BACKGROUND OF THE INVENTION

Field of Invention The invention relates generally to the field of producing pulp from wood and, more specifically, to a method for producing pulp using biopulping followed by an enzyme treatment.

Description of Related Art There are a number of processes that convert wood to wood pulp. Pulp is the fibrous slurry that is fed to a paper machine to produce paper. Mechanical, chemical and hybrid methods dominate commercial pulping plants. About 25% of worldwide pulp production is mechanical pulp. It is a high-yield process but suffers from high energy costs and damage to the wood fibers. This damage means lower strength paper. These disadvantages (cost and quality) limit the number of applications for mechanical pulp. Biopulping prior to mechanical pulping overcomes the aforementioned disadvantages. The production of pulp begins with wood chips. When a biopulping step is used, the wood chips are 'digested' with one or more fungi types prior to mechanical or chemical pulping. The fungi soften the wood chips by degrading or digesting the lignin components of the wood chips. After biopulping, the wood chips are mechanically or chemically pulped into individual fibers. The fungus and the produced enzymes are destroyed during the thermomechanical pulping process. Due, in large part, to the biochemical action of the fungi, less energy is now required to convert the chips to fibers. Some investigators claim energy savings of at least 30%. The easier conversion from chip to fiber means less damage to the wood fibers. The paper formed from these fibers is stronger. There are some drawbacks to biopulping, such as a reduction in the brightness and opacity of the resulting fibers. The production of higher quality papers is desirable. Use of biopulped fibers for this application will require improvements in brightness and opacity. Research is underway to develop strategies to address these drawbacks. Preliminary bleaching studies with hydrogen peroxide and addition of calcium carbonate to improve both brightness and opacity have met with early success. The present invention provides a method for producing pulp that addresses the above and other issues.

BRIEF SUMMARY OF THE INVENTION The present invention provides a process of producing thermomechanical pulp using a combined fungus/enzyme process in which the enzymes are produced in situ. In a first step, wood chips are treated with a lignin-degrading fungus using the biopulping process. Next, the enzymes that have been produced by the fungus are extracted from the wood chips in the form of a crude broth. After the wood chips are refined to a coarse pulp, the enzymes are used to treat the pulp prior to completing the refining process. Biopulping has been shown previously to reduce the energy requirements for mechanical pulping. Enzyme treatments have also been shown to be beneficial. This process combines these two processes by extracting and using the enzymes that are produced in the first biopulping treatment step. No additional production facilities are required to produce the enzymes. The expected benefits are energy savings and property improvements. In a particular aspect of the invention, a method for processing wood chips includes biopulping the chips, extracting at least one enzyme from the biopulped chips, performing a further pulping of the biopulped chips, and re-introducing the at least one extracted enzyme to the further pulped chips.

BRIEF DESCRIPTION OF THE DRAWINGS These and other features, benefits and advantages of the present invention will become apparent by reference to the following text and figures, with like reference numbers referring to like structures across the views, wherein: Fig. 1 illustrates the lignolytic enzyme activity change for the laccase enzyme, where thermomechanical pulping (TMP) is performed over a six hour treatment time on Picea abies (Norway Spruce) wood chips with fungal treatment using P. subserialis, T. versicolor and C. subvermispora, in accordance with the invention Fig. 2 illustrates the lignolytic enzyme activity change for the manganese peroxidase enzyme, for comparison with the results of Fig. 1, in accordance with the invention; and Fig. 3 illustrates a method for processing wood chips, in accordance with the invention.

DETAILED DESCRIPTION OF THE INVENTION The invention employs a fungus treatment and a subsequent enzyme treatment to process wood chips. In one approach, the wood chips are biopulped by inoculating them with a fungal treatment, such as a liquid mixture comprising a lignin-degrading fungus and a corn steep liquor. Enzymes which are formed as a result of the fungal inoculation are extracted from the wood chips. The enzymes can be extracted as a crude broth or pressate by applying mechanical pressure to the chips following the fungal treatment. A concentrated broth is then formed. A further pulping is performed, such as a first-stage thermomechanical pulping (TMP), to refine the biopulped chips to a coarse pulp. The resulting pulp is subsequently treated with the concentrated enzyme broth. This process has several advantages. First, the enzymes that are re-introduced into the coarse pulp are incubated on the wood and presumably adapted to reacting with the wood. Also, a separate enzyme production process is not necessary since the enzymes that are produced during the biopulping process are utilized. Finally, although mechanical energy is needed to extract the enzymes, the extraction weakens the wood chip structure, which should also reduce the necessary refiner energy in the first stage. The growth of the fungi on the wood chips is a relatively slow process compared to the normal processing time scales in the paper industry. The treatment of the wood chips with the lignin-degrading fungi can take anywhere from two to six weeks or longer depending on the degree of treatment desired. The treatment time can be shortened by using greater concentrations of fungi initially, but this would come at a higher cost. Previous related work has indicated that the inoculation amounts (5g/ton of chips) and treatment time of 2 weeks are reasonably feasible from an economic standpoint. Moreover, the use of a biological agent should not cause a concern of contamination or other health concerns relating to concentrated cultures of microorganisms since the organisms used are all naturally-occurring and limit their attacks to lignocellulosic materials. The invention is further described below based on Bartholomew, Jeremy, "A study of the lignolytic enzymes of Phlebia subserialis, and a comparative analysis of white-rot fungi on Picea abies for mechanical pulp", Masters Thesis, SONY-ESF (2003), incorporated herein by reference.

Picea abies preparation Picea abies (Norway spruce) was selected as the softwood for this example. However, different species of woods, including hardwoods and/or softwoods, can also be used. Moreover, the invention can be used with virgin wood or waste wood, including, e.g., kiln dried, air-dried and green wood from industrial, residential, sawmill, construction and demolition sources. In the present example, logs from a 79- year old tree were debarked with a 36-cm spoke shave, chipped in a Carthage 10-blade chipper, and air dried to approximately 15% moisture by spreading the chips on a tarp. The chips were then screened in a Williams classifier. All fractions were collected and the chips retained on the 15.8, 12.7 and 9.25-mm screens were pooled together and sealed in plastic bags, and stored at room temperature (approximately 24°C) for use throughout this study. TAPPI test method T- 257 cm-97 was followed for all subsequent testing and samples were taken from the pooled material as needed. TAPPI refers to the Technical Association of the Pulp and Paper Industry, Norcross, Georgia. The subject areas for TAPPI Test Methods and their numbering are: (a) Fibrous Materials and Pulp Testing, T 1-200 Series, (b) Paper and Paperboard Testing, T 400-500 Series, (c) Nonfibrous Materials Testing, T 600-700 Series, (d) Container Testing, T 800 Series, (e) Structural Materials Testing, T 1000 Series, and (f) Testing Practices, T 1200 Series. The suffix following the Test Method number indicates the category of the method. Test Method numbers consist of a capital T, followed by a space, then a number (assigned sequentially within several Test Method categories), another space, a two-letter designation of classification, a hyphen, and the last two digits of the year published. The two-letter designations for classifications are: (a) om = Official Method, (b) pm = Provisional Method, (c) sp = Standard Practice, and (d) cm = Classical Method.

Fungal Pretreatment of Wood Chips TAPPI test method T-412 om-94 was followed for moisture content determination. A 1500 g OD sample was weighed out for each bioreactor and brought up to 50% moisture content by soaking in distilled water. Bioreactors were cleaned and sterilized with a 10% (v/v) commercial Clorox bleach/90% water solution and rinsed with distilled water. Chips were layered in the reactor with 600 g on each layer; the reactor was loosely sealed with an aluminum foil cap covering the vent in the lid and then steamed for 10-minute under atmospheric conditions. The reactor was then cooled for approximately two hours until the temperature was below 30°C. The moisture content was brought up to 55% moisture by the addition 200 ml water collected during steaming plus additional distilled makeup water. Fresh fungal inoculum (2.3 ml) and 0.5% (v/v) unsterilized corn steep liquor (CSL) at 50% solids was added to the additional distilled makeup water. The fungal inoculum/corn steep liquor mixture, diluted with the distilled makeup water, was poured over the chips in the bioreactor and the cover replaced. Generally, the chips can be inoculated with the lignin-degrading fungus by providing a liquid mixture including the fungal inoculum, and applying the liquid mixture to the chips. The inoculated chips were then incubated under conditions favorable to the propagation of the lignin-degrading fungus through the chips. Specifically, the bioreactor was then placed in the incubation chamber at 27°C with forced continuous flow of warm humidified air at a rate of 0.028 cubic meters per minute. House air was measured by a flow meter and humidification was controlled by passing air through two water filled two-liter glass sidearm flasks (in series) through a fritted ground glass sparger. The sidearm flasks were immersed in a 4O0C water bath. From the hot water flasks, the warm humidified air passed though a water trap and a final filtering through a 0.2 micron Millipore air filter (for sterilization) before connecting to the individual bioreactors. «, At daily intervals, the warm humidified air flow-rate was measured and corrected if needed and the chips were checked for contamination. At weekly intervals, the water trap in the bottom of the incubation locker was emptied and one layer of chips was removed from the reactor placed in a plastic bag, sealed and frozen at -200C until further processing.

TMP Refiner Mechanical Pulp Production (KRK) Air-dried and screened Picea abies wood chips (800 g OD) were brought up to 10% moisture content and placed in the sample hopper on the pressurized refiner (Kumagai Riki Kogyo Co. Ltd., Tokyo, Japan, Model BRP45-30055). Low-pressure steam (32 kPag) softened the wood chips for three minutes. The TMP produced was sealed in a 40-liter Nalgene® carboy and refrigerated at 4°C until use.

Culture Supernatant Purification Purification involved monitoring laccase and manganese peroxidase activity and harvesting the mycelium from P. subserialis (RLG6074-sp), C. subvermispora (L- 14807 SS-3), and T. versicolor (FP-72074) on the first day after peak laccase activity. Mycelium was harvested from the liquid culture by centrifuging for 20 min at 10,000 rpm, followed by treating the crude supernatant with 10% (v/v) acetone and refrigerating for one hour at 4°C to precipitate any extracellular polysaccharide. The broth was centrifuged again for 20 minutes at 10,000 rpm and filtered through a Whatman glass microfiber GF/A 42.5-mm diameter filter. The resulting supernatant was concentrated in a DC-2 ultrafiltration unit (Amicon Corp., Danvers, Mass.) equipped with a 30-kDa molecular weight cutoff hollow fiber filter from an initial volume of 1000 ml to 100 ml. Enzyme activity was monitored at harvest time and after the final concentration.

Enzyme Treated TMP First-stage coarse thermomechanical pulp was treated with partially purified culture supernatant from P. subserialis, C. subvermispora, and T. versicolor at a dosage determined by normalizing to a manganese peroxidase enzyme activity of 1500 nkatal 1" l. Duplicate reaction vessels contained 2.0 g OD coarse refiner mechanical pulp that was suspended in 5% (w/v) 50-mM sodium acetate buffer (pH 4.5). The pulp in each reaction vessel was mixed with concentrated enzyme broth at a normalized enzyme activity of approximately 1.50 nkatal ml"1 manganese peroxidase. Laccase activity was measured and monitored throughout the experiment. For each fungus, one reaction vessel was setup in duplicate for analysis at 0, 30, 60, 90, 180 and 360-minute intervals in a constant temperature bath of 300C. Initial and final laccase and manganese peroxidase enzyme activity were measured for each time interval followed by a complete lignin analysis at each time interval to evaluate the effect of the enzymes on refiner mechanical pulp.

Soxhlet Resin Extraction TAPPI test method T-264 cm 97 details the procedure followed to report chemical analysis on an extractive free basis. Air-dried Wiley milled samples (approximately 10.0 g) of both pretreated wood samples and mechanical pulp were placed in an OD tarred 45 x 105-mm extraction thimble. The extraction thimble was placed into a 50-mm Soxhlet extractor fitted with an Allihn condenser and a 500-ml round bottom three-neck flask (Figure 11). Boiling chips were added to the boiling flask with 300 ml of the ethanol-benzene mixture. Samples were extracted for eight hours at brisk boiling with siphoning at approximately ten-minute intervals. After eight hours, the extraction thimbles were removed from the Soxhlet extractors, washed with 100% pure ethanol by placing the thimble in a 100 ml coarse ground glass crucible fitted on a 1000-ml sidearm flask. The thimble was returned to the Soxhlet extractor and extracted for four hours with 100% pure ethanol. The samples were transferred to a Buchner funnel and washed with hot water to remove the ethanol and then allowed to air dry for all subsequent carbohydrate and lignin analyses.

Enzyme Extraction from Wood Chips Picea abies chips were prepared as previously described, inoculated with Phlebia siibserialis, Ceriporiopsis subvermispora, and Trametes versicolor, and incubated for 30 days at 27°C with forced warm humidified air at a rate of 0.028 cubic meters per minute. The chips were thus incubated under conditions favorable to the propagation of the lignin-degrading fungus through the chips. Duplicate 500-g samples were removed from each bioreactor, and double-bagged in 6x9 zip lock bags. One bottom corner of the double bag was cut off with scissors. The stainless steel plates on the top and bottom pressing surfaces of the Williams press (Williams Apparatus Co., Watertown, NY) were cleaned first with soap and water and then dried with ethanol. The press was blocked up at a 45° angle and secured. The zip lock bag containing the sample was placed between the pressing surfaces and a clean 20-dram vial was placed under the cut corner of the bag. Pressure was applied (1500 psi) to the sample and the pressate was captured in the glass vial as a crude broth. Laccase and manganese peroxidase enzyme assays were performed on each vial to determine the enzyme present and enzyme concentration.

Enzymatic Treatment of TMP Extracellular lignolytic enzymes secreted into the production and growth media were identified, monitored for peak concentration within the production media, harvested for additional experimentation and finally concentrated ten-fold. The broth was centrifuged for 20 minutes at 10,000 rpm and filtered through a Whatman glass microfiber GF/A 42.5-mm diameter filter. The resulting supernatant was concentrated in a DC-2 ultrafiltration unit (Amicon Corp., Danvers, Mass.) equipped with a 30-kDa molecular weight cutoff hollow fiber filter from an initial volume of 1000 ml to 100 ml. Laboratory analysis of fungal growth established the initial growth conditions and approximate harvesting time for peak production. The enzyme concentration was then adjusted to 1.4 nkatal/ml and were used to treat lst-stage TMP as a method to reduce the amount of lignin within the pulp, reducing the electrical refining energy and thereby increasing pulp strength. This system can also be used as a first-stage biobleaching of mechanical pulp. Throughout the experiment, the enzyme activity levels were monitored, followed by a lignin analysis of the TMP. Table 1 lists the laccase and manganese peroxidase enzyme activity levels throughout the pulp treatment. Initial activity was measured from the concentrated production medium before addition to each sample and then the manganese peroxidase enzyme concentration was normalized to approximately 1.50 nkatal ml"1 for the zero-time condition. The laccase and manganese peroxidase activities were measured and monitored for the change in activity over time.

Table 1: Enzyme activity change over the 6-hour treatment time of thermomechanical pulp with partially purified lignolytic enzymes from P. subserialis, T. versicolor and C. subvermispora Initial 0 30 60 90 180 360 Activity minute minute minute minute minute minute

P. subserialis harvested at 7 days

O-SSS) 12-16 7 β3 7AS 7-55 6-67 β2β 6-95

(nkafaLp 242 1 -52 149 1 -42 1-37 1-32 1 -28

T. versicolor harvested at 10 days ΛaC??/Sen 1849.8 822.6 819.2 815.4 813.5 811.0 797.2 (nkatal/ml)

(πkatalL) 362 1-61 159 157 1 53 1 52 146

C. subvermispora harvested at 12 days / L.aCf?/Sen 864.9 864.9 865.2 862.4 858.8 854.2 852.7 (nkatal/ml) / | M+n w IN 1 -56 1.56 1.54 1.49 1.38 1.27 1.16 (nkatal/ml)

Laccase from P. subserialis showed a 22% decrease in activity while T. versicolor and C. subvermispora showed much smaller changes in activity, 3.1 and 1.4%, respectively. This difference may not be significant due to the much lower laccase activity in the enzyme broth from P. subserialis. Initial manganese peroxidase activity levels were on the same order of magnitude for all three fungal extract applications. The range in overall manganese peroxidase activity loss was from 15.8% for P. subserialis to 8.9 and 25.7% loss for T. versicolor and C. subvermispora, respectively. Figures 1 and 2 chart the enzyme activity throughout the experiment and show the decrease in activity over the life of the experiment. In particular, Fig. 1 illustrates the lignolytic enzyme activity change for the laccase enzyme, where thermomechanical pulping (TMP) is performed over a six hour treatment time on Picea abies (Norway Spruce) wood chips with fungal treatment using P. subserialis, T. versicolor and C. subvermispora. Fig. 2 illustrates the lignolytic enzyme activity change for the manganese peroxidase enzyme, for comparison with the results of Fig. 1. In Fig. 1, the horizontal axis denotes time, in minutes, from 0 to 400 minutes, while the left hand vertical axis denotes T.v. and Cs. laccase activity, and the right hand vertical axis denotes P.s. laccase activity. In Fig. 2, the horizontal axis denotes time, in minutes, from 0 to 400 minutes, while the left hand vertical axis denotes manganese peroxidase activity. Table 2 outlines the results from lignin analysis on the TMP, showing that the lignolytic enzyme treatment from C. subvermispora removed up to 3.66% of the lignin in the sample over a six-hour period, while P. subserialis and T. versicolor reduced the lignin content by similar amounts, 2.35 and 2.67%, respectively. P. subserialis showed a significant decrease in lignin content at the 90-minute sample; however, no significant change occurred after that time interval. Both T. versicolor and C. subvermispora appeared to continually decrease lignin content throughout the experiment. A longer running experiment is expected to show greater lignin losses with increased treatment time, with the enzyme activity monitored as a theoretical stopping point. These small changes in the lignin content are significant because they compare with a one to two week biopretreatment stage. Table 2: Klason lignin analysis of a Picea abies TMP treated with partially purified

enzymes from P. subserialis, T. versicolor and C. subvermispora over 6 hours

Phlebia subserialis 30 29.17 0.12 0.14 60 28.71 0.18 1.74 90 28.52 0.60 2.42 180 28.64 0.04 1.99 360 28.54 0.23 2.35

Trametes versicolor 30 28.86 0.20 1.21 60 28.61 0.53 2.10 90 28.92 0.07 1.00 180 28.35 0.25 3.03 360 28.45 0.33 2.67

Ceriporiopsis 30 29.28 0.05 -0.24 subvermispora 60 28.70 0.33 1.78 90 28.77 0.10 1.53 180 28.20 0.38 3.58 360 28.18 0.40 3.66

Lignolytic Enzyme Activity Extracted from Picea abies

Fresh Picea abies samples were treated with the three species of white-rot fungi to identify the enzymes present in the internal wood structure, measure the activity level and make comparisons with enzyme production under laboratory conditions (Table 3). A novel procedure for isolating extracellular enzymes present within the internal wood structure allowed the comparison. Specifically, duplicate 500-g samples were removed from each bioreactor, and double-bagged in 6x9 zip lock bags. One bottom corner of the double bag was cut off with scissors. The stainless steel plates on

the top and bottom pressing surfaces of the Williams press were cleaned first with soap and water and then dried with ethanol. The press was blocked up at a 45° angle and

secured. The zip lock bag containing the sample was placed between the pressing

surfaces and a clean 20-dram vial was placed under the cut corner of the bag. Pressure was applied (1500 psi) to the sample and the pressate was captured in the glass vial. The ability of P. subserialis to repeatedly produce laccase under biopulping conditions

was significant due the inability to repeatedly produce detectable activity in the

laboratory under controlled conditions with this organism. There were large variations

in detectable enzymes and activity levels under laboratory conditions and the ability to characterize the fungi under non-induced conditions, while growing in a biopretreatment environment, hold significant potential.

Table 3: Comparison of laccase and manganese peroxidase enzyme activity from P. subserialis, T. versicolor and C. subvermispora; Extracted from Picea abies and laboratory growth conditions

Picea abies Laboratory enzyme activity enzyme activity ± std. dev. at harvest time Phlebia subserialis

(nSml) 3-66 ± 0 07 4-47 @ 7 days Manganese peroxidase 0.742 ± 0.03 0.229 @ 7 days (nkatal/ml)

Trametes versicolor Laccase 676.5 @ 10 (nkatal/ml) όm ± u υu days Manganese n cr. . ~ .n peroxidase 1.25 ± 0.05 0 ^4 J 10 (nkatal/ml) αays

Ceriporiopsis subvermispora Laccase o αo + n ? 214.2 @ 12 (nkatal/ml) ZΛiZ ± v z days Manganese peroxidase 0.322 + 0.014 1.61 @ 12 days (nkatal/ml)

Fig. 3 illustrates a method for processing wood chips, in accordance with the invention. At block 300, wood chips are biopulped by inoculating the chips with a

lignin-degrading fungus, and incubating. At block 310, an enzyme is extracted from

the biopulped chips as a crude broth and/or pressate. At block 320, a concentrated

broth with the enzyme is prepared. At block 330, a further mechanical and/or chemical pulping of the chips is performed. At block 340, the extracted enzyme is re-introduced to the chips by mixing the chips with the concentrated broth. The invention has been described herein with reference to particular exemplary embodiments. Certain alterations and modifications may be apparent to those skilled in the art, without departing from the scope of the invention. The exemplary embodiments are meant to be illustrative, not Hmiting of the scope of the invention, which is defined by the appended claims.