FI115221B - Removal of resin from wood chips - Google Patents

Description

115221

The present invention relates to a method for removing pitch from chips and to a microorganism culture or inoculum according to the preamble of claim 11.

Extracted wood, "pitch", in many ways complicates pulp and paper making. Some of the wood extractants form precipitates that can cause the paper web to break and make the paper machine runnable. Extractants can still cause problems in coating and printing on paper-10, odor and taste in packaging materials, and problems in waste water treatment. In mechanical pulping, extractives remain virtually unchanged in amount and structure, whereas in chemical pulping, cooking, some of the extractant is removed, but extractives increase the consumption of chemicals.

15

The extract content and composition of the wood varies according to the species and also the growing conditions. The dry matter content of the wood is usually less than 10% but may be up to 40%. The most common groups of extractives are triglyceride, resinic acid, sterol and steryl ester groups, but the extractants also include phenolic compounds such as flavonoids and lignans. Growth conditions affect the amount of extractives and the amount of wood in different wood species. parts (for example, heartwood and surface wood) have different extractant contents.

I II ·: i i i ,,,,; Of the wood extracts, triglycerides are the most easily degradable and cause the most. ·,: Problems with mechanical pulping. Sterols and steryl esters are more difficult to decompose, although they are less present in wood. These compounds cause problems in chemical pulping. Resin acids are abundant, especially in softwood extractives. They decompose slowly and can make waste water toxic.

i * I * r 1 «»

In the past, extractants were allowed to degrade by the tree's own microbes to harmless »i ·. · · ·, To 30 levels, but removing the resin in this way by extending the storage time adds more harmful> I>, ..; changes in the wood raw material. Long-term storage of large volumes of wood entails additional costs, both in terms of degraded pulp quality and highest storage costs. An industrially appropriate shelf life would be about two weeks. Timber must be stored for at least 10 weeks in order to reduce the extract content in the wood.

Π 5221 2 An attempt has been made to find a solution to microbes that break bulk substances. Cartapip ™ (now known as Albinex ™) is commercially available. It is a pale variant of the blue sponge Ophiostoma piliferumm. It breaks down triglycerides very well, but worse than other extractants. Cartapip ™ has been reported to reduce the total amount of pine extract in two weeks by about 20% compared to the control and also works in aspen chips (Breuil et al., 1998). On the other hand, it is less suitable for the decomposition of the extracts of Finnish spruce and birch.

10 Different types of wood fungi have been studied as extractants, especially blue-green fungi (e.g. Ophiostoma and Ceratocystis) and white fungi (e.g. Ceriporiopsis subvermispora) (Martinez-Inigo et al. 1999, Dorado et al. 2000 a, b). . Effective disintegrators have also been sought for in molds and bacteria.

15 In South Africa, lipolytic activity of white fungus strains isolated from pine and eucalyptus cultures has been tested in biopulp studies (de Kokeret et al. 2000). In addition to pine and eucalyptus, the degradation of wound extracts by various types of fungi has been studied (Farrell et al. 1998, White-McDougall et al. 1998). In these studies, experiments have been carried out by growing the fungus in chips. A different process to treating wood chips is treating whole tree trunks with Phlebiopsis gigantea.

• »• · ·: The method has been developed specifically for the biological control of blue fungi, but also the '' extractants decompose, although the treatment time is long compared to the chips (Behrendt &

Blanchette 1997). Laboratory tests have found that several fungi • · · j break down up to 70% of the total amount of extractives, but comparisons are difficult because laboratory tests contain t »| *; t '' was used under varying conditions and often with quite long processing times.

The advantage of blue fungi is that they do not degrade the structural polymers of the wood, and thus "!! do not reduce the yield. The disadvantage is that they produce a dark pigment. The white fungi '^' degrade lignin and may be useful in pulping, so their use in the removal of extracts I ♦ '· · * 30 could be advantageous.

• · II ·: Blue fungi are the most effective in breaking down triglycerides, steryl inhibitors and waxes from various groups of extracts. As the sterol esters decompose, the amount of sterols increases correspondingly because the released sterols do not decompose further. Fatty acids are also degraded, but initially the proportion of free 115221 3 fatty acids increases as the triglycerides are degraded (lireuil et al. 1998, Martinez-lnigo et al. 1999). White fungi, in turn, also degrade sterols and resin acids (Martinez-Inigo et al. 1999).

5 Few studies on the microbiological degradation of spruce extracts have been published and only laboratory tests have been reported. Fiseheret et al. (1994) used the ability of three fungi (Ceriporiopsis subvermispom, Phanerochaete Chrysosporium and Ophiostoma piliferum (Cartapip ™)) to investigate the decomposition capacity of both fungi and pine.

10

The treatment of wood or wood chips with fungi has been proposed e.g. in WO 9842914 A1, WC) 9946444 A1, WO 9713025 A1, WC) 9802612 A1, WC) 97/13025, US 5,055,159, US 5,476,789, US 5,620,564, US 5,460, 697 and EP 689 625 B1.

JP 3220388 and JP 3213591 propose treating chips with microorganisms without adding nutrients with lignin acting as the sole carbon source.

Bacteria for the treatment of pulp and wood have been proposed in International Patent Publication No. WO 9636765 A1 and in U.S. Patent No. 5,711,945.

20 :: Enzymes such as Resinase® (Novozymes A / S) have also been suggested for use in reducing pitch problems. The problem with Resinase® is that it only hydrolyses' 1 triglycerides and does not affect other wood extractants. Hydrolysis products, fatty acids and '. ': Glycerol must be removed from the pulp after treatment with the enzyme.

: 1v: 25 i t <'···' Although various methods have been used to reduce the amount of pitch in the chips, microorganisms have been added to the chips, but there is no satisfactory method yet * ;;; developed by. Addition of microorganisms has been found to cause color disadvantages and * 1 '! 1 to reduce the lightness of the pulp. In addition, ligninolytic fungi can cause loss of yield f i «* ... 1 30 and due to the production of hemicellulases and cellulases, the strength of the pulp may * * *.

It is an object of the present invention to eliminate the drawbacks of the prior art and to provide an entirely novel solution for solving chip resin problems.

The invention is based on the surprising finding that chips extractants can be reduced by adding nitrogen only to the chips. Microorganisms may be added in addition to nitrogen addition, but nitrogen addition alone has been found to provide a significant reduction in extractants.

It has been known in the past that nitrogen is often a limiting factor in fungal growth in wood (Breuil et al. 1998), which can be used by fungi as inorganic ammoniacal nitrogen or in organic form as amino acids. Because of the great variety of microorganisms present in wood, it is surprising that the addition of nitrogen improves the activity of microorganisms which are capable of degrading extracts and that the activity of microorganisms increases in this direction.

The present invention thus relates to a process in which the amount of pitch in the chips is reduced by adding nitrogen to the chips. According to the method, at least 0.07 g N / kg of fresh chips is added to the chips and conditions in the chips such as humidity, temperature and aeration are adapted to the growth of microorganisms.

More particularly, the present invention relates to the character of claim 1; : the method described in the character section.

•, ': 25 In addition to nitrogen, other nutrients such as a carbon source can be added to the chips. The carbon source may be, for example, starch or a similar non-easy to use carbon source. Addition of an easy-to-use carbon source such as glucose, on the other hand, does not promote the degradation of the extractants.

t I t '·; · 'In addition to nutrients, chips can be inoculated with a culture of microorganisms which are known to * * t: 30 be able to degrade wood extracts. Preferably, such a culture of the microorganism is a fungus such as a blue fungus or a white fungus, most preferably the fungus is lignino- * *: lytic. In the context of this invention, it was found that particularly good results are obtained with:: *: strain C (i.e. strain PP / 1998 / C), which is a member of the blue fungus. The strain has been deposited in accordance with the Buda Pest Convention on a microorganism under the Budapest Treaty

S

DSMZ-Deutsche Sammlung von Microorganismen und Zellkulturen GmbH, Maschcrodcr Weg lb, D-38124 Braunschweig, November 21, 2002 under the number DSM 15309.

Thus, the invention also relates to a microorganism culture or strain according to claim 11 containing microorganisms of strain DSM 15309.

Adding nitrogen alone is technically quite simple. Nitrogen is preferably added in inorganic form, most preferably as ammonium nitrogen, for example as an ammonium salt, such as ammonium sulfate, ammonium nitrate or ammonium chloride.

With the addition of nitrogen alone, it has been found in the present invention that 20-40% of the wood extractives are removed. When in addition to nitrogen, the chips are inoculated with a microorganism capable of decomposing the wood's extracts, 10-30% of the wood's extracts can be degraded, depending on the nutrients used.

Nitrogen addition accelerates microbial growth, may produce more diverse microbial growth, and thus enhances leaching of extracts. With the addition of nitrogen alone, there is no need for a separate system for producing, transporting, and distributing the transition. Another significant advantage 20 is that the addition of nitrogen to the processing time associated with the removal of chips extracts • ,; | can be shortened to allow micro-organisms naturally occurring or added in the form of chips to produce less dye than if micro-organism transfer alone was used.

'· ", 25 The invention will now be explained in more detail with the aid of a detailed description and a few application examples.

;; Figure 1 shows the effect of different passages of strains A, B and C on fresh chips extractants.

•; 'Figure 2 shows the effect of translocations of strains A and C on fresh chips extractants * * 30 Figure 3 shows fresh and steamed chips extractants by group of extractants

Figure 4 shows the FDA activity of seed strains in fresh and steamed chips:; Figure 5 shows the decomposition of extractives in a two-week fungal treatment. Chip ί, '.: extract content 14.1 mg / g 115221 (>

Figure 6 shows the decomposition of the extractants in the two weeks of fungal treatment. Steamed chips extract content I6.7 mg / g

Figure 7 shows the temperature in the composter and outdoor air.

Fig. 8 shows the temperature in the sawdust and chips processing room 5 Fig. 9 shows the decomposition of extractives in the composter Fig. 10 shows the decomposition of the extractant in the composter 2

Fig. 12 shows the decomposition of extractives in steamed chips on strain C, pilot 1 Fig. 13 shows the decomposition of extracts on steamed chips on strain C, pilot 2 Fig. 14 shows the decomposition of extractants on strain C used in pilot experiments at different temperatures

The method of removing wood extractives according to the present invention has been applied to wood chips because wood and wood chips are normally used in the production of mechanical and chemical pulps.

"Extracts" of wood are also referred to in this application as "pitch" or "pitch". The most common groups of extractants are the triglyceride, resin acid, sterol and steryl ester groups, but the extractants also include phenolic compounds such as flavonoids and lignans. In the present invention, the extractants are determined from the wood pin by the T 204 om-88 method as an acetone soluble fraction.

According to the process of the present invention, the nitrogen is added to the wood chips at a minimum; 0.07 g N / kg of fresh chips and conditions of chips are adapted to the growth of microorganisms.

. The nitrogen may be added as organic nitrogen or inorganic nitrogen, but it is preferred to add it as inorganic nitrogen, most preferably in the form of ammonium. Nitrogen may be added as a precursor as ammonium sulfate, ammonium nitrate or ammonium chloride. Nitrogen 30 is added at least 0.07 g N / kg fresh chips, preferably at least 0.10 g N / kg fresh *; * chips. The addition is suitably between 0.07 and 0.3 g N / kg, preferably between 0.10 and 0.23 g N / kg * ... 'fresh chips, more preferably between 0.13 and 0.20 g N / kg fresh chips (0.13 g N corresponds to 0.5 g »» * ammonium chloride). More than 0.3 g N / kg can be added, but if the amount of nitrogen exceeds the tendency of microbes growing in chips 115221 7, it is economically unviable and the excess can, for example, waste the process and the environment.

The nitrogen may be added as the nitrogen salt in dry or liquid form. Most preferably, the nitrogen addition is done as a liquid which can be added to the chips by pouring, spraying, etc. When adding nitrogen, the chips can be mixed so that the nitrogen addition is uniformly applied. Nitrogen may also be added, for example, in connection with chipping, which ensures that the nitrogen is evenly distributed in the chips.

10 "Suitable conditions for the growth of chips micro-organisms" means conditions under which the factors affecting the growth of the micro-organisms, such as temperature, humidity and aeration, have been optimized. The temperature of the chips is sought to be maintained at a treatment temperature of 5 to 40 ° C, preferably 10 to 40 ° C, more preferably 15 to 30 ° C, most preferably 20 to 30 ° C. At very low temperatures, microorganisms do not grow and are unable to degrade the extracts, and at very high temperatures, they are also inhibited. Temperatures between 22 ° C and 30 ° C are most preferred if growth of mesothelial fungi is desired. Temperatures above 35 ° C begin to be too high for mesophiles. Warmed woodchips contain thermophilic microbes, fungi and bacteria at an optimum temperature of 45-50 ° C, but according to the present invention, it is most advantageous for the extraction of the extractants to maintain conditions suitable for the growth of mesophilic microorganisms.

* The moisture content of the chips is sought to be maintained at about 40-80%, preferably between 50-70%. The most preferred chip moisture for microbial activity is about 60%.

; A microorganism culture capable of reducing the amount of pitch in the chips is a culture of the microorganism capable of disintegrating the chips from the extractants by at least 10%, preferably at least 20%, as compared to the control. Microorganism • * *: The culture may be a fungus such as a blue fungus or a white fungus. The microorganism may •; · 'Be mold or bacterial. Preferably, the microorganism is such that it has no appreciable cellulase or hemicellulase activity, which would result in a decrease in mass yield.

The microorganism culture may be, for example, a commercial product such as Cartapip ™. Here: The invention found that a particularly beneficial microorganism which disintegrates wood extracts

': *' 1 is a mesophilic blue sponge represented by strain C, deposited as DSM

15309.

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115221

The microorganism culture is inoculated into chips as a mycelial powder, suspension, or liquid. The liquid may be an aqueous solution, a buffer solution or inoculation may be carried out with a culture medium optionally diluted with water or a buffer solution. Inoculation 5 can be made in combination with nitrogen addition and other nutrient supplements. Addition can be done by pouring, spraying, brushing or otherwise treating the surface of the chips. The inoculum preferably contains 10's to 10K, preferably 10 (1 to 107 microorganism cells or spores (colony forming units) / g of fresh chips. It is preferable to mix the chips with the addition of the inoculum. can also be improved during cultivation by passing air through a chips mat, such as air ducts and / or perforated plates, inoculation can also be done during chipping, in a similar way to adding nitrogen (and possibly other nutrients), such as a screw conveyor.

15

The microorganism culture may be stored for inoculation, for example, in freeze-dried or liquid form, for example in the form of a concentrated fungal mycelium suspension or a spore suspension, for example, stabilizing agents and / or preservatives may be added. The microorganism culture may also be, for example, in powder form.

Since according to the present invention, a significant proportion, 30-40%, of the wood extractants is decomposed by nitrogen alone (about 10% without nitrogen 1 parenting), chips are not necessarily inoculated at all with the wood extracting microorganisms. This, of course, greatly simplifies chip pre-treatment and reduces costs.

_! .. * 25 There is no time or space required for storing, handling and distributing the inoculum »» and no cost.

• * • »· • · · I i« *. · · ·, The processing time of the chips, ie the time of nitrogen, possible other nutrients and microorganisms

• I

* ♦ »•, which can be added from 4 days to 8 weeks, preferably 1 to 4 weeks, most preferably 1 to 2 weeks.

"· · · 'The chips to be processed may be any chips, coniferous wood such as pine or spruce or hardwood such as aspen, 115221 <) birch, alder or eucalyptus, acacia or poplar, which may be used for mechanical or chemical pulping. strain (', the treatment is well suited for spruce chips.

In the context of the present invention, it was found that steaming improves the growth of microorganism graft 5 in chips. However, since steaming destroys a substantial part of the microbes that are naturally present on the surface of the wood, steaming should not be used unless the chips are inoculated. If the chips are inoculated, suitable steaming is 100 ° C for 2 to 20 minutes, preferably 3 to 10 minutes, most preferably 3-5 minutes.

Example 1

Two experiments were performed on hex chips, one of which tested the stability of the blue fungus strains A, B and C with respect to the degradation of the extracts. It has been found that there are large differences in the disintegration of extractants per strain, from completely ineffective to excellent disintegration 15 (Farrell et al. 1998). In order to ensure that strain C was stable with respect to the degradation of the extracts, several different transitions of the strain were made and compared. In the second experiment, in addition to the degradation of the extracts, the growth potential of the fungi in fresh and steamed chips was monitored. Based on the experiment, the fungus used in the pilot experiment was selected.

20 Fresh frozen screened spruce chips (surface wood) were used for cultivation. Chips: used as such (fresh chips) and steamed. The steaming time was set to 2 minutes.

Experimenting at different times, it was found that microbial activity (as measured by the degradation of FDA, or fluorescein deacetate) is not significantly reduced if the steaming time is extended to five minutes. The chips were steamed in small portions (100 g) and when initially having a low microbial level of '^: 25, two minutes was found sufficient.

Three blue fungus strains (strains A, B and C) were grown in the chips. The reference sponge was Ίϋ Cartapip ™ (Albinex ™), a light variant of Ophiostoma piliferum. Inoculations were grown in malt broth. Strains A, B and C were inoculated with 10 6 cells / g of chips (fresh weight) and a corresponding passage was prepared from Cartapip ™ 30 according to the instructions for use.

(MM k »: · 1 Cultures were made in 250 ml conical flasks containing 30 g of chips (fresh weight) / flask. The growth temperature was 28 ° C and the humidity of the chips at the beginning of the experiment was about 60%. The flasks were aerated 3 × 115221 times a week The processing times for the chips were 4, 7, 1 () and 14 days, the stability tests were 7 and 14 days, and there were three parallel bottles.

After cultivation, the total amount of extractives from the chips was determined by extraction with 5 acetone (TAPPI standard T 204 om-88). Groups of extracts were determined from the acetone extract by gas chromatography (Örsä & Holmbom 1994). In the latter experiment, FDA assays were also performed to monitor the active biomass (FDA-fluorescein deacetate, which enzymatically releases a colored compound in proportion to its microbial activity) (Boyle & Kropp 1992). Fungal growth was also monitored by microscopy and observed for possible discoloration.

The differences between the passages were quite small for both the total digestion and for the different extraction groups, the same as for the co-digests. Figure 1 shows the breakdown of total extracts of the two passages of strains A, B and C, and Figure 2 shows two weeks of growth (strains A and C) in fresh chips for two weeks. In the inoculated control, more than 20% of the total extracts were degraded. Cartapip ™ decomposed about 40%, and strain C over 40%, strain A and B about 35%. Among the individual extracts, the triglycerides and steryl esters were clearly reduced, while the sterols increased.

20 Fresh and steamed non-inoculated chips were subjected to acetone extraction and different groups of extractives were determined. The total amount of extractives was higher in the steamed chips than in the fresh ones. Of the extraction groups, the triglycerides were many times higher in the steamed chips than in the fresh ones (Figure 3). Since triglycerides are the compounds upon which fresh wood colonizing fungi grow (Breuil et al. 1998), a significant increase in triglycerides improves the ability of these fungi. fungal growth conditions compared to fresh chips.

'' !! The experiment monitored the growth of various fungi and the breakdown of extractives in fresh and steamed wood chips. According to FDA measurements, all fungi grew better in steaming

* · '* In 30 chips (Figure 4) but with Cartapip ™ and strain C the effect of steaming on FDA

activity was much lower than others. In non-steaming chips, microbial levels remained low in both inoculated controls and inoculated flasks.

In the case of non-steaming wood chips, the low growth of inoculation strains was not due to poor competitiveness because of the very low microbial activity of the chips. Since steaming clearly contributed to the growth of all fungal strains, this could be an improvement in nutrient availability. Triglycerides are an easy-to-use carbon source, and if they are more readily available to microbes in steamed chips, steaming improves growth conditions. Steaming may also remove any inhibitory compounds present in fresh chips and volatile in the steaming. The different extract groups have less free fatty acids and resin acids in the steamed chips than in the fresh ones. Possibly, the steaming affected the structure of the wood to facilitate the growth of inocula.

The total amount of extracts was determined on 7 and 14 day cultures (Table 1). The high 10 FDA activity (Figure 4) did not always correlate with the degradation of the extracts, as both Carta pip ™ and strain C showed less growth in FDA activity than the others in the steamed chips but degraded about 30% more per week compared to the uninoculated control. The strains A and B also efficiently decomposed the extracts and these strains also had a marked increase in FDA activity in the steamed wood chips. In that strain, the FDA activity of all strains was low and there was no further degradation of extractants per week than in the inoculated control (17%), even less in strain B. Cartapip ™ treatment requires that at least 20% of the extractants disintegrate in less than three weeks compared to a sterile control (Farrell et al. 1999). This was clearly achieved at least with strains A, B and C.

In fresh chips, inoculation strains had the most efficient strain C, which decomposed 40% of the extractives in two weeks. The reference sponge product Cartapip ™ disintegrated 35% and in aged * ·,. in the control (no fungus) 27% of the extracts were degraded. In steamed chips, strain C was • · · • · *! ! clearly more effective than the reference fungus. Since the steaming chips had significantly higher levels of extractable * * · * «· 25 easily degradable triglycerides than fresh chips, · · · also had a higher proportion of extractives than fresh chips. Fungi decomposed:, ·. 40% to 50% of the total extractives, and despite a higher starting level, the final level was • · ·, · * *, even lower than in fresh chips.

t · · »• · ... 30 Table 1. Decomposition of extractives in hex chips. Breeding time 7 and 14d.

<t I

»» P - - - - .1. —.1 I ----- ----- "I

yy Fresh chips, mg / g extractor Steamed chips, mg / g extractor * /> · Sponge pd Il4d Sponge fTd Il4d 0 control 14.1 14.1 _ 0 control ~ 16.7 16.7

No fungus 11.6_10.3__No fungus | 14.6 15.6_ 115221 12

Cartapip | 11.2 Ϊ9 ^ 2 Γ ICartapip 10.4 [9 ^ 4

Strain A ΤΠβ Ϊ09 Strain A ΪΤΪ Ö2

Stock Β 12.7 9.5 Stock Β 10.3 8.8

Strain C 10.9_M__Strain C 9.7_8.1_ “Πί mushrooms” Aged Control, Cartapip Reference Sponge (modified by Ophinsttmu pilifcnmi)

Of fresh chips of free fatty acids, strain C disrupted 76%, Carta-5 pip ™ 71% within two weeks, and degraded 57% in aged control. Strain C, Cartapip ™ and control were at the same level (over 30%) in resin acid degradation. Of the sterile esters, Cartapip ™ showed the highest degradation (76%), strains Λ, B and C more than 70%, but even in the control, the degradation was almost as good. Triglycerides disintegrated well in all inoculation strains and controls. Of the total amount of extract analyzed (11.6 mg / g wood solids) in aged 10 controls, 53% was degraded in two weeks, with the best fungus strain C breaking 57% (Figure 5). In steamed chips, strain C was more effective than Cartapip ™ in breaking down steryl esters and free fatty acids. In fresh chips, the difference evened out because the decomposition of the extractants by the microbes in the chips was effective. The degradation of the resin acids appears to be largely due to the activity of the native microbes, since the degradation in the steam-chipped chips was significantly lower than in the fresh chips (Figure 6). Strain C was selected from the tested fungi for pilot experiments because it grows well in fresh chips and no heat treatment of the chips is required, it degrades 10-20% more than the so-called total extract in two weeks. in aged control, the strain efficiently degrades spruce extracts and the yield of fungal transplants to the chips to be processed is relatively easy due to the yeast-like growth method.

. . Example 2. · · · T Blue fungus strain C, selected on the basis of laboratory tests, was piloted with two different hex chips. Both included Cartapip ™ as a reference strain.

.:. 150 and 25 kg batches of chips were processed in the tests.

* i t. L t For pilot test 1, chopped in spring (April) and fresh frozen ,,,,; spruce surface wood. The second batch was chipped in the autumn (October) for a second pilot test, 30 and used fresh. For laboratory tests, the chips were screened, but in the pilot tests, '' un screened chips were used. The chips were used either as such (“fresh chips”) or steamed.

115221 13

The pilot cock was made in 450 l open air compartments (pilot 1, uninsulated and socket ('inoculated fresh chips)') and indoors in 80 l lids with plastic lids with holes to facilitate ventilation (pilot 1 and 2). product and steamed seed strain C. For pilot experiments, the steam treatment was carried out in an autoclave: the chips were heated to 100 ° C and immediately cooled to 80 ° C for a total treatment time of about 20 minutes.

Transfer of strain C was grown in malt broth on a shaker for 2-3 d. Cartapip ™ (now known as Albinex ™) transfer was made according to the manufacturer's instructions. Strain C and Cartapip ™ were inoculated with 106 cells / g chips (fresh weight).

About 150 kg of wood chips / composter was processed in the composter and about 25 kg / churn in the scraps.

The moisture content of the chips was adjusted to about 60% of the fresh weight. The temperature of the compost inoculated strain C was continuously monitored, as was the outdoor temperature. Temperatures were measured daily for indoor receipts. Extracts and composters were sampled for extracts after 7d and at the end of the experiment after 16d.

Total substances were determined by the Tappi standard (T 204 om-88), but the extraction solvent was: acetone. The extraction groups were determined according to Örsä and Holmbom (1994).

In the first experiment, the chips were thawed two days before the start of the experiment, but thawing in larger bags was slower than expected, and the chips were still cool when inoculated. The temperature of the composters did not rise above 20 degrees during the experiment, but remained warmer than the outside air during the second week of the experiment (Figure 7). The temperature in the scraps was the same as the room temperature except for the steamed chips, which ';;; at the beginning of the experiment was warmer due to steaming. The receipts were placed in the device space, '! 'Whose temperature declined markedly in the middle of the experiment (Figure 8).

: ···: 30

Less than 10% of the acetone-soluble extract disintegrated compared to the aged control. The differences between strain C and Cartapip ™ were quite small (Table 2). Changes '': by extractant group are shown in Figures 9 and 10. In the composter, low temperature influenced the degradation result. On the other hand, the temperature in the boot cultivation was quite high at the beginning of Experiment I151521, and over 30% of the extracts were also degraded in the aged control. Strain C was slightly more effective than the reference strain compared to Cartapip ™ extract group.

5 Table 2. Acetone-soluble solvents mg / g (% lion run) in pilot test l._

Composters _Incubation Time_ _Od 7d 16d

Check "__ 17 /) __ 13.5 (21) __ 14.7 (14) _

Base C ___ 12.5 (26) __ 13.2 (22) _

You received _lncubation time_ _ Od 1 7d 16d

Check "__ [7,0__12,3 (28) __ 11,2 (34) __

Cartapip ___ 11.9 (30) _______ 10.2 (40)

Strain C__11.0 (35) "" 10.6 (36) _

Strain C + steaming 17.6__11.8 (33) __ 10.6 (36) _ "aged control (natural growth) 10

In experiment 2, fresh (non-frozen) chips were used and the test was done indoors. The experimental setup was the same as in Experiment 1. The temperature throughout the experiment was about 20 ° C. A clear increase was observed on the fifth day from the start of the experiment. The decomposition of the acetone-soluble extractants 15 is shown in Table 2 and the changes by group of extractants in Figure 11.

Table 3. Acetone-Soluble Extract mg / g (% degraded) in Pilot Test 2.

5 Handling Incubation Time! "Night p7d [Tid '! Check3 23/7 17.4 (27) 18.2 (23) I Cartapip 24 / Ϊ 15.3 (35) ·] KantaC 15.7 (34) 13.4 (43)

Strain C + 16 ^ 6 17/7 13.1 (21) .:. steaming; ': 3 aged control (natural growth) ,,,,: 20 There was a big difference in the extract content of the chips: in test 1 17.0 mg / g and in test 2 23.7 mg / g. Test 1 used wood chopped in spring and test 2 used wood chopped in autumn, both surface wood. The extract composition of the chips by group is shown in Fig. 115221 15, Part 14. According to the literature, the fresh content of fresh spruce is about 2.2% (Fcngel & Wegener, 1989).

The proportion of inoculum strains in total digestion lysis compared to the aged 5 control was greater than in Experiment I, in part because lysis in control was less effective.

The degradation of strain C can be considered quite good: 20% compared to the aged control (over 40% of the total extracts) and the resin acid and steryl esters were clearly reduced. Strain C 10 was more effective than Cartapip ™ in disrupting all groups of extracts analyzed.

Strain C grew very well in the steamed wood chips, but the decomposition of the extractives did not promote steaming. The abundant grafting produced the same result as promoting the competitiveness of the graft strain by heat treatment of the chips. The effect of strain C on the digestate of steamed 15 chips is shown by groups of extracts in Figures 12 and 13.

Example 3

Nutrient tests were performed in 250 ml conical flasks, 30-50 g chips (fresh weight) / flask. The humidity of the Hak-20 was adjusted to approximately 60% of the fresh weight at the beginning of the rearing. Fresh chips and various nutrients with and without strain C were used in nutrient tests. The growth temperature was, depending on the experiment, either room temperature (20-22 ° C) or 28 ° C and the growth time was 7 or 14 d. In the temperature test, steamed chips were used at various temperatures (22.28,; 32 and 37 ° C). The culture flasks were aerated three times a week with sterile compressed air.

25

In nutrient test 1, standard microbiological growth media such as. . TSB (= Trypticase Soy Broth) and ME (= Malt Extract), the former used 4 ml / 30 g> 1 · chips and the latter 0.2 ml / 30 g chips. The growth temperature was 28 ° C and the test duration was 14 h d. The fresh chips had an extractant content of 17.6 mg / g. In the control chips, almost equal proportions (23% and 26%) of the extractants in the pilot and nutrient tests were degraded; in the experiment, TSB supplementation decomposed 41%. The addition of malt nuts gave a few percent '..' improvement over control chips. Inoculation strain C still provided a few '1'% improvement over nutrient supplement alone. The results show that the nutrients had a clear effect on the decomposition of acetone-soluble extracts (Table 4).

115221 K)

Table 4. Effect of standard microbiological culture media on decomposition of equipment. The content of fresh chips in the extract is I7.6 mg / g.

Nutrient Supplement Extractinect mg / g Extracted%

Water 13.0 26 ~ ME ΓΠ8 33 TSB KU 41 ME + Strain C KU 39 TSB + Strain C ~ 9A "47 ':" 5

In nutrient test 2 (Table 5), individual nutrients and combinations thereof were added to the chips. Growth was done at room temperature but otherwise as in Experiment 1.

The fresh chips had an extractant content of 14.0 mg / g. In both inoculated and inoculated chips, the most effective degradation was obtained by the addition of ammonium nitrogen or high-nutrient medium (THG), which was included as a positive control. The proportion of strain C in degradation was in the same order as in Experiment 1, less than 10%. Under the influence of glucose, the decomposition of the extracts was almost prevented in the inoculated chips. Starch, on the other hand, had a clear positive effect both alone and in combination with ammonium nitrogen.

!. ·: »» · · T *. Table 5. Individual nutrients and their combinations. Concentration of fresh chips / t. 14.0 mg / g.

* * · · ./ Inoculated Inoculated (strain C)

Ravinnelisäys

Extractives Extractives mg / g degraded% mg / g degraded% "Water Ϊ23 12 lp 14 Starch 1% Tp 19% Γ 35

Glucose 1% lp Ϊ6 lp 2 ′ Ammonium Nitrogen (N) 8.6 39 8.1 8.1 Starch + N lp 21 p 39 115221 17 I Sandalwood 0.02% ΓΪΤ, 3 ΓΪ9 8.7 ΓΤχ Starch. ι N i yeast extract 9.7 31 9.0 36 THG ^ P ^ "36 p" ™ 44

Nitrogen (N) content 0.13 g / kg fresh chips; THG, tryptone-yeast extract-glucose medium

In Experiment 3, the degradation of the extracts was investigated for one week in culture (28 ° C).

5 The most common microbial culture media were used as a nutritional supplement and dosed at 4 ml / 50 g chips. The most nutritious addition again produced the most effective breakdown. The effect of dilute NB (1/4 NB) in the inoculated chips was more pronounced than in the non-inoculated chips. The size of the passage did not affect the lysis result (Table 6).

10

Table 6. Effect of nutrients on short-term chips processing.

Inoculated Inoculated (C)

Addition Extractives Extractives mg / g Decomposed% mg / g Decomposed%

Water LM) 7 Ϊ23 12 ME + yeast extract 0.01% Ϊ7 / 7 16 ΪΤ, 6 17 ··; ~ m lp 29 p 34] 1/4 NB LL4 19 ~ '% 9 29 NB + iso transfer 9.3 34 "pd 12 ^ 8 9 ip 22 •» NB, nutrient broth; PD, potato dextrose; iso transfer, 5 times the number of cells compared to conventional 15 passages.

»*» · * * · • »

The results clearly showed that the nutrients of the inoculum affect the degradation of the extracts.

'[*. The addition of nitrogen allows the growth of microbes that have not adapted to the scant environment of wood 4a. The effect of starch on strain C inoculated with strain C (nutrient test 2) was very clear. Starch is an important carbon source for wood colonizing microbes. The effect of nitrogen on the decomposition of the extractives was evident in strain C inoculated chips at significantly lower doses of nitrogen than in inoculated chips, and the differences between the various 115221 18 additions were quite small. With high nutrient and nitrogenous additions (culture media TSB, TUG, NB), the chips' own microbes efficiently decomposed the extracts and strain C provided only a few percent improvement. The effect of strain C was more pronounced when less nitrogen was added to the chips (dilute medium (1/4 NB, 5 yeast wool). Glucose had very little effect and strain ('lysis of extracts appeared to be almost inhibited by glucose. however, because of the large inoculation, competitive strain C does not use extractants as its carbon source when glucose is exceptionally available, however, in wood, lipids are a more economical source of energy than soluble sugars with much less H) (Fleet et al. 1999). in very young cultures, extracellular lipolytic enzymes with nitrogen-related yeast extract and carbon-olive oil are added, whereas peptide-glucose medium has a markedly delayed production of enzymes (George et al. 1999).

15 The effect of different sources of carbon and nitrogen on the color of mycelium of the Ophiostoma piceue fungus has been studied in solution cultures: starch and ammonium chloride have produced white mycelium, with many other carbon-nitrogen combinations varying degrees of brown and gray, even black (Eagen et al. 1999). In experiment 2, with the addition of starch-ammonium chloride, the chips remained light, but no significant darkening was observed in the others.

20 experiments in chips, whether inoculated or not. The darkening of the mycelium is influenced by e.g. microbial competition (Yang, 1999).

The amount of acetone-soluble extracts was reduced by 20-30% over two weeks in the control; Ί compared to simply adding nutrients to the chips. Inoculation strains require n.

• ·;> t: 25 20% improvement in extract degradation. Inoculation strain C increased degradation by less than 10% when nutrients were added at high levels. Strain C provided a greater increase when nutrients were added. . was less. Chips' own microbes can be more versatile to break down extracts. But at the same time the risk of degradation of the structural polymers increases.

Growth of strain C was monitored over a temperature range of 20-37 ° C. Steamed chips were used in the experiment. The decomposition of the aggregates at various temperatures is shown in Figure 15. Strain · ... · C has a typical mesophilic growth area.

* * · 115221 ΙΟ

References:

Behrendt, C.J. & Blanehettte, R.A. 1997. Biological processing of pine logs for pulp and paper production with Phlehiopsis gigantea.Appi environments Microbiol. 63 (5): 1995-2000.

5 Boyle, C.D. & Kropp, B.R. 1992. Development and comparison of methods for measuring growth of filamentous fungi on wood. Can. J. Microbiol. 38: 1053-1060.

Breuil, C., Iverson, S. & Gao, Y. 1998. Fungal treatment of wood chips to removeextractives, pp. 541-565. Environmentally Friendly Technologies for the Pulp and Paper Industry. Young, R.A. & Akhtar, M. Eds. John Wiley & Sons, New York.

10 Dorado, J., Claassen, F.W., van Beek, T.A., Lenon, G., Wijnberg, J. & Sierra-Alvarez, R. 2000a. Elimination of Softwood extractives by white rot fungi. J. Biotechnol. 80: 231-240. Dorado, J., Claassen, F.W., Lenon, G., van Beek, T.A., Wijnberg, J. & Sierra-Alvarez, R. 2000b. Degradation and detoxification of softwood extracts by sapstain fungi. Bioresource Technology 71: 13-20.

15 Eagen, R., Brisson, A. & Breuil, C. 1999. Syntheses the sap stain fungus Ophiostomapiceae in different types of melanin in different growth media. Can. J. Microbiol. 43: 592-595.

Farrell, R.L., Hata; K. & Wall, M.B. 1998. Solving pitch problems in pulp and paper processes by the use of enzymes or fungi, pp. 197-212. Advances in Biochemical 20 Engineering Biotechnology, Vol Biotechnology in Pulp and Paper Industry. Eriksson, K.-; E. L., Ed. Springer, Berlin.

: * 'Farrell, R.L., Hadar, Y., Wendler, A. & Zimmermann, W. 1999. US Patent 5,998,197.

Dec.7, 1999. (Fungi for pitch reduction and their preparation.), '·· Fengel, D. & Wegener, G. 1989. Wood: Chemistry, Ultrastructure, Reactions. Berlin, de '• j 25 Gruyter.p. 182. ISBN3 11-012059-3.

, · Fischer, K., Akhtar, M., Blancette, R.A., Bumes, T.A., Messner, K. & Kirk, T.K. 1994.

Reduction of resin content in wood chips during experimental Biological processes.

. : Holzforschung 48: 285-290.

Fleet, C., Breuil, C. & Uzunovic, A. 2001. Nutrient consumption and pigmentation of deep 30 and surface colonization of sapstain fungi in Pinus contorta. Holzforschung 55: 340-346.

George, E., Tamerler, C., Martinez, A., Martinez, M.J. & Keshavarz, T. 1999. Influence of '*: Growth Medium Composition on the Lipolytic Enzyme Activity of Ophiostoma Piliferum' (Cartapip ™). J. Chem. Technol. Biotechnol. 74: 137-140.

115221 20 dc Koker.T.II., Zhao, J., Allsop, S.F. & Jansc, BJ.il. 2000. Isolation and enzymatic characterization of South African white-rot fungi. Mycol. Res. 104: 820-824.

Hatakka, A., Mettälä, A., Elimen, J. & Maijala, P. 2000. Evaluation of fungi lor bio-Kraft Pulping of Softwood. Proceedings of the 2000 TAPPI Pulping Conference, Boston, ΜΛ, 5 USA, November 5-9, 2000. 6 p. (CD-ROM)

Martinez-lnigo, M.J., lmmerzeel, P., Gutierrez, A., del Rio, J.C. & Sierra-Alvarez, R. 1999. Biodegradabilityof extracts in sapwood and heartwood from scots pine by sapstain and white rot fungi. Holzforschung 53: 247-252.

White-McDougall, W.J., Blanchette, R.A. & Farrell, R.L. 1998. Biological control of blue 10 stain fungi on Populus tremuloides using selected Ophiostoma isolates. Holzforschung 52: 234-240.

Yang, D-Q. 1999. Staining ability of various sapstain fungi on agar plates and on wood wafers. Forest Prod. J. 49 (11-12): 78-90.

Örsä, F. & Holmbom, B. 1994. A convenient method for the determination of wood 15 extractives in Papermaking process waters and effluents. J. Pulp Paper Sci. 20: J361-366.

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Claims (11)

1. A process for reducing the amount of wood extraction agent in wood chips, characterized in that 5. the chips are added to nitrogen in an amount of at least 0.07 g N / kg fresh wood chips (fresh weight) and the conditions in the wood chips are made suitable for the growth of the naturally occurring microorganisms in wood chips, - the chip treatment is completed from 4 days to 8 weeks. 10 Process according to claim 1, characterized in that the chips are inoculated by a microorganism culture which is capable of reducing the amount of wood extraction agent in the chip. Method according to claim 2, characterized in that the chips are grafted by means of a microorganism culture or graft containing microorganisms of the strain DSM 15309. Method according to claim 1, characterized in that the chip is not applied to a microorganism culture or graft. 20 A process according to any of the preceding claims, characterized in that the moisture content, temperature and air conditioning of the chips are made suitable for the growth of microorganisms. 25 6. A method according to any of the preceding claims, characterized by the temperature of the chips, it is sought to be maintained at 5-40 ° C during the treatment. preferably at 10 - 40 ° C, most preferably at 20 - 30 ° C. * 1 A process according to any of the preceding claims, characterized in that the chip treatment is completed from one week to four weeks, preferably from one week. tili two weeks. ) i t Method according to any of the preceding claims, characterized in that the moisture content of the chips is poured between about 40 and 80%, preferably between 50 and 70%. 115221 Process according to any of the preceding claims, characterized in that in addition to nitrogen, the chip is supplied with a suitable carbon cold, such as starch. 5 Process according to any of the preceding claims, characterized in that the nitrogen is supplied in the form of ammonium, such as ammonium sulphate, ammonium nitrate or ammonium chloride. Microorganism culture or graft, characterized in that it contains microorganisms of strain DSM 15309. 1