Extractives of wood, "the resin", impede pulping and papermaking in many ways.

Some extractives of wood form deposits, which may cause breaks in the paper web and hamper the runnability of the paper machine. The extractives can further cause problems in paper coating and printing, detrimental taste and odour in packaging materials, and problems in effluent treatment. In mechanical pulping, the extractives remain almost unchanged as to their quantities and structures, whereas in chemical pulping, i. e. , cooking, part of the extractives exit; however, the extractives increase the consumption of chemicals.

The content and composition of wood extractives vary in accordance with the vari- ety of tree and also according to its growing conditions. As regards the dry weight of wood, the contents of extractives is generally less than 10 %, but it can be as high as 40 %. The most common extractive groups are triglyceride groups, resin acid groups, sterol ester and steryl ester groups, but the extractives also include phenolic compounds, such as flavonoids and lignanes. The growing conditions of the various types of tree have an effect on the amount of extractive, and the content of extrac- tives in the different parts of wood (e. g. , heartwood and sapwood) is different.

Of wood extractives, triglycerides are the easiest to decompose and they cause the most problems in mechanical pulping. Sterols and steryl esters are more difficult to decompose, even though their quantities in the wood are smaller. These compounds cause problems in chemical pulping. Resin acid quantities are high in the extractives of softwood in particular. They decompose slowly and may render wastewater toxic.

Extractives have earlier been allowed to decompose under the effect of the wood's own microbes so as to reach a harmless level, but the removal of resin by extending

the storage time in this way increases the number of harmful changes in the raw wood material. Long-term storage of large quantities of wood causes additional costs both in the form of deteriorated pulp quality and in the form of higher storage costs. A suitable storage time for industrial purposes would be two weeks.

However, to decrease the extractive content of the wood, the wood should be stored for at least 10 weeks.

Attempts have been made to find a solution to the problem of resin by means of mi- crobes that decompose extractives. There is a product commercially available and called Cartapip (Albinex at present). It is a colorless variant of a blue stain fungus Ophiostoma piliferum. It is good in decomposing triglycerides, but not so good in decomposing other extractives. The Cartapip product has been reported to reduce the total quantity of pine extractives by about 20% in two weeks com- pared with a reference, and also to work e. g. in aspen chips (Breuil et al. , 1998). But it is not so well suited for decomposing the extractives of Finnish spruce and birch.

Different types of wood-inhabiting fungi have been studied as decomposers of ex- tractives: the blue stain fungi in particular (e. g. , the Ophiostoma and Ceratocystis genera) and white-rot fungi (e. g., Ceriporiopsis subvermispora) (Martinez-Inigo et al. 1999, Dorado et al. 2000 a, b). Effective decomposers of extractives have also been sought in moulds and bacteria.

In connection with bio pulping research in South Africa, the lipolytic activity of white-rot fungal strains isolated from pine and eucalyptus cultures was tested (de Koker et al. 2000). In addition to pine and eucalyptus, the decomposition of aspen extractives by different types of fungi has been studied (Farrell et al. 1998, White- McDougall et al. 1998). The tests in these studies were made by means of growing fungus in wood chips. The method differing from the treatment of wood chips is treatment of whole tree-trunks with the Phlebiopsis gigantea fungus. The method was actually developed for biological control of blue stain fungi, but extractives also decompose, although the time of treatment is long compared with wood chips (Behrendt & Blanchette 1997). It has been found in laboratory tests with several fungi that up to 70 % of the total quantity of extractives will decompose, but it is

difficult to make a comparison, because varying conditions have been used in the laboratory tests as well as rather long treatment times in many cases.

Blue stain fungi have the advantage that they do not decompose the structural polymers of wood and thus they do not reduce the yield. They have the drawback that they produce dark pigment. White-rot fungi decompose lignin and may be to advantage in pulping, so their use in the removal of extractives could be advanta- geous.

Of the different extractive groups, triglycerides, steryl esters and waxes are decom- posed most efficiently by blue stain fungi. As steryl esters are decomposing, the quantity of sterols increases correspondingly, because the freed sterols will not de- compose any further. Fatty acids also decompose, but in the beginning the propor- tion of fatty acids will grow as the triglycerides decompose (Breuil et al. 1998, Mar- tinez-Inigo et al. 1999). White-rot fungi for their part also decompose sterols and resin acids (Martinez-Inigo et al. 1999).

Only few reports have been published concerning the microbiological extractives of spruce, and there are reports on laboratory tests only. Fischer et al (1994) used both spruce and pine in their study of the ability of three fungi (Ceriporiopsis subvermis- pora, Phanerochaete chrysosporium and Ophiostoma piliferum (CartapipTM) to de- compose extractives. Irrespective of the type of wood species or fungus, 25-30 % of the extractives decomposed in a two-week treatment.

Treatment of wood or wood chips with fungi has been proposed in the following printed patent specifications, among others: WO 9842914 Al, WO 9946444 Al, WO 9713025 Al, WO 9802612 Al, WO 9713025, US 5,055, 159, US 5,476, 789, US 5,620, 564, US 5,460, 697 and EP 689 625 Bl.

Printed patent specifications JP 3220388 and JP 3213591 propose treatment of wood chips with micro-organisms without any addition of nutrients, because the lignin functions as the principal carbon source.

International printed patent specification WO 9636765 Al and US patent 5,711, 945 propose the use of bacteria in the treatment of pulp and wood.

Also enzymes, such as Resinase (Novozymes A/S), have been proposed for use in order to reduce resin problems. A problem with the Resinases product is that it hy- drolyses triglycerides only, but it does not affect the other extractives of wood. The products of hydrolysis, fatty acids and glycerol must be removed from the pulp after the enzyme treatment.

Some printed publications have studied the effect of an addition of nutrients on the growth of fungi. Labosky et al. (1991) studied the effect of an addition of different nitrogen levels on the decomposition of lignin in chips made of red oak under the effect of the Phanerochaete chrysosporium fungus. It was found that an addition of nitrogen improves the ability of the P. chrysosporium fungus to decompose lignin.

Wang et al. (1996) for their part studied how an addition of nitrogen (ammonium nitrate) and other nutrients affected the growth of the Ophtostoma piceae and Le- cythophora sp. fungi in aspen chips. It was found that growth of the said fungi is improved by adding ammonium nitrate and D glucose. It was mentioned in a re- search programme of the Forestry Branch (2002) that an extra source of nitrogen was essential for inoculated fungi and bacteria to reach a high rate of growth in chips. Patent publication US 5,055, 159 described treatment of wood chips with the Ceriporiopsis subvermispora fungus before the making of mechanical pulp. It was pointed out in the publication that owing to the performed treatment the consump-

tion of energy was reduced in mechanical pulping and it was found that an addition of small nitrogen quantities improved the penetration of fungi into the wood chips.

Of the above-mentioned publications no one studied the effect of an addition of nu- trients on a reduction of the quantity of wood extractives, or if they did, they found no essential reduction in the quantity of extractives.

Although efforts have been made to reduce resin substances in wood chips by vari- ous methods, wherein micro-organisms have been added to the chips, no satisfac- tory method has been developed to date. The addition of micro-organisms has been found to cause colour problems and to reduce the brightness of pulp. In addition, ligninolytic fungi may cause losses of yield and the pulp strength may suffer be- cause they produce hemicellulases and cellulases.

The purpose of the present invention is to eliminate at least some drawbacks relat- ing to the known technology and to provide a solution of a new kind to solve the resin problems of wood chips.

The invention is based on the surprising finding that extractives of wood chips can be reduced by adding only nitrogen to the chips. Micro-organisms may be added besides the addition of nitrogen, but just an addition of nitrogen has been found to result in a considerable reduction in the quantity of extractives.

It was already known that nitrogen is often a factor limiting the growth of fungi in wood materials (Breuil et al. 1998), as the fungi are able to use nitrogen as inor- ganic ammonium nitrogen or in organic form as amino acids. Since wood naturally contains very many various micro-organisms, it is surprising that an addition of ni- trogen improves the activity of those very micro-organisms, which are able to de-

compose wood extractives, and that the activity of the micro-organisms increases exactly in this direction.

Thus, the present invention concerns a method, wherein the quantity of resin sub- stances in wood chips is reduced by adding nitrogen to the chips. According to the method, nitrogen is added to the wood chips preferable at least 0.07 g N/per fresh kg of wood chips, and the conditions in the chips, such as moisture, temperature and aeration, are made suitable for the growth of micro-organisms.

In more exact terms, the present invention concerns the method presented in the characterizing part of claim 1.

Besides the nitrogen source, other nutrients may also be added to the wood chips, such as a carbon source. The carbon source may be, for example, starch or some other such not-easy-to-use carbon source. On the other hand, an addition of an "easy"carbon source, such as glucose, will not promote the decomposition of ex- tractives.

Besides nutrients, wood chips may be inoculated by such a micro-organism culture, which is known to be able to decompose extractives. Such a micro-organism culture is preferably a fungus, such as blue stain fungus or white-rot fungus, most prefera- bly the fungus is ligninolytic. It was found in connection with the present invention that especially good results are achieved with strain C (that is, strain PP/1998/C) belonging to the blue stain fungi. The strain is stored in accordance with the Buda- pest agreement in a micro-organism collection DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braun- schweig, on the 2lest day of November 2002 carrying number DSM 15309, accord- ing to the Budapest agreement.

Thus, the invention also concerns a micro-organism culture or inoculation in accor- dance with claim 14 and containing micro-organisms of strain DSM 15309.

Adding just nitrogen is quite simple technically. Of course, the nitrogen is added in such a form, which the micro-organisms occurring naturally in the wood chips are able to use as their nitrogen source. The nitrogen is preferably added in an inorganic form, preferably as ammonium nitrogen, for example, as ammonium salt, such as ammonium sulphate, ammonium nitrate or ammonium chloride. The nitrogen may also be added in such a form, from which it can turn into ammonium nitrogen, such as urea or ammonia.

It has been found in the present invention that just by adding nitrogen 20-40 % of the wood extractives can be removed. When in addition to the nitrogen the chips are inoculated with a micro-organism, which is able to decompose wood extractives, 10 - 20 more % of the wood extractives can be decomposed, depending on the nutri- ents used.

The addition of nitrogen speeds up the start of microbial growth, it may produce a more diversified microbial growth and in that way make the decomposition of ex- tractives more effective. When using only an addition of nitrogen, there is no need for any separate inoculation production, transportation and distribution system. It is also a significant advantage that by adding nitrogen it is possible to shorten the time of treatment relating to the removal of wood extractives, whereby the micro- organisms existing naturally in the wood chips or added as inoculation thereto have the time to produce less colouring matter than if using a micro-organism inoculation only.

In the following, the invention will be studied more closely with the aid of a de- tailed description and some application examples.

Figure 1 shows the effect of different inoculations of strains A, B and C on extrac- tives of fresh wood chips.

Figure 2 shows the effect of inoculations of strains A and C on extractives of fresh wood chips.

Figure 3 shows extractives of fresh and steamed wood chips by extractive group.

Figure 4 shows the FDA-activity of fungal strains in fresh and steamed wood chips.

Figure 5 shows decomposition of extractives in a two-week fungus treatment. The extractive content of wood chips is 14.1 mg/g of chips.

Figure 6 shows decomposition of extractives in a two-week fungal treatment. The extractive content of steamed wood chips is 16.7 mg/g of chips.

Figure 7 shows the temperature in composting devices and out of doors.

Figure 8 shows the temperature in tubs and in the chips treatment room.

Figure 9 shows decomposition of extractives in composting devices.

Figure 10 shows decomposition of extractives in tubs.

Figure 11 shows decomposition of extractives in a pilot test 2.

Figure 12 shows decomposition of extractives in steamed wood chips with strain C, pilot 1.

Figure 13 shows decomposition of extractives in steamed wood chips with strain C, pilot 2.

Figure 14 shows the extractive composition of wood chips used in the pilot tests.

Figure 15 shows decomposition of extractives with strain C at different tempera- tures.

The method according to the present invention for removing wood extractives is applied to wood chips for the reason that wood chips are normally used when mak- ing mechanical and chemical pulp.

As used herein, wood chips means pieces of chips normally obtained in chipping, the length of which is approximately 20-40 mm. The wood chips can be sorted by removing too big and too thick chip pieces.

In this application, "extractives"of wood are also called"resin"or"resin matter".

The most usual extractive groups are the triglyceride, resin acid, sterol and steryl ester groups, but extractives also include phenolic compounds, such as flavonoids and lignanes. In the present invention, extractives are defined as a fraction dissolv- ing from wood into acetone by a method in accordance with the Tappi Standard T 204 om-88.

According to the method in accordance with the present invention, nitrogen is added to wood chips at least 0.07 g N/kg of fresh chips, and the conditions of the chips are made suitable for the growth of micro-organisms.

The nitrogen may be added as organic nitrogen or as inorganic nitrogen, but it is preferable to add it as inorganic nitrogen, most preferably in ammonium form. The nitrogen may be added, for example, as ammonium sulphate, ammonium nitrate or ammonium chloride or, for example, as urea or ammonia. It is recommended to add the nitrogen at least 0.07 g N/kg of fresh chips, preferably at least 0.10 g N/kg of fresh chips. The addition is suitably in a range of 0.07-0. 3 g N/kg, preferably in a range of 0.10-0. 23 g N/kg of fresh chips, more preferably 0.13-0. 20 g N/kg of fresh chips (0.13 g N is equal to 0.5 g as ammonium chloride). It is possible to add more than 0.3 g N/kg, but if the quantity of nitrogen exceeds the requirement of the

microbes growing in the wood chips, it is not economically profitable and any sur- plus may be a load on the process and on the environment, for example, through the wastewater.

Nitrogen may be added, for example, as nitrogen salt, either dry or in liquid form. It is most advantageous to do the nitrogen addition as a liquid, which can be added to the wood chips by pouring, spraying etc In connection with the addition of nitro- gen, the wood chips may be agitated, so that the addition of nitrogen arrives uni- formly in the wood chips. Hereby the wood chips may be agitated, for example, by a screw conveyor. Nitrogen may also be added in connection with the chipping, and in this way make sure that the nitrogen will be distributed evenly into the wood chips.

Conditions suitable for the growth of micro-organisms in wood chips means such conditions wherein the factors affecting the growth of micro-organisms, such as temperature, humidity and aeration, have been set to be as favourable as possible.

During the time of treatment, the temperature of wood chips is preferably at 5- 40°C, more preferably at 10-40°C, still more preferably at 15-30°C, most pref- erably at 20-30°C. Micro-organisms won't grow at very low temperatures and are not able to decompose extractives, while at very high temperatures their growth is also prevented. In order to promote the growth of mesophilic fungi the most favour- able temperatures are in a range of 22-30°C. Temperatures over 35°C are already too high for mesophiles. In warmed-up piles of wood chips thermophilic microbes, fungi and bacteria occur, whose optimum temperature is in a range of 45-50°C, but according to the present invention it is most favourable for the decomposition of extractives to keep the conditions suitable for the growth of mesophilic micro- organisms. As it is preferable to keep the dead time of wood chips at the plant as short as possible before taking the chips to the pulping process, the temperature will not necessarily have the time to rise to a level suitable for thermophiles, which

means that the temperature need not be adjusted. On the other hand, high tempera- tures are probably not harmful in the late stage of treatment, when a significant part of the extractives has already decomposed.

The humidity of wood chips should be kept in a range of 40-80 %, preferably in a range of 50-70 %. For microbial activity, the most favourable humidity of wood chips is approximately 60 %. The humidity percentage means the relation of the difference in weight between fresh wood chips and oven-dry wood chips to the weight of fresh wood chips expressed as a percentage.

When agitating the wood chips, the supply of oxygen is also improved in the pile of wood chips. The aeration in the pile of wood chips can also be improved during the treatment by conducting air through the chip bed. The chip pile may be equipped, for example, with air conditioning tubes and/or with perforated plates. If required, heat may also be supplied into the wood chips along with the airflow, or the chip pile may be cooled. The airflow may also be used for controlling the humidity of the chip pile. The chip pile may be covered in order to maintain a suitable tempera- ture and humidity.

It is advantageous to add the nitrogen into fresh chips, which means chips"as such", that is, wood chips containing the micro-organisms existing naturally therein, which have not been destroyed, for example, by steaming, irradiating or drying the wood chips. Generally speaking, such chips can be regarded as being fresh, which essentially contain those micro-organisms, which exist naturally in wood chips. Be- sides newly made chips, such chips may also be regarded as being typically fresh, which have stayed at the plant from chipping to the process treatment. The time usually varies from a few days to about a couple of weeks. Of course, the time may also be longer, if, for example, the chip pile freezes in between and no significant microbiological changes thus occur in the wood chips. The nitrogen quantity of

"0.07 g N/kg of fresh wood chips"means 0.07 g N kg per fresh weight of the chips.

If the humidity of the chips is set at, for example, 60 %, the quantity of nitrogen then corresponds with 0.11 g N/kg calculated per dry weight.

Since according to the present invention a considerable part, 30-40 %, of wood extractives is made to decompose just by using nitrogen (without any addition of nitrogen about 10 %), it is not necessary to inoculate the wood chips at all by micro- organisms decomposing the wood extractives. Of course, this considerably simpli- fies the pre-treatment of wood chips and reduces costs. No time or premises need to be used for storing, treating and spreading of the inoculation, and thus no costs will be incurred.

A micro-organism culture able to reduce the quantity of resin in wood chips means such a micro-organism culture, which is able to decompose no less than 10 % of the chip extractives, preferably no less than 20 % compared with a reference. The mi- cro-organism culture may be some fungus, such as blue stain fungus or white-rot fungus. The micro-organism may be a mould or bacterium. The micro-organism is preferably one having no noteworthy cellulase or hemicellulase activity, which would result in a reduced pulp yield. The micro-organism culture may, for example, be a commercial product, such as Cartapip. It was found in the present invention that a particularly advantageous micro-organism for decomposing wood extractives is a mesophilic blue stain fungus represented by strain C, which is stored carrying the number DSM 15309.

The micro-organism culture is inoculated into the wood chips as a mycelium pow- der, as a suspension or as a liquid. The liquid may be an aqueous solution, a buffer solution or the inoculation may be made along with the growing solution, which is possibly diluted with water or with a buffer solution. The inoculation may be made together with the addition of nitrogen and the addition of other nutrients. The addi-

tion may be made by pouring, spraying, brushing or by treating the surface of the wood chips in some other, manner. The inoculum preferably contains 105-108, preferably 106-107 of micro-organism cells or spores (units forming colonies) /g of fresh wood chips (per fresh weight). It is advantageous to agitate the wood chips while adding the inoculation. When agitating the wood chips, the supply of oxygen is also improved in the pile of wood chips. The aeration of the chip pile can also be improved during the cultivation by leading air through the chip bed. The chip pile may be equipped with, for example, air-conditioning tubes and/or with perforated plates. The inoculation may also be made in connection with the chipping, in a way similar to the addition of nitrogen (and possibly of other nutrients). The wood chips can hereby be agitated by a screw conveyor, for example.

The micro-organism cultivation may be stored for inoculation, for example, in cold- dried or liquid form, whereby it may be, for example, a concentrated mycelial sus- pension or a spore suspension, and e. g. stabilizing agents and/or preserving agents have possibly been added to it. The micro-organism cultivation may also be, for example, in powder form.

The time for treatment of the wood chips, that is, the time from adding nitrogen, other possible nutrients and micro-organisms until further treatments of the wood chips, may vary from 4 to 8 weeks, the preferable time being 1-4 weeks, most preferably 1-2 weeks. The time for treatment of the wood chips thus means the time, in which the nitrogen (or other possible nutrients) is allowed to affect the mi- cro-organisms existing naturally in or added to the wood chips.

The wood chips to undergo treatment may be any wood chips for use in the making of mechanical or chemical pulp, softwood, such as pine or spruce, or hardwood, such as aspen, birch, alder, eucalyptus, acacia or poplar. If strain C is used in the

treatment, then the treatment is very suitable for softwood chips, especially for spruce chips.

It was found in connection with the present invention that steaming improves the growth of the inoculated micro-organism in the wood chips. However, as steaming destroys an essential part especially of microbes existing naturally on the wood sur- face, it is not advantageous to use steaming.

In the following the invention will be examined with the aid of a few non-limiting application examples.

Example 1 Using spruce chips two tests were performed, in one of which the stability of blue stain fungus strains A, B and C was studied in regard to the decomposition of ex- tractives. It has been found that great strain-specific differences exist in the decom- position of extractives, from entirely inefficient to excellent decomposition (Farrell et al. 1998). Since the aim was to make sure that strain C is stable in regard to the decomposition of extractives, several different inoculations were made of the strain, and the inocula were compared with one another. In the other test the decomposi- tion of extractives was observed as well as the growing ability of fungi in fresh and steamed wood chips. Based on the test, a fungus for use in a pilot test was chosen.

Screened spruce chips (sapwood) deep-frozen when fresh were used in the cultiva- tions. The wood chips were used as such (fresh wood chips) and in steamed form.

The steaming time chosen was 2 minutes. A suitable steaming is normally at 100°C for 2-20 minutes. By experimenting with different times it was found that micro- bial activity is not reduced significantly (measured by decomposition of FDA, that

is, fluoresceindiacetate), if the steaming time is prolonged to five minutes. The wood chips were steamed in small batches (100 g) and since it had a low microbe level originally, two minutes were found to be a sufficient time.

Three strains of blue stain fungi (strains A, B and C) were cultivated in the wood chips. The reference fungus was Cartapip (AlbinexTM), a light-coloured variety of the Ophiostoma piliferum fungus. The inoculations were cultivated in malt extract medium. Strains A, B and C were inoculated 106 cells/g of wood chips (fresh weight) and a corresponding inoculation was made of the Cartapip product in accordance with the instructions for use.

The cultivations were made in conical bottles of 250 ml containing 30 g of wood chips (fresh weight)/bottle. The cultivation temperature was 28°C, the humidity of the chips was about 60 % in the beginning of the test. The bottles were aerated 3 times a week by filter-sterilized air. The times for treatment of wood chips were 4, 7,10 and 14 days, in the stability test 7 and 14 days. There were three parallel bot- tles.

The total quantity of extractives in the wood chips was determined by extracting with acetone (TAPPI Standard T 204 om-88). The extractive groups in the acetone extractive were determined using a gas chromatograph method (Orsa & Holmbom 1994). FDA determinations were also made in order to observe the active biomass (FDA = fluoresceindiacetate, from which a coloured compound is released enzy- matically in a quantity related to the microbial activity) (Boyle & Kropp 1992). The growth of fungi was also followed microscopically and possibly occurring changes in colours were observed.

The differences between inocula were small as regards both the total decomposition and the decomposition of different extractive groups, of the same magnitude as be- tween parallels. Figure 1 shows the decomposition of total extractives in two differ- ent inocula of strains A, B and C, and Figure 2 shows it by extractive group (strains A and C) in a two-week cultivation in fresh wood chips. Of total extractives, over 20 % decomposed in a non-inoculated control, Cartapip decomposed about 40 %, and strain C over 40 %, strains A and B about 35 %. Of the individual extractive groups, triglycerides and steryl esters were reduced clearly, whereas the quantity of sterols increased.

An acetone extraction was made of fresh and steamed non-inoculated wood chips and the different extractive groups were defined. The total quantity of extractives was higher in the steamed wood chips than in the fresh wood chips. Of the extrac- tive groups, the quantity of triglycerides was many times higher in the steamed wood chips compared with the fresh wood chips (Figure 3). Since triglycerides be- long to those compounds, on which the growth of fungi colonising fresh wood relies (Breuil et al. 1998), a considerable increase of triglycerides improves the growing conditions of the concerned fungi compared with fresh wood chips.

The tests observed the growth of different fungi and the decomposition of extrac- tives in fresh and steamed wood chips. Based on FDA measurements, all fungi grew better in the steamed wood chips (Figure 4), but with the CartapipTM product and with strain C the effect of steaming on the FDA activity was much smaller than with the others. In non-steamed wood chips, the microbe level remained low both in non-inoculated controls and in inoculated bottles. In the non-steamed wood chips, the slight growth of inoculated strains was not due to any poor competitive ability, because the microbe activity of the wood chips was very low. Since steaming clearly promoted the growth of all fungus strains, it may be a matter of improved supply of nutrients.

Of the extractives the total quantity was determined in cultivations of 7 and 14 days (Table 1). A high FDA activity (Figure 4) did not always correlate with the decom- position of extractives, since both CartapipTM and strain C grew less strongly than the others in the steamed wood chips judging by the FDA activity, but they decom- posed about 30 % more of the extractives in a week compared with the non- inoculated reference. Strains A and B also decomposed extractives efficiently, and with these strains the FDA activity had also increased clearly in the steamed wood chips. In the fresh wood chips, the FDA activity of all strains was low, and of the extractives no more had decomposed than in the non-inoculated reference (17 %), with strain B even less. It is required of the Cartapip Tm treatment that at least 20 % of the extractives will decompose in less than three weeks compared with a steril- ized reference (Farrell et al. 1999). This was clearly achieved at least with strains A, B and C.

Strain C was the most effective one of the inoculation strains in fresh wood chips decomposing 40 % of the extractives in two weeks. Cartapip, the reference fun- gus product, decomposed 35 % and in the aged control (no fungus) 27 % of the ex- tractives decomposed. In the steamed wood chips, strain C was clearly more effec- tive than the reference fungus. Since there were clearly more extractives, especially easily decomposing triglycerides, in the steamed chips than in the fresh chips, a greater proportion of the extractives also decomposed than in the fresh chips. The fungi decomposed 40-50 % of the total extractives, and in spite of the higher start level the final level was even lower than in the fresh chips. Table 1. Decomposition of extractives in spruce wood chips. Cultivation time 7 and 14 days. Fresh wood chips, extractives mg/g Steamed wood chips, extractives mg/g Fungus 7 days 14 days Fungus 7 days 14 days 0 reference 14. 1 14. 1 0 reference 16. 7. 16. 7 No fungus 11. 6 10. 3 No fungus 14. 6 15. 6 Cartapip 11. 2 9. 2 Cartapip 10. 4 9. 4 Strain A 11. 6 10. 9 Strain A 11. 1 9. 2 Strain B 12. 7 9. 5 Strain B 10. 3 8. 8 Strain C 10. 9 8. 4 Strain C 9. 7 8. 1

"No fungus"= aged reference, Cartapip is the reference fungus (modified Ophio- stomapiliferum.

Strain C decomposed 76 % of the free fatty acids in fresh wood chips in two weeks, Cartap iptm decomposed 71 %, and in the aged reference 57 % decomposed. In the decomposition of resin acids, strain C, Cartapip and the reference were on the same level (over 30 %). Of steryl esters Cartap iptm decomposed the most (76 %), strains A, B and C decomposed over 70 %, but the decomposition was almost as good also in the control. Triglycerides decomposed well with all, both with the in- oculation strains and in the control. 53 % of the total quantity of analysed extrac- tives (11.6 mg/g dry matter of wood) in the aged control decomposed in two weeks, the best fungus, that is, strain C, decomposed 57 % (Figure 5). In the steamed wood chips, strain C was more effective than the CartapipTM product in decomposing steryl esters and free fatty acids. The difference levelled out in fresh wood chips, because the decomposition of extractives by the chips'own microbes was efficient.

It seems that the decomposition of resin acids was largely due to the activity of

natural microbes, because in the steamed wood chips the decomposition was clearly poorer than in the fresh wood chips (Figure 6). Of the tested fungi strain C was cho- sen for the pilot tests, because it grows well in fresh wood chips and no heat treat- ment of the wood chips is needed, of the total extractives it decomposes in two weeks 10-20 % more than in the so-called aged control and it decomposes the ex- tractives of spruce efficiently. The yield of fungus inoculation with strain C is rela- tively easy for the chip batches to be treated owing to the yeast-like manner of growth.

Example 2 Using blue stain fungus strain C chosen on the basis of laboratory tests pilot tests were made with two different spruce wood chips. Cartapip was used as reference strain in both tests. Wood chip batches of about 150 and 25 kgs each were treated in the tests.

For pilot test 1 spruce sapwood chipped in the spring (in April) and deep-frozen fresh were used. One batch was chipped in the autumn (in October) for one pilot test 2, and it was used when fresh. For laboratory tests the wood chips were screened, but unscreened wood chips were used for the pilot tests. The wood chips were used either as such ("fresh wood chips") or in steamed form.

The pilot tests were done out of doors in composting devices of 450 1 (pilot 1, non- inoculated wood chips and fresh wood chips inoculated with strain C). and indoors in plastic tubs of 80 1 provided with a lid, in which tubs holes were drilled to facili- tate ventilation (pilots 1 and 2). In the tub tests fresh non-inoculated wood chips were used, as well as fresh wood chips inoculated with strain C and with the Car- tapipTM product and steamed wood chips inoculated with strain C. For the pilot

tests, the steam seasoning was done in an autoclave: the wood chips were heated to 100°C and cooled immediately to 80°C. The whole treatment lasted for about 20 minutes.

The inoculum of strain C was cultivated in malt extract medium on a rotary shaker for 2-3 days. The Cartapip inoculation was made in accordance with the manu- facturer's instructions. Strain C and Cartapip were inoculated 106 cells/g wood chips (fresh weight). The cell densities of the inocula were determined as colony forming units (CartapipT"") or in a calculation chamber e (strain C).

The composting devices were used for treating about 150 kgs of wood chips/composting device and the tubs for about 25 kgs/tub. The humidity of wood chips was controlled in the inoculation to about 60 % of the fresh weight. The tem- perature of the composting device inoculated with strain C was observed constantly as well as the temperature of the outside air. The temperature was measured daily in the tubs located indoors. Samples were taken for extractives from tubs and com- posting devices after 7 days and at the end of the test after 16 days. Total extractives were determined according to the Tappi Standard (T 204 om-88), but acetone was used as the extracting solution. The extractive groups were defined in accordance with Orsa and Holmbom (1994).

In the first test, the wood chips were taken to melt two days before starting the test, but melting proceeded more slowly than anticipated in the rather big sacks, and the wood chips were still cool at the time of inoculation. During the test the temperature did not rise above 20 degrees in the composting devices, but it remained above the temperature of outside air as the air cooled in the second week of the test (Figure 7).

In the tubs, the temperature was the same as the room temperature, except as re- gards the steamed wood chips, which were warmer than the others in the beginning

of the test due to the steam seasoning. The tubs were placed in a room space, where the temperature dropped clearly halfway through the test (Figure 8).

Of the extractives dissolving in acetone, less than 10 % decomposed compared with the aged control, and the differences between strain C and the CartapipTM product were quite small (Figure 2). Changes by extractive group are presented in Figures 9 and 10. The low temperature in the composting devices affected the decomposition result. In the tub cultivations again the temperature was quite high in the early part of the test, and more than 30 % of the extractives decomposed even in the aged con- trol. Strain C was clearly more efficient than the reference strain Cartapip com- pared by extractive group.

Table 2. Extractives dissolving in acetone mg/g (% decomposed) in pilot test 1.

Composting devices Time of incubation 0 days 7 days 16 days Controla 17. 0 13. 5 (21) 14.7 (14) Strain C 12.5 (26) 13.2 (22) Tubs Time of incubation 0 days 7 days 16 days Controla 17. 0 12. 3 (28) 11.2 (34) Cartapip 11. 9 (30) 10.2 (40) Strain C 1. 1. 0 (35) 10.6 (36) Strain C + steaming 17. 6 11. 3 (33) 10. 6 (36) a aged control (natural growth)

Fresh wood chips (not deep-frozen) were used in test 2 and the test was done in tubs indoors. The test arrangement was the same as in test 1. The temperature was about 20°C all through the test. A distinct growth of microbes was noticed on the fifth day after the start of the test. Table 2 shows the decomposition of extractives dissolving in acetone and Figure 11 shows the changes by extractive group.

Table 3. Extractives dissolving in acetone mg/g (% decomposed) in pilot test 2. Treatment Time of incubation 0 days 7 days 14 days Controla 23. 7 17. 4 (27) 18.2 (23) Cartapip 24. 1 15. 3 (35) Strain C 15. 7 (34) 13.4 (43) Strain C+ steaming 16. 6 17. 7 13. 1 (21) a aged control (natural growth) There was a big difference in the extractive content of wood chips: 17.0 mg/g in test 1 and 23.7 mg/g in test 2. Wood that had been chipped in the spring was used in test 1 while wood chipped in the autumn was used in test 2, both being sapwood. Figure 14 shows the extractive composition of the wood chips by group. According to in- formation in the literature, the acetone extractive content of fresh spruce wood is about 2.2 % (Fengel & Wegener, 1989).

In the decomposition of total extractives, the proportion of inoculated strains com- pared with the aged control was greater than in test 1, partly because the decomposi- tion was of a lower efficiency in the control.

The decomposition of strain C can be regarded as quite good: 20 % compared with the aged control (over 40 % of total extractives) and the resin acids steryl esters were reduced clearly. In the decomposition of all analysed extractives groups, strain C was more efficient than Cartapip.

Strain C grew very well in the steamed wood chips, but the steam seasoning did not promote the decomposition of extractives. By plentiful inoculation the same result was achieved as by promoting the competitive ability of the inoculation strain by thermal treatment of the wood chips. Figures 6 and 7 show the effect of strain C on the extractives of steamed wood chips by extractive group.

Example 3 Nutrient tests were done in conical bottles of 250 1, with 30-50 g of wood chips (fresh weight)/bottle. The humidity of the wood chips was controlled in the begin- ning of growths at about 60 % of the fresh weight. In the nutrient tests fresh wood chips and different nutrients were used together with strain C and without it. De- pending on the test, the incubation temperature was either room temperature (20- 22°C) or 28°C and the incubation time was 7 or 14 days. In the temperature test, steamed wood chips were used at different temperatures (22,28, 32 and 37°C). The incubation bottles were aerated three times a week by sterile pressurized air.

In nutrient test 1, ordinary microbiological nutrient solutions were added to the wood chips, such as TSB (= Trypticase Soy Broth) and ME (= Malt Extractive), of which the former was used to a quantity of 4 ml/30 g wood chips and the latter to 0.2 ml/30 g wood chips. The incubation temperature was 28°C and the test lasted for 14 days. The extractive content of the fresh wood chips was 17.6 mg/g. In the

control wood chips, an almost equal proportion (23 and 26 %) of the extractives decomposed in the pilot test and in the nutrient test, whereas in the nutrient test 41 % decomposed by an addition of TSB. By an addition of malt extractive an im- provement by a few percent was achieved compared with the control wood chips.

With inoculation strain C a further improvement by a few percent was achieved compared with the addition of nutrient only. The results show that the nutrients had a clear effect on the decomposition of extractives dissolving in acetone (Table 4).

Table 4. Effect of ordinary microbiological cultivation media on the decomposition of extractives. Extractive content of fresh wood chips 17.6 mg/g. Nutrient addition Extractives mg/g Decomposed extractives % Water 13. 0 26 ME 11. 8 33 TSB 10. 3 41 ME + strain C 10. 8 39 TSB + strain C 9. 4 47 In nutrient test 2 (Table 5), individual nutrients and nutrient combinations were added to the wood chips. The incubation was done at room temperature, but other- wise in the same way as in test 1. The extractive content of the fresh wood chips was 14.0 mg/g. Both in the non-inoculated wood chips and in the inoculated wood chips the most efficient decomposition was achieved by adding ammonium nitrogen or a medium with plenty of nutrients (THG), which was used as a positive control.

The share of strain C of the decomposition was of the same magnitude as in test 1, less than 10 %. Due to the effect of glucose, the decomposition of extractives was almost prevented in the inoculated wood chips. With starch again a clear positive effect was obtained both alone and together with an addition of ammonium nitro- gen. Table 5. Individual nutrients and their combinations. Extractive content of fresh wood chips 14.0 mg/g.

Non-inoculated Inoculated (strain C) Addition Extractives Decomposed % Extractives Decomposed % of nutrient mg/g mg/g Water 12. 3 12 12. 1 14 Starch 1 % 11. 3 19 9. 1 35 Glucose 1 % 11. 7 16 13.. 7 2 Ammonium 8.6 39 8.1 42 nitrogen (N) Starch + N 11. 0 21 8. 5 39 Yeast extract 11.3 19 8.7 38 0. 02% Starch + N + 9. 7 31 9. 0 36 yeast extractive THG 9. 0 36 7. 8 44 Quantity of nitrogen (N) 0.13 g/kg of fresh wood chips; THG = tryptone-yeast ex- tractive-glucose medium.

The decomposition of extractives in an incubation of one week was examined in test 3 (28°C). As the nutrient addition the most usual general microbial cultivation me- dia were used, which were dosed to 4 ml/50 g of wood chips. The addition of the

highest quantity of nutrients again produced the most efficient decomposition. In the inoculated wood chips, the effect of a weak nutrient broth NB addition (1/4 NB) was clearer than in the non-inoculated wood chips. The size of the inoculum did not affect the decomposition result (Table 6).

Table 6. Effect of nutrients in a treatment of wood of short duration. Non-inoculated Inoculated (strainC) Addition Extractives Decomposed % Extractives Decomposed % mg/g mg/g Water 13. 0 7 12. 3 12 ME + yeast 11. 7 16 11. 6 17 extract 0. 01 % NB 10. 0 29 9. 2 34 1/4 NB 11. 4 19 9. 9 29 NB + large 9. 3 34 inoculum PD 12. 8 9 10. 9 22

NB, nutrient broth; PD, potato dextrose ; large inoculation, 5 times the cell quantity of an ordinary inoculation.

The results show clearly that the nutrients of the inoculum affect the decomposition of extractives. The addition of nitrogen allows growth even of such microbes, which have not adapted to the environment scarce of

nitrogen in the wood. The effect of starch in wood chips inoculated with strain C (nutrient test 2) was very clear. Starch is an important carbon source for the mi- crobes colonising wood. The effect of nitrogen on the decomposition of extractives was obvious in wood chips inoculated with strain C with clearly smaller nitrogen doses than in the non-inoculated wood chips, and the differences between the dif- ferent additions were quite small. With additions containing plenty of nutrients and with nitrogenous additions (cultivation media TSB, THG, NB), the chips'own mi- crobes decomposed extractives efficiently, and an improvement by just a few per- cent was achieved with strain C. The effect of strain C was clearer when less nitro- gen was added to the wood chips (weak incubation base 1/4 NB, yeast extract). Glu- cose had a very small effect and with strain C the decomposition of extractives seemed to be almost prevented by the effect of glucose. The decomposition of ex- tractives, which was less than in the control, may be due to the circumstance that even due to the large inoculation the competitive strain C does not use the extrac- tives as its carbon source when glucose is exceptionally available. However, in wood, lipids are a more advantageous energy source than soluble sugars, of which there is much less in wood (Fleet et al. 1999). Cartap iptm has been found to produce extracellular lipolytic enzymes even in very young cultivations, when the cultiva- tion medium contains yeast extract as the nitrogen source and olive oil as the carbon source. On a peptone glucose base, the yield of enzymes starts clearly later (George and others 1999).

In liquid cultures the effect of various sources of carbon and nitrogen on the colour of the mycelium of the Ophiostoma piceae has been studied: using e. g. starch and ammonium chloride, white mycelium have been produced, with several other com- binations of nutrients various degrees of brown and grey, even black mycelium.

(Eagen and others 1999). In test 2 the wood chips remained bright with an addition of starch-ammonium chloride, but no significant darkening was noticed even in the other wood chips of the test in question, irrespective of whether they were inocu-

lated or not. The darkening of the mycelium is also affected by the competition be- tween the microbes (Yang, 1999).

During a time of two weeks, the quantity of extractives soluble in acetone was de- creased by 20-30 % compared with the control, just by adding nutrients to the wood chips. An improvement by approximately 20 % on the decomposition of ex- tractives is required of inoculation strains. Strain C increased the decomposition by less than 10 %, when plenty of nutrients were added. With strain C a higher increase was achieved when less nutrients were added.

The growth of strain C was observed in a temperature range of 20-37°C. Steamed wood chips were used in the test. Figure 9 shows the decomposition of total extrac- tives at different temperatures. Strain C has a typical mesophilic area of growth.

Example 4 The effect of nitrogen on the decomposition of extractives in softwood chips was studied in test 4. Sapwood chips of spruce and pine chips (block chips) were used in the test. The chips were chosen by hand and the maximum piece length was about 30 mm. Chipping had been done in early September 1-2 weeks before the test started.

Nitrogen was added to the wood chips as ammonium chloride (NH4C1). The nitro- gen levels were: 0,0. 5 and 1.0 g of ammonium chloride/kg of wood chips (per fresh weight).

Wood chips were used to a quantity of 50 g/bottle (250 ml Erlenmeyer), and there were three parallel samples. The humidity of the wood chips was set at about 60 % of the fresh weight at the beginning of the test. The wood chips were incubated at room temperature, they were aerated 3 x week. The time of incubation was 2 weeks.

A part of the wood chips was inoculated with micro-organism strain C (DSM 15309). The density of the inoculum was 106 cells/g of fresh wood chips (fresh weight). The inoculum was incubated in 2 % malt extract medium for 2-3 days on a rotary shaker.

The reference or zero (0) sample was untreated (non-incubated) wood chips.

Of the wood chips total extractives were defined according to the Tappi Standard 204 om-88. Acetone was used as the extracting solvent.

Table 7 shows the quantities of extractives mg/g of wood dry matter (average value and standard deviation of parallels) and how much of the extractives has decom- posed compared with the untreated control (decomposition calculated from the av- erage value).

Table 7. SPRUCE No inoculum Inoculum DSM15309* N added g/kg Extractives Decomposed % Extractives Decomposed % mg/g mg/g 0 13. 0A0. 3 10. 9i1. 2 15 0. 13 9.7~0 24 10.7~0.2 16 0.26 10.5~1.1 18 11.0~0.5 14 Extractive content of untreated spruce wood chips 12. 80. 2 mg/g. PINE No inoculum InoculumDSM15309* N added Extractives Decomposed Extractives mg/g Decomposed g/kg mg/g % % 0 30. 56. 5 9 27. 61. 9 17 0. 13 20. 93. 9 37 23. 73. 0 29 0.26 20.0~2.0 40 24.6~3.6 26 Extractive content of untreated pinewood chips 33. 4-+0 mg/g.

*The DSM 15309 inoculation strain is the same as the strain C used in the earlier tests.

The extractive content of spruce was somewhat lower than in the earlier tests and its natural microbe activity was small in view of the time of chipping. In the non- inoculated wood chips, the effect of nitrogen addition was in the same direction as in the earlier tests, in other words, the decomposition of extractives became more effective with a small addition of nitrogen, but in this test the inoculation strain had a boosting effect in those wood chips only, where nitrogen was not added.

In the pinewood chips, the effect of nitrogen was considerable, especially in the non-inoculated wood chips, but there was a rather big deviation between the paral-

lels. The wood chips were darkened in part even at the beginning of the test and thus their natural growth of microbes was plentiful.

Based on the pine results, the effect of nitrogen seems obvious. In the tests of this example, no additional improvement in the decomposition of extractives was achieved with the inoculation strain in either kind of wood chips.

It seems in fact that in several cases the microbes occurring naturally in wood chips are sufficient for bringing about decomposition of extractives, when the conditions of the microbes are improved by adding nitrogen and by making the conditions suitable for the growth of microbes also in other respects. Hereby no significant ad- ditional improvement in the decomposition of extractive is achieved by any inocula- tion of micro-organisms. If for some reason the microbes of wood chips are excep- tionally scarce or of few varieties, inoculation may be used to make more effective the decomposition of extractives brought about by microbes existing naturally in the wood chips.

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