GB1560021A - Biological degradation of lignocellulose - Google Patents

Description

(54) BIOLOGICAL DEGRADATION OF LIGNOCELLULOSE (71) We, GENERAL ELECTRIC COM PANY, a Corporation organized and existing under the laws of the State of New York, United States of America, of 1 River Road, Schenectady 12305, State of New York, United States of America, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following state ment: - The present invention relates to the biological pre-treatment of lignocellulosic materials to at least partially remove the lignin significantly.

The bulk of renewable organic matter on earth consists of lignocellulose. The cellulose component of this material is a linear polymer of glucose, and in a pure or relatively pure form it can be converted to a variety of useful products such as paper, meat, milk, sugar, ethanol and methane. Except in the form of cotton and some bacterial polymers, cellulose does not occur pure naturally but is present in the tissue of land plants complexed with lower molecular weight, alkalisoluble polysaccharides collectively termed hemicelluloses and with lignin, a high molecular weight three-dimensional random polymer of phenylpropane alcohols. Lignin protects cellulose from enzymatic hydrolysis to soluble sugars. The greater the lignin content of lignocellulose, the more resistant is its cellulose component to enzymatic attack.

Cellulose can be freed of lignine by physical means, e.g. fine grinding, and by chemical extraction at elevated temperatures, but both methods are expensive relative to the value of the final product A number of mold spedes have been shown to degrade lignin enzymatically, but their use has not been commercially attractive because they grow so slowly. Typically, two months or more are required to effect degradation of 50% of the lignin in a woody substrate. The lignin-degrading molds previously described have been mesophilic; that is, they grow best at temperatures of between 20--30"C.

The present invention relates to a thermo tolerant mold which has been found to be a rapid degrader of lignin. Also, it has been found that the lignin is degraded appreciably only under damps opposed to submerged conditions.

The present process uses the thermotolerant mould, Crysaspvorium pruinosum, which was obtained from the American Type Culture Collection, Rockville, Maryland, U.S.A. In the art Chrysosporium prnosum is considered an equivalent of the thermotolerant molds Phanerochaete chrysosporium and Sporotri- churn taru1entum.

Briefly stated, the present process for de grading lignocellulose comprises providing a substrate composed of lignocellulose dampened with nutrient mineral solution at a pH from 4 to 5, said substrate being comprised of 10% by weight to 80% by weight of lignocellulosic solids based on the total amount of ligno cellulosic solids and nutrient solution, inocu lating said substrate with Chrysosporium pruinosum, maintaining the inoculated sub strate at a temperature ranging from 20"C to 45"C to grow said Chrysosporium pncino- sum which produces enzyme systems that de grade said lignocellulose, and allowing said mold to grow at said temperature until at least a significant amount of said lignocellulose is degraded.

Those skilled in the art will gain a further and better understanding of the present invention from the detailed description set forth below, and forming a part of the specification, in which: Figure 1 shows lignin and cellulose de gradation by Chrysosporium pruinosum grow ing on a submerged substrate; and Figure 2 shows lignin and cellulose degrada tion by Chrysosporium pruinosurn growing on a damp substrate.

In the present process any lignocellulosic maaterial is useful. Representative of the lignocellulosic materials are wood, manure fiber, paper, straw and agricultural wastes.

Preferably, the lignocellulosic material is ground by any suitable means, preferably to a particulate size less than 5 mm. to increase its surface area and thereby significantly or substantially increase the rate of degradation.

Specifically, the more surface area provided by the lignocellulose, the quicker is the invasion process by the mold mycelium.

The particular nutrient mineral solution used is not critical except that it must have a pH of F5 to be operable for growing the mold. Specifically, the solution is largely inorganic comprised of a number of minerals in solution to provide the major nutrient ions such as sodium, potassium, phosphate, sulfate, magnesium and iron and usually includes an organic chelating agent to keep iron from precipitating and thiamine which is a necessary vitamin for Chrysosporium pruinosum mold growth and is included in the nutrient solution if it is not already present in the lignocellulosic substrate or if not present in sufficient amount to promote growth of the mold. The absolute concentrations of the nutrients in the solution are not critical as long as they are present in adequate amounts for the Chrysosporium pruinosum cells to grow but not so high as to inhibit growth. Standard bacteriological growing media are useful here- in as nutrient mineral solution because they all contain the major ions necessary for mold growth. and the exact formulation may be modified in the standard manner depending on the composition of the particular lignocellulosic mass, i.e. the extent to which the nutrients are already contained in the substrate.

In carrying out the present process the lignocellulosic mass is dampened by the nutrient solution to produce the substrate on which the Chrysorponum pruinosum mold is grown.

This can be done by a number of conventional techniques such as dropping or spraying the solution onto the lignocellulosic mass. When the mass is in particulate form, it can be admixed with the nutrient solution if desired.

After addition of the solution to the lignocellulosic mass, a short period of time should be allowed for the solution to equilibrate, i.e.

spread itself through the mass.

The lignocellulosic mass is contacted with an amount of nutrient solution to produce a damp substrate comprised of from about 10% by weight to about 80% by weight of lignocellulosic solids based on the total amount of lignocellulosic solids and amount of nutrient mineral solution. Amounts of lignocellulosic solids less than about 10% by weight result in no significant degradation of lignine whereas with amounts of solids higher than 80% by weight the mold does not grow. Satisfactory results are achieved at 20% by weight lignocellulosic solids concentration, i.e. 20 grams of lignocellulosic solids/80 grams of mineral solution+20 grams of lignocellulosic solids.

As used herein by a damp lignocellulosic substrate it is meant a substrate comprised of 10% by weight to 80% by weight of lignocellulosic solids based on the total amount of lignocellulosic solids and nutrient solution present. This is in contrast to a submerged substrate or condition wherein typically about 0.5% by weight of lignocellulosic solids is used based on the total amount of lignocellulosic solids and nutrient solution, i.e. 0.5 grams of lignocellulosic solids/99.5 grams of nutrient solution+0.5 gram of lignocellulosic solids.

The damp substrate may be inoculated with Chrysosporium pruinosum cells or with spores of the Chrysosporium pruinosum cells.

The inoculation can be carried out by a number of techniques which allow as much contact as possible between the Chrysosporitzm pruinosum cells and the substrate. Preferably, the spores or Chrysosporium pruinosum cells are initially suspended in a liquid medium such as water usually at room temperature and the suspension dropped or sprayed onto the substrate.

The inoculated substrate is then incubated with sufficient aeration at a temperature ranging from 20"C to 45"C to grow the Chrysosporium prunwsum cells. The incubation can be carried out by a number of techniques which maintain the required growing temperature and also the required solids concentration such as a hot air incubator or by contacting the inoculated substrate with flowing hot air.

Preferably, for fastest growth of the mold, the growing temperature ranges from 38"C to 40"C.

While the mold is growing, i.e. incubating, it is consuming lignocellulose and producing enzyme systems indicated to be lignase for degradation of the lignin and cellulase for degradation of the cellulose.

The period of time that the inoculated substrate is incubated, i.e. the period that the mold is grown at a particular temperature is determinable empirically and depends on the extent of degradation of the particular mass of lignocellulose desired. The extent of degradation of the lignocellulosic mass can be determined in a conventional manner, generally by determining the amounts of lignin and cellulose remaining.

The resulting product which is at least significantly delignified, lignin-depleted or degraded can then be used as a source of cellulose-or sugar-rich animal feed or fermentation substrate. The process will also produce soluble derivatives of lignin which may be useful as chemical feedstocks.

Besides being required for the process to work, the damp condition allows greater amounts of substrate to be treated per unit volume of culture relative to conventional submerged microbial processes.

The invention is further illustrated by the following experiments where materials and methods were as follows: Organism Chrysosporium pruinosum (Gilman et Abbot) Carmichael, ATCC 24782, was used in all experiments and was obtained from the American Tvpe Culture Collection, Rockville, Maryland, U.S.A. This organism has recently been identified as the imperfect state of the fungus Pharerochaete chrysosporium.

Media The nutrient mineral medium solution of the following composition was used: 'NH4)2SOf, 5.0 grams; KH2PO4, 6.04 grams; Na2HPO4, 0.85 grams; "trace elements" solu tion, 10 ml; distilled water added to 990 ml.

The trace elements solution had the following composition: MgSO4 . 7H2O, 5.0 g; ZnSO4 . 7H2O, 0.2 g.; FeSO4 . 7H,O, 0.5 g.; MnSO4 . 4H2O, 0.5 g.; CaC12, 0.5 g.; versenol, 5.0 g.; distilled water to 250 ml.

The pH of the medium was set at 5.0 before autoclaving by adding a few drops of concentrated HCl. An aliquote of a filter-sterilized solution of thiamine HCl (1 mg/ml) was added to the medium after autoclaving to give a final thiamine concentration of 1 ag/ml.

Glucose-mineral-agar medium was prepared by adding 40 ml of a sterile 25% glucose solution to 960 ml of the hot sterile mineral medium described above containing 15 g. of agar.

Preparation of the Lignocellulosic Substrate Two hundred grams of dry manure were added to 2 litres of distilled water in a one gallon Waring blender and shredded at low speed for 15 seconds. The mixing was repeated three times with 15 second intervals between mixings. The suspension was transferred to a 30 cm. diam. X 61 cm. high glass jar and diluted 1:1 with distilled water. This sus pension was mixed with a propeller connected to a shaft and variable speed motor. The motor speed was set so that all material was suspended, and the suspension was stirred for 1.5 hours. The mixing speed was then reduced for 0.5 hr. to allow sand to settle while the lighter fiber particles remained suspended.

With the motor running at reduced speed, the suspended particles were siphoned off and caught on a 20 mesh (0.238 mm openings) screen. This material was washed on the screen with distilled water. The stirring, settling, screening process was repeated three times, and the resulting fibers were dried at 65 C for three days. The dried fibers were ground in a meat grinder to redisperse the particles, and these were stirred in glass jars.

Fiber was dried in a 65"C oven for at least two days prior to use until a constant weight was obtained. Since the material is somewhat hygroscopic in air, it was found convenient to place it in a covered petri dish on the surface of a hot plate while weighing samples. The hot plate surface was maintained at 100"C.

Dry particulate manure fiber prepared in this manner had an average size of 1-3 mm in length and 0.5 mm in width. It contained 14+1.5% by weight ash, 37 + 2% by weight reducing sugar (cellulose + hemicellulose), 37+1.5 % by weight lignin and these are the amounts on which % by weight solubilized in Figures 1 and 2 were based.

Preparation of Inoculum Cultures of C. pruinosum were grown at 38"C for 5 days on glucose-mineral agar plates. Ten ml of sterile distilled water was added to each plate, and the surface growth was gently suspended with a glass spreader.

This suspension, which contained mainly spores and some fragments of vegetative mycelium was used as the inoculum for all cultures.

Conditions of Growth Cultures were grown on submerged substrates in mineral medium solution in shake flasks and on damp substrates on the surface of mineral agar plates.

Submerged (shake flask) cultures contained 50 ml of mineral medium solution and 50 mg of dry, washed manure fiber in 259 ml erlenmeyer flasks. The flasks were autoclaved for 20 minutes and inoculated after cooling with 0.1 ml of the spore suspension described above. Cultures were incubated at 38"C at 80% relative humidity with rotary shaking at 230 rpm in a New Brunswick model G26 incubator-shaker.

Damp (agar surface) cultures were prepared in the following manner. Sterile Nuclepore membrane filters (5 y pore size, 47 mm diameter) were placed, dull side down, on the surface of 25 mm X 150 mm petri plates containing approximately 200 ml of mineral agar medium, composed of the nutrient mineral medium solution thickened with 1.2% by weight agar. One filter was used per plate.

Two hundred mg of dry, sterile manure fiber was spread on the surface of each filter. The 200 mg fiber samples were dispensed from 16 mmX 125 mm screw-capped Pyrex (Registered Trade Mark) tubes. This was equivalent te a 20% by weight solids concentration, i.e.

20% bv weight fiber/20% by weight fiber+ 80 % by weight nutrient mineral solution.

Each pile of fiber was inoculated at the periphery with two drops (0.1 ml) of spore suspension prepared as described above. All cultures were incubated at 38"C. Relative humidity in the incubator was maintained at 80% using water-filled trays.

Analytical Methods 1. Measurements of residual organic and inorganic constituents of fiber and fiber cultures for Figures 1 and 2.

a. Agar plate cultures (Damp Cultures).

Filters containing fiber or fiber plus mycelium were removed from the agar plates, and the fiber-mycelial mats were scraped off the filters and washed with distilled water into tared crucibles. The crucibles were dried at 65"C to a constant weight and then ashed overnight in an oven at 550"C. The organic fraction was defined as that material which vaporized at 5500 C.

b. Shake flask cultures (Submerged Cul- tures).

The contents of a pair of shake flasks were combined and vacuum - filtered through an 0.4 ,a, 47 mm diameter Nuclepore filter to trap particulate materials. Residue remaining on the sides of the flasks was washed onto the filter with a minimal amount of distilled water. The filtered residue was transferred to tared crucibles and dried and ashed as described above.

2. Analysis for cellulose, hemicellulose and lignin for Figures 1 and 2.

Samples of insoluble material were collected as described above except that they were transferred to 25 mm i.d.X50 mm high glass weighing vials and dried at 65"C.

Dry samples of fiber cr fiber plus mycelium were mixed with 10 ml of 72% sulfuric acid and allowed to stand for three hours with hourly mixing for a few seconds. These were then diluted to 50 ml with distilled water and allowed to stand overnight. Each sample was then vacuum - filtered through a tared 0.4 u, 47 mm Nuclepore filter. Aliquotes of the filtrate were retained for carbohydrate (reducing sugar) assays. In this assay 0.1 ml of filtrate was mixed with 3.9 ml of a reagent containing 1.0% thiourea and 0.05% anthrone in 72% H2SO4. The mixture was heated to 95"C for 10 minutes, cooled to room temperature, and the optical density at 610 nlu was measured. Glucose was used as a standard.

These assays were routinely performed using a Technicon model AA-1 autoanalyzer.

The residue on the filter was washed with distilled water until the filtrate pH reached five as measured with pH paper. The filter plus residue was dried overnight at 65"C and weighed. The filters and samples were then transferred to tared crucibles and ashed overnight at 550"C. Lignin was defined as the fraction of the sample (excluding the filter) which vaporized. The filters contained less than 0.5 mg ash.

Results Figures 1 and 2 illustrate the present invention. In Figures 1 and 2, two by weight solubilized is based on initial content in the fiber sample.

Figure 1 illustrates the degradation of manure fiber constituents by Chrysospmium pruinosum growing in submerged (shake flask) cultures. Each data point represents the combined contents of two shake flasks. Single points indicate that replicates give identical values. Dashed lines indicate uninoculated controls.

Figure 1 shows the loss of various fiber components with time in submerged (shake flask) cultures and that little or no lignin is solubilized, i.e. degraded, by C. pruinosum under submerged conditions relative to the un- inoculated controls over the thirty-dav period of active degradation. About 50% of the carbohydrate (cellulose and hemicellulose) fraction and 40% of the total organics are solubilized in this period.

Figure 2 illustrates the degradation of manure fiber constituents by Chrysosporium pruinosum growing in damp fiber on the surface of mineral agar plates. Each data point represents the contents of one plate. All experiments were run in duplicate. Dashed lines indicate uninoculated controls.

Figure 2 shows the loss of various fiber components with time in damp fiber cultures on the surface of mineral agar plates and illustrates that extensive degradation of lignocellulose occurs preferentially on non-submerged substrates i.e. damp substrates, and that a solids concentration of about 20pyx by weight allows rapid lignocellulose degradation.

Specifically, fifty percent of the lignin, 80% of the carbohydrate (cellulose and hemicellulose) and 75 of the total organics are solubilized after 12 days of incubation after which degradation appears to stop. Less material is solubilized in the uninoculated controls under these conditions than in the shake flasks.

Table I shows lignin and cellulose degradation rates for Polyporus versicolor, a wellstudied mesophilic lignin-degrading mold, and for C. pruinosum the present thermo-tolerant lignin-degrading mold as illustrated in Figure 2.

TABLE I Lignocellulose Degradation by a Mesophilic Mold (Polyporous Versicolor) and a Thermotolerant Mold (Chrysosporium Pruinosum) % Cellulose % Lignin Organism Degraded Time Degraded Time P. versicolor'^' 76 245 days 50 168 days C. pruinosum 80-90 12 days 50 12 days ' & ) E. B. Cowling, Comparative Biochemistry of the Decay of Sweetgum Sapwood by White-Rot and Brown-Rot Fungi. U.S.D.A. Tech. Bull. W1258, (1961).

The data indicate that under appropriate cultural conditions enough lignin can be removed biologically to make 8590% of the cellulose susceptible to degradation by cellulase. The rate and yield data for cellulose degradation shown in Figure 2 are minimum values because the cell wall sugar residues contribute to the cellulose values obtained, and the cultures used were started from small spore inocula. Using a larger inoculum of cellulase and "lignase"induced vegetative cells, i.e. the C. pruinosum, would presumably shorten the incubation period required to reach maximum cellulose degradation.

Figure 2 shows that significant amounts of lignin can be degraded, and 8090% of the substrate's cellulose content can be exposed to the action of cellulase enzymes. The manure fiber used in the experiments outlined in Figures 1 and 2 is rich in lignin relative to other natural organic materials. Cellulose digestability is inversely correlated with lignin content, suggesting that the present thermotolerant mold's performance on a resistant substrate such as manure fiber indicates equal or better digestive capacity on substrates containing less lignin such as wood. In fact, additional experiments have shown good lignin and cellulose digestion using newsprint as a growth substrate. Newsprint is derived largely from lignified wood fibers.

Our co-pending British Patent Application No. 35156/77 (Serial No. 1,560,022) claims a process for degrading lignocellulose which comprises providing a substrate composed of lignocellulose dampened with nutrient mineral solution of a pH from 4 to 5, said substrate being comprised of 10% by weight to 80% by weight of lignocellulosic solids based on the total amount of lignocellulosic solids and nutrient solution, inoculating said substrate with Chrysosporium pntmosum, maintaining the inoculated substrate at a temperature ranging from 20"C to 45"C to grow said Chrysos ponum pruinosum which produces enzyme systems that degrade said lignocellulose, and elevating said temperature to a temperature ranging from 50"C to 70"C, at which elevated temperature said Chrysosporium pruinosum stops growing but at which degradation of said lignocellulose continues significantly.

WHAT WE CLAIM IS:- 1. A process for degrading lignocellulose which comprises providing a substrate composed of lignocellulose dampened with nutrient mineral solution at a pH from 4 to 5, said substrate being comprised of 10% by weight to 80% by weight of lignocellulosic solids, based on the total amount of lignocellulosic solids and nutrient solution, inoculating said substrate with Chrysosporium pnunosum, and maintaining the inoculated substrate at a temperature ranging from 20"C to 45"C to grow said Chrysosponum prumosum which produces enzyme systems that degrade said lignocellulose, and allowing said mold to grow at said temperature until at least a significant amount of said lignocellulose is degraded.

2. A process according to claim 1 wherein said substrate is inoculated with a liquid suspension of spores of said Chrysosporium prza7zesum.

3. A process according to claim 1 wherein said substrate is comprised of about 20% by weight of lignocellulosic solids based on the total amount of said lignocellulosic solids and nutrient solution.

4. A process according to claim 1 wherein said inoculated substrate is maintained at a temperature of 38"C to 40"C to grow said Chrysosponum pnunos urn.

5. A process according to claim 1 wherein said lignocellulose is in particulate form.

6. A process according to Claim 1 and substantially as hereinbefore described with reference to Fig. 2 of the accompanying Drawings.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (6)

**WARNING** start of CLMS field may overlap end of DESC **. The data indicate that under appropriate cultural conditions enough lignin can be removed biologically to make 8590% of the cellulose susceptible to degradation by cellulase. The rate and yield data for cellulose degradation shown in Figure 2 are minimum values because the cell wall sugar residues contribute to the cellulose values obtained, and the cultures used were started from small spore inocula. Using a larger inoculum of cellulase and "lignase"induced vegetative cells, i.e. the C. pruinosum, would presumably shorten the incubation period required to reach maximum cellulose degradation. Figure 2 shows that significant amounts of lignin can be degraded, and 8090% of the substrate's cellulose content can be exposed to the action of cellulase enzymes. The manure fiber used in the experiments outlined in Figures 1 and 2 is rich in lignin relative to other natural organic materials. Cellulose digestability is inversely correlated with lignin content, suggesting that the present thermotolerant mold's performance on a resistant substrate such as manure fiber indicates equal or better digestive capacity on substrates containing less lignin such as wood. In fact, additional experiments have shown good lignin and cellulose digestion using newsprint as a growth substrate. Newsprint is derived largely from lignified wood fibers. Our co-pending British Patent Application No. 35156/77 (Serial No. 1,560,022) claims a process for degrading lignocellulose which comprises providing a substrate composed of lignocellulose dampened with nutrient mineral solution of a pH from 4 to 5, said substrate being comprised of 10% by weight to 80% by weight of lignocellulosic solids based on the total amount of lignocellulosic solids and nutrient solution, inoculating said substrate with Chrysosporium pntmosum, maintaining the inoculated substrate at a temperature ranging from 20"C to 45"C to grow said Chrysos ponum pruinosum which produces enzyme systems that degrade said lignocellulose, and elevating said temperature to a temperature ranging from 50"C to 70"C, at which elevated temperature said Chrysosporium pruinosum stops growing but at which degradation of said lignocellulose continues significantly. WHAT WE CLAIM IS:-

1. A process for degrading lignocellulose which comprises providing a substrate composed of lignocellulose dampened with nutrient mineral solution at a pH from 4 to 5, said substrate being comprised of 10% by weight to 80% by weight of lignocellulosic solids, based on the total amount of lignocellulosic solids and nutrient solution, inoculating said substrate with Chrysosporium pnunosum, and maintaining the inoculated substrate at a temperature ranging from 20"C to 45"C to grow said Chrysosponum prumosum which produces enzyme systems that degrade said lignocellulose, and allowing said mold to grow at said temperature until at least a significant amount of said lignocellulose is degraded.

2. A process according to claim 1 wherein said substrate is inoculated with a liquid suspension of spores of said Chrysosporium prza7zesum.

3. A process according to claim 1 wherein said substrate is comprised of about 20% by weight of lignocellulosic solids based on the total amount of said lignocellulosic solids and nutrient solution.

4. A process according to claim 1 wherein said inoculated substrate is maintained at a temperature of 38"C to 40"C to grow said Chrysosponum pnunos urn.

5. A process according to claim 1 wherein said lignocellulose is in particulate form.

6. A process according to Claim 1 and substantially as hereinbefore described with reference to Fig. 2 of the accompanying Drawings.