DE2746873A1 - BIODEGRADATION OF LIGNOCELLULOSE - Google Patents

Description

Biodegradation of lignocellulose

The invention relates to the biological pretreatment of lignocellulosic materials in order to at least partially remove the lignin to remove.

Most of what is left on earth has to be reprocessed organic material consists of lignocellulose. The cellulose part of this material is a linear glucose polymer, and in pure or relatively pure form it can be converted into a variety of useful products, such as • Paper, meat, milk, sugar, ethanol and methane. Excepted in the form of cotton and some bacterial polymers, cellulose does not naturally occur in its pure form, but is present in the tissue of land plants, complexed with low molecular weight, alkali-soluble polysaccharides, which are collectively referred to as hemicelluloses, and with lignin, which is a high-molecular, three-dimensional is a random polymer of phenylpropane alcohols.

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Lignin protects the cellulose from enzymatic hydrolysis to soluble sugars. The greater the lignin content of the lignocellulose is, the more resistant is the cellulose component to enzymatic intervention.

Cellulose can be physically freed from the lignin, e.g. by fine grinding and chemical extraction at elevated levels Temperatures, but both processes are expensive relative to the value of the final product.

While a number of mold species have been shown to enzymatically degrade lignin, theirs has been more commercial Not given because they are growing so slowly. It usually takes two or more months to break down effect of 50% of the lignin in a woody substrate. The previously known, lignin-degrading mold species have been described as mesophilic, i.e. they grow best at temperatures between 20 and 30 C.

The present invention now uses a thermostable mold that rapidly degrades lignin. It was also found that the lignin is only noticeably degraded under steam - in contrast to the submerged state.

The method according to the invention makes the lignocellulose freed from lignin, making the cellulose accessible to fermentative degradation.

'In the method according to the invention, the thermostable Mold Chrysosporium pruinosum, that of the American

used,

Culture collection point in Rockville, Maryland, USA, \ The mold Chrysosporium pruinosum was regarded as equivalent to the thermostable molds Phanerochaete chrysosporium and Sporotrichum pulverulentum .

The method according to the invention for breaking down lignocellulose comprises preparing a substrate composed of lignocellulose moistened with nutrient solution, whose pH value is between 4 and 5. The substrate is made

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from about 10 to about 80% by weight of lignocellulosic solids, based on the total amount of lignocellulose solids and nutrient solution. The soaked substrate is made with Chrysosporium pruinosum inoculated, and the inoculated substrate kept at a temperature of about 20 to about 45 ° C to give the Chrysosporium pruinosum, which creates enzyme systems that break down lignocellulose, then the temperature is raised 50 to 70 C, at which the growth of Chrysosporium pruinosum stops, but at which the lignocellulose degradation continues significantly,

Exemplary of the lignocellulosic materials that can be used in the method according to the invention are Wood, manure fiber, paper, straw and agricultural waste. The lignocellulosic material is preferably with a ground suitable device, preferably to a particle size of less than 5 mm, in order to enlarge the surface and to markedly or considerably increase the rate of degradation. The more surface the lignocellulose has, the faster the mold mycelium penetrates.

The nutrient solution used in the individual case is not critical, with the exception that they have a pH value of 4 to 5 must in order to favor the growth of the mold. The solution consists of a number of minerals that are the main nutrients, such as sodium, potassium, phosphate, sulfate, magnesium and iron, and it usually contains an organic, Chelating agent to keep iron from precipitating and also contains thiamine, which is essential for growth of Chrysosporium pruinosum is a necessary vitamin. The latter is only added to the nutrient solution if it is not already or insufficiently present in the lignocellulosic material. The absolute concentrations of the nutrients in the solution are not critical as long as adequate amounts are present for Chrysosporium pruinosum cells to grow but the concentration must not be so high that it hinders growth. Standard bacteriological media can be used as a nutrient mineral solution, as they are all

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Meal growth contain necessary main ions and the exact Formulation of the medium can be modified depending on the composition of the respective lignocellulose can, i.e. to the extent that the nutrients are already present in the lignocellulosic material.

To carry out the method according to the invention, the Lignocellulosic material moistened with the nutrient solution, to obtain a substrate on which the Chrysosporium pruinosum mold can grow. This moisturizing can be done by a number of conventional techniques, such as dripping or spraying the solution onto the lignocellulosic mass. If this mass is in the form of particles, it can be mixed with the nutrient solution. After adding the Solution to the lignocellulosic mass, the solution is allowed to equilibrate, i.e., disperse, for a short period of time the solution through the mass.

Bring the lignocellulosic mass in such an amount Nutrient solution in contact that the wetted substrate is about 10 to about 80 wt .-% lignocellulosic solids consists, based on the total amount, of lignocellulose solids and NMhrminerallösung. For quantities less than 10% by weight of lignocellulose solids does not result in any noticeable degradation of the lignin, while with more than 80% by weight Lignocellulose solids the mold does not grow. Satisfactory Results are obtained at a lignocellulosic solids concentration of 20% by weight, i.e. with 20 g lignocellulose solids in a mixture of 80 g mineral solution and 20 g lignocellulose solids.

The moistened one used in the process according to the invention Lignocellulosic substrate differs from an immersed substrate, which usually contains 0.5% by weight

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Lignocellulosic solids are present based on that Total amount of lignocellulose solids and nutrient solution, e.g. 0.5 g of lignocellulose solids for a mixture of 99.5 g nutrient solution and 0.5 g lignocellulose solids.

The moistened substrate can with Chrysosporium pruinosum cells or inoculated with spores of such cells. Inoculating can be done using a number of techniques, which allow maximum contact between the Chrysosporium pruinosum cells and the substrate. Preferably the spores or cells are suspended in a liquid medium, such as water, usually at room temperature, and the suspension is dripped or sprayed onto the substrate.

The inoculated substrate is then incubated with adequate ventilation at a temperature in the range of 20 to 4b C, to grow the Chrysosporium pruinosum cells. Incubation can be carried out by a number of techniques, which maintain the required growth temperature and the necessary solids concentration, such as by means of a hot air incubator or by bringing the inoculated substrate with flowing hot air. The fastest growth of the mold takes place at temperatures of 38 to 40 ° C.

When growing, i.e. when incubating, the mold consumes lignocellulose and creates enzyme systems called lignase 'to break down the lignin and cellulase to break down the cellulose.

After the desired growth of the mold is achieved, increases the temperature to 50 to 70 ° C. At this elevated temperature, mold growth stops, but it works Lignocellulose degradation continues. A temperature of 50 to 60 ° C is preferred for the fastest degradation. The mold can be At this elevated temperature, the substrate is no longer used, but the lignose and cellulose enzymes can continue to work. the

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elevated temperature is maintained until a suitable amount of the lignin is removed. Cellulose is also used at the same time reduced.

The length of time during which the inoculated substrate is incubated, i.e. the time during which the mold at a certain Temperature is grown as well as the time for which the incubated substrate is at that particular elevated temperature is maintained, can be determined empirically and depends on the desired degree of degradation for the respective lignocellulose mass. In particular, after 3 days of incubation, the temperature can be increased and the incubated substrate can be increased for 3 days, for example Temperature are maintained. The degree of degradation of the lignocellulosic mass can be determined in a conventional manner, generally by determining the remaining amounts of lignin and cellulose. This cycle can be repeated and the optimal time and temperature for the degradation of a certain lignocellulosic material can be determined graphically, so that a maximum amount of free or for Cellulase sensitive to cellulose.

The product obtained, which is at least noticeably freed from lignin or degraded, can then be used as a source of cellulose or high-sugar animal feed or as a fermentation substrate. The process also produces soluble ones Lignin derivatives which may be useful as chemical starting materials.

By the mere moistening of the lignocellulosic material, as opposed to immersion, larger amounts of lignocellulose can be released per unit volume of culture.

Chrysosporium pruinosum (Giiman and Abbot) Carmichael, ATCC 24782 used. This organism was recently identified as the incomplete state of the Phanerochaete mushroom chrysosporium identified.

The following composition was used as the nutrient mineral medium:

5 g (NH ₄ J ₂ SO ₄ / 6.04 g of KH ₂ PO ₄ / Na ₂ HPO ₄ 0.85 g / 10 ml of

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Solution with trace elements as well as distilled water to 990 ml. The solution with the trace elements had the following Composition:

5 g MgSO ₄ -7H ₂ O / 0.2 g ZnSO ₄ -TH ₃ O, - 0.5 g FeSO ₄ -TH ₂ O / 0.5 MnSO ₄ ¹ 4H _? 0; 0.5 g CaCl, 5 g Versenol and distilled water to 250 ml. The pH of the medium was adjusted to 5.0 by adding a few drops of concentrated hydrochloric acid before it was introduced into the autoclave. A fraction of a filter-sterilized solution of thiamine HCl (1 mg / ml) was added to the medium after the treatment in the autoclave in order to have a thiamine concentration of 1 µg / ml. A 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.

To produce the lignose cellulose material, 200 g of dry manure was added to 2 liters of distilled water in a 3.785 Added a full Waring mixer and held for 15 seconds cut up at low speed. Mixing was repeated three times, keeping between mixes took a 15 second break. The suspension was then transferred to a glass vessel with a diameter of 30 cm and a height of 61 cm and diluted 1: 1 with distilled water. This suspension was by means of a variable speed adjustable motor, which was connected to a shaft and propeller, mixed. The engine speed was adjusted so that all of the material was suspended and the suspension was stirred for 1 1/2 For hours. The mixing speed was then slowed down for 1/2 hour to allow the sand to settle while the lighter fiber particles remained suspended. While the engine continues to operate at reduced speed stayed, they were. suspended particles drawn off by siphons and placed on a sieve with a mesh size of 0.238 mm transferred. The material was washed on the sieve with distilled water. Stir, allow to settle and Seven was repeated three times, and then the obtained faeries were dried at 65 ° C. for three days. The dried ones

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Fibers were chopped in a meat chopper to redisperse the particles, and these particles were cut into Glass vessels stored.

The fibers were in an oven heated to 65 C for at least Dried 2 days before use until constant weight was obtained. Because the material in air something is hygroscopic, it was locked in a lock when weighing

Plate.

closed Petri dish on the surface of a 100 ⁰ C hot

The dry particulate manure fibers produced in this way had an average size of 1-3 mm in diameter Length and 0.5 mm in width. The fibers contained 14 + 1

% By weight ash, 37 + 2% by weight reducing sugars (cellulose and hemicellulose) and 37 + 1.5% by weight lignin.

The abovementioned quantitative data are those on which the% by weight data of solubilized material are shown in the figures 1 and 2 are based on of which

FIG. 1 shows the degradation of lignin and cellulose by Chrysosporium pruinosum mold on a submerged substrate, FIG. 2 the degradation of lignin and cellulose by Chrysosporium pruinosum on a soaked substrate and FIG. 3 shows the degradation of lignin and cellulose by growing and shows elevated (non-growing) cultures of Chrysosporium pruinosum.

Cultures of Chrysosporium were used to prepare the inoculum pruinosum grown on glucose mineral agar plates at 38 ° C for 5 days. 10 ml of sterile, distilled water was added to each Plate was added and the surface growth was slightly suspended with a glass spreader. This suspension, which mainly contained spores and some fragments of vegetative mycelium, was used as inoculum for all cultures.

The cultures were both on in mineral nutrient solution in shake flasks submerged substrates as well as on moistened substrates

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Substrates grown on the surface of mineral agar plates. The submerged cultures contained in shake flasks 50 ml mineral medium solution and 50 ml dry, washed manure fibers in 250 ml Erlenmeyer flasks. The pistons were sterilized for 20 minutes and, after cooling, inoculated with 0.1 ml of the spore suspension described above. the Cultures were incubated at 38 ° C and 80% relative humidity with rotating shaking at 230 revolutions per month Minute on a New Brunswick Model G26 incubator shaker.

The soaked cultures on the agar surface were as follows Prepared: Sterile Nuclepore Membrane Filters (5 µm pore

size, 47 mm diameter) were placed with the matt side down on the surface of Petri dishes (25 150 mm), which contained about 2OO ml of mineral agar medium composed of the nutrient mineral solution thickened with 1.2 wt .- * agar was. One filter was used per plate. 200 mg of dry, sterile manure fibers were placed on the surface of each Filters spread out. The 200 mg fiber samples were taken from Pyrex tubes with screw caps and dimensions of 16 χ 125 ml taken. This corresponded to a 20% strength by weight solids concentration, i.e. 20% by weight fibers based on a mixture of 20% by weight fibers + 80% by weight nutrient solution. Every pile of fibers was inoculated on the periphery with 2 drops (0.1 ml) of the spore suspension as described above, and then the cultures incubated at 38 ° C. The relative humidity in the incubator was determined using water-filled troughs 80% held.

The determinations of the remaining organic and inorganic components of the fibers and fiber cultures for the figures 1 and 2 took place as follows:

a. For the agar plate cultures (moist cultures): The filters containing fibers or fiber + mycelium were removed from the agar plates and the fibrous mycelium mats from the Filters scraped off and washed with distilled water in tarred crucibles. The TiegeJ. were at 65 ° C until reaching of constant weight and then incinerated in an oven at 550 ° C overnight. The organic part

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was defined as the material that evaporated at 550 C, b. Shake flask cultures (submerged cultures): The contents of a pair of shake flasks were combined and through a nucleopore filter with pore openings of 0.4 μm and 47 mm diameter vacuum filtered to retain the solid particles. The one left on the sides of the pistons Remainder was washed onto the filter with a small amount of distilled water. The filtered residue was poured into the Transferred the covered crucible, dried and ashed as described above.

The analysis for cellulose, hemicellulose and lignin for Figures 1, 2 and / was carried out as follows: Samples of the insoluble material were collected as described above except that they were placed in glass jars with a diameter of 25 mm and a height of 50 mm and dried at 65 ° C. Dry samples of Fibers or fibers + mycelium were mixed with 10 ml of 72% sulfuric acid and left for 3 hours standing, mixing for a few seconds every hour. The whole was then made up to 50 ml with distilled water diluted and left to stand overnight. Each sample was then filtered using a tarred Nuclepore filter with 0.4 ^ m pore size and 47 mm diameter in a vacuum. Fractions of the filtrate were retained for carbohydrate (reducing sugar) experiments. In this attempt 0.1 ml of the filtrate was mixed with 3.9 ml of a reagent containing 1% thiourea and 0.05% anthrone in 72% sulfuric acid contained. The mixture was heated to 9'5 ° C. for 10 minutes, cooled to room temperature and the optical density was measured Density at 610 nyu. Glucose was used as the standard. These Experiments were routinely performed on a Technicon Model AA-1 autoanalyzer.

The residue was washed on the filter with distilled water until the ~ filtrate pH reached 5, which was measured with pH paper. The filter + residue were then dried overnight at 65 ° C and weighed. Filters and samples were transferred to tarred crucibles and incinerated at 550 ° C. overnight. Lignin was defined as that part of the sample (except in the filter) that evaporated. The filters contained more than 0.5 mg of ash. 809817 / 07Ö?

For the results of FIG. 3, moistened, inoculated fiber cultures were prepared on agar plates as described above. After 3 days, a number of cultures were compared that were found in the growth temperature of 38 ° C was left.

The solubilized constituents plotted in% by weight in FIGS. 1, 2 and 3 are based on the original content the fiber sample.

Figure 1 illustrates the degradation of the components of the manure fibers by Chysosporium pruinosum, which is submerged in Cultures grown in shake flasks. Each point represents the combined contents of two shake flasks. Individual points show that the repeated samples gave identical values. The dashed lines apply for the non-inoculated comparison samples.

Figure 1 shows the loss of various fiber components over time in submerged cultures, and it results. that under these conditions little or no lignin is made soluble, i.e. broken down, by Chrysosporium pruinosum, compare with the non-inoculated control samples, and this during a period of 30 days of active degradation. While that time were 50% of the carbohydrates (cellulose and hemicellulose) and 40% of the total organic components made soluble.

FIG. 2 shows the degradation of the manure fiber components by Chrysosporium pruinosum, which is grown in soaked fibers on the surface of mineral agar plates. Each point represents the content of a disk. All experiments were carried out in duplicate. The dashed lines apply to the non-inoculated comparison samples.

Figure 2 shows the loss of various fiber components over time in soaked fiber cultures on the surface of mineral agar plates and shows that a breakdown of lignocellulose takes place preferably on non-submerged substrates, ie on soaked substrates, and that a solids concentration of about 20 wt. -% allows a rapid degradation of lignocellulose. After 12 days of incubation were

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50% of the lignin, 80% of the carbohydrates (cellulose and hemicellulose) and solubilized 75% of the total organics. After this time, the degradation seems to stop. Under the above conditions, less material is solubilized in the non-inoculated control samples than in the shake flask.

The following table compares the degradation rates for lignin and cellulose by Polyporus versicolor, a well-studied, lignin-degrading, mesophilic mold and by Chrysosporium pruinosum, that in the present Invention used, lignin-degrading, thermostable mold specified.

TABEL Time , Dismantled Time Dismantled (Days) Lignin (%) (Day organism Cellulosed) 245 50 168 P. versicolor 76 12th 5O 12th C. pruinosum 80-90

(a) E.B. Cowling, Comparative Biochemistry Of The Decay Of Sweetgum Sapwood By White - Rot and Brown - Rot Fungi. U.S.D.A. tech. Bull No. 1258, (1961).

Figure 3 shows that significant degradation of lignin and cellulose continues at a temperature of 55 ° C, i.e. at a Temperature at which the mold cannot grow.

The information in the table above shows that enough lignin can be removed biologically under suitable culture conditions can to make 85 to 90% of cellulose degradable by cellulase. The speed and yields for the Cellulose degradation according to FIG. 2 are minimum values, since the cell wall sugar residues contribute to the cellulose values obtained and the cultures started from small spore inocula. Under Use of a larger inoculum of cellulase and lignase introduced by vegetative cells, i.e. the Chrysosporium pruinosum mold, would presumably die for the maximum Cellulose degradation shortens the incubation time required.

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Figure 3 shows that despite the heating of the cells to temperatures at which they can not grow (55 C), the enzymatic breakdown of lignin and cellulose in the absence more viable Cells went on. Cellulase studies have shown that the optimal cellulase activity is above 55 ° C. This shows that when the temperature is increased according to the method according to the invention, the cell metabolism ends and the saccharification is continued.

FIG. 2 shows that significant amounts of lignin can be broken down and that 80 to 90% of the cellulose content of the substrate exposed to cellulase enzymes. The manure fibers used in the experiments were relatively rich in lignin compared to other natural organic materials. The cellulose degradability is reversed proportional to the lignin content, and this suggests that the degradability of the used in the process according to the invention heat-resistant mold that was present on a resistant substrate such as manure fibers Substrates with less lignin content, such as wood, should be the same or better. More attempts actually have one shown good lignin and cellulose degradation in newsprint as a growth substrate. Newsprint is becoming big Part made from lignin-containing wood fibers.

In another German patent application filed on the same day by the applicant, for which priority de: “US patent application Serial No. 7 34 32 5 of October 20 , 1976 , there is disclosed growing a thermally stable mold on a soaked lignocellulosic substrate at a temperature of 20 ° C to 45 ° C, the mold producing enzymes that degrade the substrate.

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Claims (5)

4389-RD-947O GENERAL ELECTRIC COMPANY claims 1. Process for breaking down lignocellulose, characterized by the following stages: Manufacture of a lignocellulosic substrate soaked in a nutrient solution with a pH of 4 to 5 wherein the substrate comprises about 10 to about 80 weight percent lignocellulosic solids based on the total consists of lignocellulose solids and nutrient solution, inoculating the substrate with Chrysosporium pruinosum and Maintaining the inoculated substrate at a temperature of about 20 to about 45 ° C to protect the Chrysosporium pruinosum to breed that produces enzyme systems that degrade the lignocellulose that is then heated to 50 to 70 ° C, at which Temperature the Chrysosporium pruinosum stops growing, but the breakdown of lignocellulose continues. 2. The method according to claim 1, characterized in that that the lignocellulosic substrate with a liquid suspension of spores of Chrysosporium pruinosum is inoculated. 3. The method according to claim 1, characterized in that that the substrate consists of 20 wt .-% lignocellulose solids, based on the total amount Lignocellulose solids and nutrient solution is composed. 4. The method according to claim 1, characterized in that that the inoculated substrate for growing the Chrysosporium pruinosum at a temperature of about 38 to about 40 ° C. 5. The method according to claim 1, characterized in that that the elevated temperature between 809817/079? 5O and 60 ° C.