end of this period and allowed to cool to room temperature before inoculation with selected fungal test cultures, which is described below.

Nutrient supplementation, inoculation and incubation Sterile wood chips were supplemented with nutrients. Test cultures were sub- cultured on malt extract plates, and mycelia aseptically harvested after mycelial lawns had covered the surface of the media in Petri dishes. Biomass, of each respective test culture, was collected as mycelia from the media surface of 5 Petri dishes, homogenized in the nutrient solution to raise the inoculum potential and was used to inoculate the contents of the relevant bioreactor. Bioreactors were then transferred to an incubator, and incubation room for four weeks at 29°C.

Storage of treated wood chips The contents of bioreactors were examined at the end of the four-week period.

Wood chips were removed from the bioreactors and qualitatively assessed for contamination by unwanted micro-organisms. Wood chips from uncontaminated bioreactors were retained, and were washed with water to remove excess biomass before placed in plastic bags and stored in a freezer at-4°C until further use.

Preparation of Extractive-free Wood Chips To investigate the effect of the removal of extractives content of wood chips on the alkaline pulping properties, extractive-free wood chips were prepared by hot

water extraction. Thus, for each respective wood species, a suitable amount of wood chips was subjected to hot water extraction to remove some of the alkali consuming compounds. An equivalent of 1 500g oven dry wood chips was boiled in 10 litres distilled water for 3 hours. Extracted wood chips were removed at the end of this period, washed with clean water, and stored in a freezer at-4°C until further use.

Alkaline Pulping Experiments Wood Chip Preparation In a preliminary investigation to determine the repeatability of small-scale pulping trials using microbomb digesters, it was noted that relative large variations in pulp yield were obtained within samples. It was thought that variations in moisture contents of a given wood chips inventory were responsible for the variation. As relatively small wood chip samples were used in pulping trials, i. e., 80g oven dry, small errors in calculated dry mass charged to micro-digesters led to large errors in the calculated pulp yield, as yield was calculated as the oven dry mass charged to the micro-digesters. To accurately determine the mass of wood chips samples charged to micro-digesters. It was decided to dry wood chips before mass was determined on an electronic balance.

After treated and untreated wood chips were removed from storage in freezer prior to pulping trials, chips were placed in a drying oven at 104°C for 24 hours.

Chips were removed after this period, transferred to plastic bags, and placed in a desiccator at room temperature for 24 hours. Mass of wood chips was then gravimetrically determined on an electronic balance, and 80g samples were weighed. The 80g wood chip samples were transferred to 500ml plastic beakers, and submerged in water for 24 hours to adjust to the moisture content to approximately 55%. This procedure was followed to displace air enterained in wood chips with water, as air pockets in wood are known to deter penetration of cooking liquor resulting in uneven cooking. After soaking chips in water, the mass\'of samples were again determined to calculate the amount of water absorbed by wood chips. Therefore the amount of water to be added to adjust the liquor to wood ratio during cooking was accurately calculated.

Alkaline Pulping Procedures All wood chips samples selected for pulping trials were pulped in precision stainless steel micro-digesters, each with a volume of 500ml. It was decided to use small-scale micro-digesters in the comparative alkaline pulping evaluations for the following reasons: in view of the large number of pulping trials required for this study, the use of micro-digesters allowed multiple pulping trials to be conducted simultaneously during a single cooking cycle inside a 15 dm3 laboratory- type digester, and of equal importance is that this arrangement allowed two different pulping processes, i. e., Kraft and Soda AQ pulping, to be conducted simultaneously at exactly the same pulping conditions. Therefore half the micro-digesters of untreated and treated wood chips could be allocated for Kraft pulping, and the other half to Soda AQ pulping trials.

For each cooking cycle, eight micro-digesters were charged with the equivalent of 80g oven dry wood chips samples. Therefore four micro-digesters were allocated for Kraft pulping, and were charged with differently treated wood chips samples of the same wood species. This arrangement was duplicated for the four micro- digesters allocated to Soda AQ pulping. Each fungal pre-treated wood chips sample was pulped in triplicate for the two respective pulping processes.

Wood chips were thus placed in the micro-digesters, cooking liquor added, and digesters sealed with stainless steel screw caps. The eight micro-digesters were then placed inside the 15dm3 batch-type laboratory digester. Water was added to the digester until all eight micro-digesters were sub-merged, the pressure lid of the digester was sealed, and the cooking cycle initiated.

At the end of each cooking cycle, the digester was allowed to blow-off for 30 minutes until atmospheric pressure was reached inside the digester before pressure lid was removed. Micro-digesters were removed and immediately transferred to a cold water bath for a further 30 minutes to allow to cool to room

temperature. Micro-digesters were then opened, and black liquor decanted into sample bottles for analyses. Cooked wood chips from each micro-digester were quantitiatively removed and washed through a 12-mesh screen onto a 150-mesh screen. Matter that remained on the 12-mesh screen was considered as pulp rejects, while pulp on the 150-mesh screen was regarded as accept pulp.

Analysis of Pulping Properties The effect of the fungal pre-treatment of wood chips for the three wood species on the respective Kraft and Soda AQ pulping properties were experimentally determined as described below. Pulp yield was calculated as the total pulp yield, and thus included screened rejects, and as screened pulp yield. Extent of delignification of pulp was determined as the residual lignin in pulp, or Kappa number, according to TAPPI Standard Test Method No. T236om-85 (1978).

Chemical consumption of cooking liquor was determined as the residual active alkali (RAA) of the black liquor, according to TAPPI Standard Test Method No.

T625 85-om (1978).

Pulping Selection Factor In order to facilitate the screening of test cultures based on the results of the above analyses for use in larger scaled bio-pulping trials, it was decided to combine the three parameters that defined pulping properties, viz., pulp yield, Kappa number and RAA value, as a Selection Factor and thus combine these properties in a single ratio as a rapid screening technique to select fungal treatments that were thought to suitably enhance these pulping properties.

The increase in pulp yield was considered the most important criteria in the determination of the Selection Factor. The reduction in residual lignin content, and reduction in chemical consumption was considered to be of secondary financial importance. It is proposed that the pulp yield would contribute 70% to the total importance, while Kappa No. and RAA values contributed 15% each.

The equation was expressed as a Pulping Selection Factor (PSF), and was calculated from:

where: PY % Increase in pulp yield Kn = % Reduction in Kappa No.

RAA % Increase in Residual Active Alkali It was felt that such a factor would : simplify screening of suitable cultures with desired effects on alkaline pulping properties of wood chips treated with these cultures, would express the different contributions as weighted factors into a single factor, and be directly comparable with the magnitude of the desired property of the pulp product, i. e., high pulp yield and low residual lignin contents while reducing chemical consumption during pulping.

Although it could be argued that more definitive statistical procedures, e. g., multiple regression techniques, could have been followed rather than the robust PSF technique, it was felt that the primary aim of the PSF was to rapidly screen the desired fungal pre-treatments from numerous other pre-treatments. The PSF equation was specifically designed to firstly highlight the financially important contribution of increases in pulp yield. Therefore pulp yield was chosen as the numerator. Secondly, the combined fractional changes in Kappa number and RAA were chosen as the denominator. By using this arrangement, reductions in Kappa number and increases in RAA values would have the overall effect of increasing the PSF, thus indicating a positive effect on the overall desired pulping properties.

Results and Discussion The optimum growth temperatures of the two most promising fungal co-cultures X9 (Pycnoporis sanguineus) and Y1 (Aspergillus flavipes) were experimentally determined.

Pulping Selection Factors (PSF) Pulping Selection Factors of Treated Eucalyptus Wood Chips The Pulping Selection Factors calculated for Kraft pulping of Eucalyptus wood chips are listed in Table 1, and of Soda-AQ pulping in Table 2. Included in the tables are the relative increases in pulp yields, the reductions in Kappa numbers, and the increases in RAA\'s to indicate the relative contributions of these properties to the PSF. Table 1 : Kraft Pu1 in Selection Factors S of treated Eucal tus wood chi s, % Increase. % Reduction % Increase MF Culture Yield K2pps RAA 1 CONTROL\'- 2 P. chUysosporium (28°C) 2.38 5.05-11. 39 9 3 P. chrysosporium (39°C) 3.80 12 4 C. subvermispora 4. 75 5.13 8. 23 12 5 C. versicolor 5. 70 8.94-5. 38 1 4. 75 4. 21 8. 39 12 7 P. eryngii 1.66 3.89 12.66 4 8 L. edodes 0. 48 0. 64 X9 4. 04 6. 08 20.09 11 10 Z6 4. 04 2. 46 16.14 10 11 Z7-1. 90-2. 03-17. 09-4 -6. 49 6 13 Z9-3.09-0.83-9. 34-7 14 Z10 4. 99 5.72 21.20 1z 15 Yl-X9 co-culture 8. 08 9.77 32.44 24. 16 ao-extracted wood-0.24 11.01 46.36-1 Table 2 : Soda A Pul in Selection Factors S of treated tus. is # Culture @/o Incresse % Reducdon % Increase PSF # Culture % ZMr

Table 3: Kraft Pulping Selection Factors (PSF) of treated Wattle wood chips # Culture % Increase % Reduction % Increase PSF Yield Kappa # RAA I CONTROL--- 2 P. chrysosporium (28°C) 0.46 4.54 2.14 1 3 P. chrysosporium (39°C) 0.92 5.11 11.39 2 4 C. subvermispora 4.85 7.87 66. 55 18 5 C. versicolor 0. 23 5. 37-5. 69 1 6 P. radiata 1. 85 2.27 8.54 7 P. eryngii 4. 62 8.63 61. 21 17 8 L. edodes 0. 69 1.66-0.36 2 9 X9 3. 46 6. 32-2. 14 8 10 Z6 4. 62 5.68 32.74 13 11 Z7-0.46-0. 45-23. 49-1 12 Z8 4.16 5.33 61.92 15 13 Z9-1. 15-2.61-21.71-2 14 Z10 3. 23 2.50 64.06 11 15 Yl-X9 co-culture 6. 47 12. 86 30 16 HO-extracted wood 0. 92 11.28 127. 40 7 Table 4: Soda-AQ Pulping Selection Factors (PSF) of treated Wattle wood chips # Culture % Increase %Reduction % Increase PSF Yield Kappa # RAA 1 CONTROL - - - - 2 P. chrysosporium (28°C) 3.85 8.07 2.89 10 3 P. chrysosporium (39°C) 4.54 8. 74 10.69 12 4 C. subvermispora 7.94 12. 15 54. 34 28 5 C. versicolor 0. 23 13.58-7.23 1 6 P. radiata 5. 22 7. 27 8. 67 13 7 P. eryngii 5. 44 5. 30-4.91 8 L. edodes 5. 90 1. 89-2.31 14 9 X9 4. 99 9. 84 2.60 12 10 Z6 6. 80 7. 15 18. 21 18 11 Z7 -1. 81-5.84-16.18-4 12 Z8 7. 71 9. 04 61.85 28 13 Z9-1.81-5.97-26.88-4 14 Z10 0.45 2. 94 47.69 1 15 Y1-X9 co-culture 9.52 16. 14 81.50 43 16 H2O-extracted wood 4. 54 15. 93 111.85 29 Pulping Selection Factors of Treated Pine wood Chips The Pulping Selection Factors calculated for Kraft pulping of Pine wood chips are listed in Table 5 and for Soda-AQ pulping in Table 6.

Table 5: Kraft Pulping Selection Factors (PSF) of treated Pine wood chips # Culture % Increase % Reduction % Increase Held Kappa # RAA 1CONTROL. 2 P. chrysosporium (28°C) 0, 52-3,34-1, 39 3 P. chrysosporium (390C) 4,13 3,28 12,90 4 C. subvermispora 5,17 3,89 6, 40 10 5 C. versicolor 0,52 -7, 00-18,44 1 6 P. radiata 4,39 4,81 45,10 7 P. eryngii 0, 26 0,75-30,28 1 8 L. edodes 0, 00-0,44 15. 57 0 9 X9 1, 03-0, 17 1,92 2 10 Z6-0,52-8,74-21,75-1 11 Z7 2,07 5,63 41,58 12 Z8-2,07-5,77-41. 47 13 Z9 5, 43 6,66-4,69 13 14 Z10-6,72 0,51 8. 21-16 15 Y1-X9 co-culture 6, 20 7,13 23, 03 16 ELOextracted wood 3,88 13,04 46,59 13 Table 6. Soda A Prrl in Selection Factors S of treated Pine wood chips # Culture % Increase % Reduch\'on YO Increase PSF ) CultureTacrM % jRcJcM % c/- F Yield Kappa RAA 1 CONTROL 2 P. ch7ysosporium (280C)-3. 20 0.17 2.10-8 3 P. chrysosporium (390C) 3.20 0.59 16.18 8 4 C. subvermispora 1.23 4.98-23.40 3 5 C. versicolor-2. 96 2.04-1.86-7 6 P. radiata-1. 23 5.47 29. 22-3 7 P ; eryngii-2. 71 2.80-5.36-6 8 L. edodes-2. 71 0.48 7.33-7 9 X9-2. 96-1.25 3.14-7 10 Z6-6. 16-6.71-1.05-14 11 Z7 3. 69 4.12 57. 04 12 12 Z8-7.64-1.49-19.21-16 13 Z9 2. 96 7. 09 6.52 7 14 Z10-10. 59-0.10 10.01-26 15 Y1-X9 co-culture 5.17 6.47 32.48 15 16 H, 0-extracted wood 7.39 10.97 55. 76 26

For the different fungi used, variations in the proportions of cellulose, hemicellulose and lignin, provided important insight into the alkaline pulping properties of wood chips treated by these cultures. Moreover, it was thought that other physical and chemical changes brought about in the wood matrix, e. g. increased porosity and cell wall thinning, also contributed to changing the pulping properties of wood. In the above search for cultures that (i) extended delignification, (ii) increased pulp yield and (iii) reduced chemical consumption, it was found that pre-treatment of wood chips with the synergistic liginolytic and xylanolytic co-culture Y1-X9 effected the greatest increase in all three of the above pulping properties.

It was also found that the hardwood species, viz., Eucalyptus grandis and Acacia mearnsii, were more amenable to fungal pre-treatments than the softwood species, viz., Pinus patula. Moreover, it was established that Soda-AQ pulping benefited more from the fungal pre-treatment of wood chips than did Kraft pulping and more importantly, c-culture Y1-X9 synergistically improved Soda-AQ pulping, since the greatest benefits in pulping properties were obtained for this combination. This result was particularly important, as the alkaline pulping process was thus enhanced without the use of sulphur compounds.

The higher Kraft pulp yield after fungal pre-treatment was attributed to the higher cellulose content of wood chips charged to the digester. In addition, the wood chips also contained less lignin, and more importantly, less hemicelluloses.

Removal of hemicelluloses was important as retention of the hemicelluloses content has been shown to be negatively correlated with (i) pulp yield, and (ii) rate of delignification. As a result of the fungal colonization of wood chips, it was thought that enhanced penetration of alkali allowed a higher pulp yield as (i) lower screened rejects were obtained, and (ii) more homogeneous cooking occurred, thus less over-and undercooking of chips resulted. Pulp yield of Soda-AQ pulping was improved by the suppression of the peeling reaction by AQ. It was thought that penetration of AQ was enhanced as a result of the fungal pre- treatment which left the wood matrix more porous.

Hemicelluloses removal was also thought to improve delignification, as (i) the pore sizes in the wood cell walls increased, and (ii) the enhanced topochemical effect both facilitated ligning removal. The lower Kappa number was also explained by lower reprecipitation of lignin onto cellulose fibres as a result of lower deposition of xylan onto fibres. Moreover, the extended delignification was attributed to the ligninolytic attack on wood, whereby the lignin content was thus reduced, and the residual lignin was left more fragmented and also more soluble.

It was thought that the similar reactions that occur during lignin bio-degradation and during alkaline delignification, complemented each other and thus contributed to higher overall delignification rates. Extended delignification was also attributed to enhanced penetration of alkali owing to physical changes brought about by fungal colonization, i. e., (i) the wood matrix was rendered more porous, (ii) cell walls were thinner, and (iii) the wood matrix swelled. Finally, delignification was thought to be hampered by higher guaiacyl lignin content, particularly in the case of Pine, as it is known that softwoods contain more guaiacyl lignin than syringyl ligning. Delignification during Soda-AQ pulping has been shown to be proportional to the cellulose contents of wood and the enhanced delignification of fungal treated wood was thus partly attributed to the higher cellulose contents of wood charged to the digester. Therefore more AQ was also available for delignification due to the degradation of extractives, which would have otherwise consumed alkali. As a result of the ligninolytic attach of wood, lignin was left more fragmented, and thus more reactive sites were available for the AQ-AHQ redox reactions, thus further increasing the delignification rate.

The lower chemical consumption during Kraft and Soda-AQ pulping was attributed to less alkali consumed by lignin, hemicelluloses and extractives due to the reduction of these components effect by the fungal pre-treatment. Moreover, the presence of excess alkali during bulk delignification has been shown to be critical and this in part explained the extended delignification. The lower chemical consumption during Soda-AQ pulping was in part explained by the (i) high cellulose contents, and (ii) the low and fragmented lignin contents of wood

charged to the digester bombs, as it is known that these increase the AQ-AHQ redox reactions during pulping.

The summary above explains why the synergistic ligninolytic and xylanolytic co- culture improved on previous efforts of bio-alkaline pulping. The results obtained for alkaline pulping of wood chips treated with reference cultures that has been used in similar work reported in the literature, confirmed that co-culture Y1-X9 was superior in enhancing Kraft and Soda-AQ pulping. Similarly, other test cultures also significantly improved the status of bio-alkaline pulping.

It is noteworthy that hot water extraction of wood chips prior to pulping also had a beneficial effect on the Pulping Selection Factor, particularly in the case of Pine and Wattle chips. From Table 3 it is seen that a PSF of 7 was calculated from Kraft pulping of water-extracted Wattle chips, while a PSF of 29 (Table 4) was calculated for Soda-AQ pulping of such treated wood chips. Similarly, a PSF value of 26 was obtained for Soda-AQ pulping of Pine chills (Table 6) that were treated by water extraction prior to pulping. Interestingly, Eucalyptus wood chips subjected to water extraction did not benefit significantly compared to Pine and Wattle chips. It was thought that Pine and Wattle wood chips therefore contained more extractives that were easier to remove during such a hot water extraction stage.

The positive influence of water extraction on the calculated PSF values were mainly owing to the effect on the extent of delignification and on chemical consumption during pulping. These aspects are clearly illustrated in Tables 5 and 6, as water extraction resulted in a reduction in Kappa number of 13,04 % for Kraft pulping of Pine chips and a reduction of 10,97 % by Soda-AQ pulping.

Similarly, the RAA value increased by 46,59 % for Kraft pulping and by 55,76 % in the case of Soda-AQ pulping of Pine chips.

Although these results indicate that water extraction of wood chips prior to pulping could significantly enhance alkaline pulping properties, it should be noted that the primary aim of this work was to investigate means of enhancing alkaline pulping by suitable fungal pre-treatments of wood chips. The effect of hot water extraction prior to pulping was included in this work to predict the effect of

extractives removal by fungal cultures on these pulping properties. In view of the positive results obtained however, it is proposed that future work should investigate the financial benefits of water extraction prior to bio-alkaline pulping.

From the work discussed and summarized here it was concluded that: 1. All test cultures were confirmed as mesophillic furigal cultures, as the optimum growth rates recorded were between 28-30°C.

2. Molasses at a minimum concentration of 0,5% (5g/k) enhanced the formation of fungal biomass compared with standard laboratory liquid nutrient media. As higher concentrations of molasses did not increase the formation of fungal biomass, a concentration of 0,5 % was regarded as the optimum concentration for inoculum production. Moreover, as molasses is an inexpensive byproduct of the sugar industry, it was felt that costs could be considerably lowered to prepare fungal inocula.

3. At a concentration of 0,5 % molasses, it was calculated that urea should be added at a concentration of 0,28 g/k to provide the desired carbon to nitrogen ratio of 20: 1 on wood chips.

4. Chemical sterilization of wood chips, by sodium sulphite or boric acid solutions, proved unsuccessful as growth of four common contaminants persisted while growth of the desired test cultures was inhibited at a given concentration of these compounds. Sterilisiation by pressurized steam in an autoclave however was confirmed as a suitable technique to ensure homogeneous growth of the desired fungal cultures at bench-scale.

5. The calculated 0,5 g/ton (dry mass) inoculum, applied with the formulated nutrient supplement solution, resulted in homogeneous fungal growth on wood chips. It was therefore concluded that this quantity of fungal biomass provided adequate inoculum potential on condition that asepsis was practiced during inoculation.

6. Co-culture Y1-X9 was the most suitable fungal pre-treatment to enhance alkaline pulping.

7. The Pulping Selection Factor (PSF) was a suitable equation to screen fungal pre-treatments for their ability to improve alkaline properties and moreover, results obtained using this equation correlated closely with the Final Selection Factor (FSF) to screen test cultures for their ligninolytic and hemicellulolytic potential.

8. P. chrysosporium should definitely be included in further pulp tests as a reference culture because it performed well in non-sterile conditions.

SODA A. Q.-PULPING Material and methods (Control) Soda A. Q. pulping of the tree wood was done in a 15-litre batch type laboratory digester. The equivalent mass of wood chips was used for each digestion. A programmable logic controller (PLC) controlled the digester, thus a certain digestion schdule was followed during the digestion of the chips. The wood chips were screened prior to pulping and soaked in water until a moisture content of 60% was obtained.

Pulping conditions In Hardwood pulping 14% Sodium Hydroxide, 0.1% anthraquinone to 1500g oven dry wood chips was used. This was made into a solution with water using a ratio of 4.5: 1 liquid to solid ratio. The initial pulping temperature was 50°C, after 90 minutes the temperature was about 155°C and blow off was initiated until a temperature of about 135°C was obtained. The digester then heated the chips to a temperature of 170°C, which is the digesting temperature of the digester, this temperature was maintained for 25 to 30 minutes. After this the digester blow off was initiated and a final temperature of 100°C was obtained. After cooking cycle was complete the chips were removed from the digester and black liquor was collected for chemical analysis. The cooked chips were blown to pulp using a high-pressure nozzle on a hose pipe through a 10-mesh steel screen and pulp was collected on a 150-mesh screen. The graph of the pulping cycle is given below.

Pulping of Hardwood Graph 1: Temperature vs. Time graph of a Hardwood cooking cycle

In Softwood pulping 14% Sodium Hydroxide, 0.1% anthraquinone to 1500g oven dry wood chips was used. This was made into a solution with water using a ratio of 5.4: 1 liquid to solid ratio. The initial pulping temperature was 50°C, after 90 minutes the temperature was about 155°C and blow off was initiated until a temperature of about 135°C was obtained. The digester then heated the chips to a temperature of 170°C, which is the digesting temperature of the digester, this temperature was maintained for 130 minutes. After this the digester blow of was initiated and a final temperature of 100°C was obtained. After cooking cycle was complete the chips were removed from the digester and black liquor was collected for chemical analysis. The cooked chips were blown to pulp using a high-pressure nozzle on a hose pipe through a 10-mesh steel screen and pulp was collected on a 150-mesh screen. The graph of the pulping cycle is given below.

Pulping of Softwood Graph 2: Temperature vs. Time graph of a Softwood cooking cycle

Pulping t.

Three different wood species were used in the pulping procedure, they were Pinus Patula, eucalypt Grandis and wattle. The wood chips of these three species were first soaked in water until they had a moisture content of 60%. This was done so that chemical for the pulping could easily be absorbed into the chips by diffusion as it was observed that it was very difficult to pulp dry wood chips.

The wood chips were pulped in triplicate and the average of all the data obtained was taken. Pulp yield, percentage rejects, shive content, chemical consumption, Kappa number, pulp response to beating and freeness of pulp was obtained and recorded using TAPPI standard methods.

Pulp yield The pulp yield was measured as the amount of pulp that was obtained during the pulping of each species. It was given as the mean percentage of three pulping cycles. The following table 7 shows the results of the pulping trials in relation to pulp yield.

Table 7: Percentage rejects Wattle 39.9% Eucalypt Grandis 47.2% Pinus Patula 38.1 % Rejects from the pulping cycle were collected and dried for 24 hours in an oven at a temperature of 105°C. The dry rejects were placed in a dessicator for 24 hours before being weighed and percentage rejects calculated from the total mass of the dry chips. The following table 8 shows the results of the pulping trials in relation to percentage rejects: Table 8: Chemical consumption Wattle 10.9% Eucalypt Grandis 1.7% Pinus Patula 12.6%

The chemical consumption of the cooking cycle was determined by calculation of the residual active alkali present in the black liquor as per TAPPI Standard Test Method no T625 om-85. The table 9 below shows results obtained: Table 9: Shive content Wattle 1.7 Eucalypt Grandis 2.89 Pinus Patula 2.96 Kappa number Wattle 2.15% Eucalypt Grandis 1.89% Pinus Patula 5. 13%

The Kappa number is the amount of lignin left in the pulp after the pulping cycle.

The Kappa number is determined using the TAPPI Standard Test Method no cm- 85. The results are found in the following table 10: Table 10: Water extraction Waffle 22.4 Eucalypt Grandis 23.2 Pinus Patula 39.5

Wood chips of the three species were placed in the digester and water was added without the presence of chemicals. The chips were cooked using the pulping cycle for hardwoods. These chips were rinsed in hot water to remove any excess extracts on them. The wood chips were pulped in triplicate and the average of all the data obtained was taken. Pulp yield, percentage rejects, shive content, chemical consumption, Kappa number, pulp response to beating and freeness of pulp was obtained and recorded using TAPPI standard methods. The results were evaluated in the same way as that of the. wood chips that were not extracted and can be seen below : Table 11 : Pulp Yield Wattle 44% EucalyptGrandis\'53% Pinus Patula 40% Table 12: Percentage rejects Wattle 0.8% Eucalypt Grandis 0% Pinus Patula 3% Table 13: Chemical consumption

Wattle 3.5% Eucalypt Grandis 4.7% PinusPatula 5% Table 14: Shive content Wattle 0.32% Eucalypt Grandis 0. 09% Pinus Patula 3.37% Table 15: Kappa number Wattle 8 Eucalypt Grandis 8. 3 Pinus Patula 15. 4

Beating (Hardwoods) Beating of the pulp was done by taking about 800g dry mass of pulp and adding 20 litres of water to it. The pulp was mixed well before adding to a Voith overhead beater. The pulp was beaten for 1 minute per beating cycle. Results obtained are given in the table 16 below : Table 16: EUCALYPT GRANDIS TIME °SR Omin 20 1 min 33 2min 42 3min 51 Table 17: WATTLE TIME °SR Omin 23 1 min 37 2min 53 3min 75 Table 18 : PINUS PATULA For the Softwoods beating was done for 5 minutes per interval.

TIME °SR Omin 13 5min 19 10min 25 15min 33 20min 44 25min 53 The readings for the water extracted wood chips are seen in the following tables : Table 19: EUCALYPT GRANDIS TIME\'SUR Omin 27 1 min 33 2min 41 3min 47 Table 20: WATTLE TIME °SR Omin 24 1 min 39 2min 46 3min 60 Table 21: PINUS PATULA For the Softwood beating was done for 5 minutes per interval.

TIME °SR Omin 15 5min 23 10min 32 15min 41 20min 45 25min 56 STRENGTHTESTING For the strength tests, hand sheets were made and these hand sheets were evaluated for various strength properties. The hand sheets were cut and conditioned for 48 hours at 55% humidity and 21 OC. The paper was then tested for tensile, burst and tear strength using the TAPPI standards T404 om-87, T403 om 91 and T414 om 88. The results of these tests follow in the following tables :

Table 22: EUCALYPTUS Beating °SR Grammage Breaking Tear Index Burst Time Length Index (min) (degrees) (g/m2) (km) (mN. m2/g) (kPa. m2/g) 0 20 91.3 4.498 5.67 1.39 1 33 101.7 7.00 9.18 3.48 2 42 108.8 9.57 9.23 3.81 3 51 103.7 12.07 10.24 5.75 Table 23: WATTLE Beating °SR Grammage Breaking Tear Index Burst Time Length Index (min) (degrees) (g/m2) (km) (mN. m2/g) (kPa. m2/g) 0 23 83.56 4.947 4.32 1.22 1 37 71.72 6.219 5.21 1.86 2 53 83. 74 7.113 5.99 2.72 3 75 78. 52 12.07 8.27 5.53 Table 24: PINUS PATULA Beating °SR Grammage Breaking Tear Index Burst Time Length Index (min) (degrees) (g/m2) (km) (mN/m2/g) (kPa. m2/g) 0 13 90.5 4.51 11.44 1.88 5 19 103 9.77 20.31 4.96 10 25 79.9 12.42 21.60 5.89 15 33 98.1 12.76 21.85 6.93 20 44 102 13.53 20.51 8.04 25 53 100 18. 93 24. 05 8. 40

The readings for the water extracted wood chips are seen in the following tables : Table 25: EUCALYPTUS Beating °SR Grammage Breaking Tear Index Burst Time Length Index (min) (degrees) (g/m2) (km) (mN.m2/g) (kPa. m2/g) 0 27 77.67 3.585 4.68 1.13 1 33 78.4 4.632 6.10 1.71 2 41 83.2 6.359 6.34 2.66 3 47 92.9 8.38 6.78 3.34 Table 26: WATTLE Beating °SR Grammage Breaking Tear Index Burst Time Length Index (min) (degrees) (g/m2) (km) (m N. m2/g) (kPa. m2/g) 0 24 99.8 1.75 2.45 0.48 1 39 89.6 4.132 5.13 1.68 2 46 98.5 6.33 5.62 2.17 3 60 100.3 6.46 5.47 2.41 Table 27: PINUS PATULA Beating °SR Grammage Breaking Tear index Burst Time Length Index (min) (degrees) (9lm2) (km) (m N. m2/g) (kPa. m2/g) 0 15 75.23 6.24 16.68 3.05 5 23 81.8 9.77 19.82 5.22 10 32 79.8 12.43 20.32 5.40 15 41 89.5 12.76 20.45 5.78 20 45 79.96 17.26 18.96 6.75 25 56 85.3 18.93 23.3 6.92

To improve on current bio-pulping achievements to date, the use of a consortium of fungal cultures to pretreat wood chips is disclosed. The invention involves the isolation and screening of suitable ligninolytic and hemicellulolytic mono-cultures and to then combine these cultures as co-cultures. In previous investigations using conventional microbiology techniques, most fungal interactions have been shown to be\'antagonistic. One of the most important findings\'of the invention is the disclosure of synergism between a ligninolytic (Pycnoporous sanguineus) and xylanolytic (Aspergillus flavipes) co-culture using a novel screening technique/method in vitro. For the selection of suitable fungal pre-treatments for bio-pulping studies based on their ability to delignify wood chips, a unique index (Final Selection Factor) was formulated based on the relative ratios of wood components. The invention furthermore discloses that a high Final Selection Factor calculated for the suitable fungal pre-treatments also gives a good correlation with improved alkaline pulping properties of such treated wood chips.

The pulping properties investigated were the (a) pulp yield ; (b) extent of delignification (Kappa number); and (c) extent of consumption of cooking chemicals.

Wood chips treated with co-cultures of Pycnoporous sanguineus and Aspergillus flavipes showed the highest response to alkaline pulping properties.

The invention also shows that fungal pretreatment of wood chips with co-cultures shows a higher pulp yield, and a lower residual lignin content of pulp, or Kappa number. The resultant unbleached pulp produced from the fungal co-culture treatment also shows improved beating response and produces a stronger paper as well.

It is foreseen that this fungal pretreatment process of wood chips can also be practiced du ring transport in the holds of freight ships. The major costs associated with this process would be: the conversion of holds of freight ships into suitable packed-bed bioreactors, and the control system to provide optimum

conditions for growth of desired fungal cultures. This may not be expensive in relation to the revenues from a premium on the price of such wood chips.

The results disclosed have provided information of enhancing alkaline pulping and the up scaling thereof. Together with pulp mills the fungal pretreatment of wood chips with the first successful disclosed co-culture, should be further tested on an industrial pilot scale operation. The results disclosed confirm the better pulping performance of synergistic fungal pretreated wood chips with Soda-AQ delignification. Alkaline delignification without sulphur containing chemical additives will be of additional benefit to pulp mills, as this will lessen environmental pollution and easier recovery of pulping chemicals will be possible.

EXAMPLE 3 The following preferred laboratory scale embodiment as indicated in Figure 3 was applied: The described technique of co-cultural fungal pretreatment of wood chips before alkaline pulping, was up-scaled in a pilot plant bioreactor 1 designed and constructed to allow a solid substrate fermentation of not less than 0,5 tons of wood chips (dry mass) 3. In the up-scaling studies a suitable process control system 9 was designed to allow optimum growth conditions of the co-cultures within the bioreactor 1. Controlled oxygen supply occurred through the aeration of the packed wood chip bed by means of an air pump 4. The temperature was controlled to a set value of not more than 38°C by pumping hot water 5 through copper pipes 2 by means of a water pump 7. The control system 9 comprises thermocouples 6, a personal computer 11 and an analog to digital converter 10 and an element 8 to heat the water 5. This up-scaling confirmed that the inoculation of Eucalyptus grandis wood chips with co-culture Pycnoporus sanguineus and Aspergillus flavipes resulted in superior pulping properties of both the Kraft-and Soda-AQ alkaline pulping processes, as (a) significantly higher pulp yields; (b) lower Kappa numbers; and

(c) lower chemical consumption were recorded, as shown in Table 28 hereunder. In Table 28 the fungus X9 refers to Pycnoporous sanguineus while the fungus Y1 refers to Aspergillus flavipes. Phanerochaete chrysosporium is also included in the results because this fungus was : (a) used to optimize the pilot plant bioreactor as represented in Figure 3; and (b) disclosed by previous inventions, as well as in the literature, as proven white-rot fungi for pulping purposes.

TABLE 28 Table 28 (a): Pulp yields from Kraft and Soda-AQ pulping of Eucalyptus grandis wood chips after 4-week incubation in pilot plant bioreactor. Pulping Treatment Screened Shives Pulp Method Rejects Content yield (%) (%) (%) KRAFT Control. 42.35 49.9 Contaminated wood. 93 3.69 48. 1 chips Co-culture X9-Y1. 16.43 54.1 P. chrysosporium. 23.63 52.2 SODA-AQ Control 1.12.52 52.1 Contaminated wood 1.29 4.01 47.6 chips Co-culture X9-Y1. 56.33 55.1 P. chrysosporium. 65.69 53.4

Table 28 (b) : Kappa Number of pulp from Kraft and Soda-AQ pulping of Eucalyptus grandis wood chips after 4 week incubation in pilot plant bioreactor. Treatment KRAFT Pulping SODA-AQ Pulping Kappa no. Kappa No. Control 17.50 16.96 Contaminated wood 16.48 16.03 chips Co-culture X9-Y1 15.25 14.47 P. chrysosporium 16.11 15.80 Table 28 (c): RAA of black liquor for Kraft and Soda-AQ pulping of Eucalyptus grandis wood chips after 4 week incubation in pilot plant bioreactor. Treatment KRAFT Pulping SODA-AQ Pulping RAA (gel) as Na20 RAA (gull) as Na20 Control 17.50 16.96 Contaminated wood chips 16.48 16.03 Co-culture X9-Y1 15.25 14.47 P. chrysosporium 16.11 15.80

Moreover, such treatment also improved the strength and beating properties of the paper pulp produced, with enhanced bleaching characteristics. These strength and bleaching properties are shown in Tables 29 (a) and (b) hereunder.

TABLE 29 Table 29 (a): Strength properties of Kraft and Soda-AQ pulps from treated Eucalyptus grandis wood chips after 4 week incubation in pilot plant bioreactor at constant freeness (38°SR). Pulping Treatment Tear Index Breaking length Burst Index Method (mN. m2/g) (km) (kPa. m2/g) KRAFT Control 7.47 7.43 2.35 Contaminated wood 6.20 5.60 1.60 chips Co-culture X9-Y1 6.95 10.35 3.08 P. chrysosporium 7.55 9.42 2.92 SODA-AQ Control 7.24 7.00 3.24 Contaminated wood 5.92 6.47 1.95 chips Co-culture X9-Y1 6.57 10.35 3.33 P. chrysosporium 7.06 8.68 2.86 Table 29 (b) : Consumption of bleach chemicals after bleaching of Kraft and Soda-AQ pulps of treated Eucalyptus grandis wood chips after 4 week incubation in pilot plant bioreactor. Pulping Treatment Cl2 Cl2 C102 C102 Method Contents Consumed Content Consumed (gel) as Cl2 (%) (gull) as Cl2 (%) KRAFT Control. 456 92.4 0 100 Contaminated wood. 338 93.57 0 100 chips Co-culture X9-Y1. 824 88.26 0.094 98.43 P. chrysosporium. 403 93.28 0 100 SODA-AQ Control. 339 94.35 0 100 Contaminated wood. 214 96.43 0 100 chips Co-culture X9-Y1. 691 88.48 0.024 99.60 P. chrysosporium. 386 93.57 0 100

EXAMPLE 4 A unique method (Final Selection Factor) was formulated and applied for the selection of suitable fungi for bio-pulping and is described in the following preferred embodiment: Previous attempts to demonstrate interactions between co-cultures resulted only in the observation of antagonism, manifested as deadlock or as one organism overgrowing the other. However, such interactions were studied on non-selective nutrient media, e. g. malt extract agar media. It was thus decided that a medium more closely representative of native wood, e. g. milled wood media, would give more realistic indications between wood decay fungi as they occur in nature.

Interactions on Synthetic Media.

In order to study the interactions of the selected mono-cultures in co-cultures, a novel screening technique was adapted from tests originally designed to investigate antibiotic bacterla. The technique included the use of artificial wood media and milled wood media. The study was initiated by firstly determining the growth rates of each mono-culture at 29°C in triplicate on each medium in order to accommodate different lag phases during subsequent co-culture applications.

Liquid suspensions of spores and viable propagules were prepared in physiological saline. The surfaces of the media in all plates were dried by pre- incubation at 29°C for 48 hours to permit rapid absorption of liquid in inocula and thus ensure rapid immobilization of propagules at locations inoculated. Cultures were then drop-inoculated by streak-inoculation using sterile Pasteur pipettes passed across diameters of respective plates. The lag phase of each mono- culture on each medium was recorded. Mean growth rates were calculated every 24 hours as factors of the increase in width of the inoculated streak. The mono- cultures were then grouped according to lag phases, growth rates and physiological profiles.

Suspensions of each mono-culture of each c-culture to be investigated were prepared as above. One mono-culture of a given c-culture was then inoculated as one of the bars of an L-shape streak and the other mono-culture of that co- culture was inoculated as the second bar of the L-shape as shown in Figure 4.

Taking into account their lag phases and radial growth rates, the mono-cultures were applied at times calculated to best permit simultaneous expiration of their respective lag phases and synchronous initiation of growth of each member of given co-cultures, to produce simultaneous colonisation of the center of the zone contained by the L-shape streak. Plates were then placed in plastic bags to prevent excessive dehydration of media, incubated at 29°C and inspected for interactions on a daily basis.

Interaction of Selected Co-cultures on Wood Chips.

Suitable co-cultures obtained from the above investigation on interactions were then used to inoculate wood chips and submitted for quantitative analysis. Co- cultures were inoculated simultaneously and incubated for a 4 week period.

Secondary selections of test Cultures.

In the process of selection of suitable mono-cultures and co-cultures it was felt that it was important to examine the glucose and xylose + mannose data holistically in conjunction with the corresponding lignin values to interpret the above mentioned properties meaningfully. The reason for this was that, for example, a given mass of decayed wood which had lost lignin but not cellulose would appear to contain a higher percentage of cellulose compared with a mass of wood which had lost both cellulose and lignin, or with wood which had lost neither of these components. Therefore, percentage residual values of each component would be unrealistic if interpreted separately. Moreover, the corresponding weight losses required being included in such examinations, in order to interpret the data meaningfully.

The selection factor was calculated from the following ratio: SF = (% Glucose) (% Klason lignin) + (% Xylose + % Mannose) To simplify this principle, the selection factor SF was normalised by converting it to unity and can be expressed as follow : FSF = Sfsam pie Sfcontrol