UA106042C2 - Method for fermentation of low molecular sugar - Google Patents

Abstract

The invention relates to the method for fermentation of low molecular sugar, comprising mixing of low molecular sugar and fermentative microorganisms in the presence of modified biomass, which was subjected to irradiation.

Description

All patents, patent applications, and publications referenced herein or attached to this document as Appendices A-E are hereby incorporated by reference in their entirety.

The field of technology

This invention relates to the processing of biomass and products obtained from it.

Technical level

Various carbohydrates, such as cellulosic and lignocellulosic materials, for example in fibrous form, are produced, processed and used in large quantities in many fields of application.

Often, such materials are used once and then thrown away as waste or simply considered waste, such as sewage, cake, wood sawdust and straw.

Various cellulosic and lignocellulosic materials, their use and fields of application are described in US patents MoMo 7307108, 7074918, 6448307, 6258876, 6207729, 5973035 and 5952105; and in various U.S. patent applications, including "EIVKOOZ MATEKIAI 5 AMO SOMROZITE5", PCT/OZ2006/010648, filed March 23, 2006, and ""IVKOOZ MATEKIAI 5 AMO SOMROZITE5", US Patent Application Publication Mo 2007/0045456.

Since cellulosic and lignocellulosic materials are widely available, and waste cellulosic and lignocellulosic materials require disposal, it would be beneficial to find good uses for such materials. Cellulosic and lignocellulosic materials are being considered for the production of biofuels such as ethanol, but have not yet been commercially implemented on a large scale.

The essence of the invention

In particular, described herein are carbohydrate-containing materials (eg, biomass material or biomass-derived materials), such as starch materials, cellulosic materials, lignocellulosic materials, or biomass material, which are low molecular weight sugars (eg, monosaccharides, disaccharides, or trisaccharides) or include their significant amounts, and methods of obtaining and processing such materials to change their structure, for example, to functionalize these materials with one or more desired types and quantities of functional groups. Products obtained from structurally modified materials are also described. For example, many of the methods described herein can provide cellulosic and/or lignocellulosic materials that have a lower molecular weight and/or crystallinity relative to the starting material. Many of the methods described herein provide materials that can be more readily utilized by various microorganisms to produce useful products such as hydrogen, alcohols (e.g., ethanol or butanol), organic acids (e.g., acetic acid), hydrocarbons, coproducts (eg proteins) or mixtures of any of them.

In some cases, functionalized biomass is more soluble and more easily utilized by microorganisms compared to biomass that is not functionalized. In addition, many of the functionalized materials described herein are less susceptible to oxidation and may have improved long-term stability (eg, oxidation in air under ambient conditions). Many of the resulting products, such as ethanol or n-butanol, can be used as fuel to power cars, trucks, tractors, ships or trains, for example as fuel for internal combustion engines or as feedstock for a fuel cell.

Many of the resulting products can also be used to power aircraft such as jet-powered aircraft or helicopters. In addition, the products described in this document can be used to generate electrical energy, for example, in conventional steam generator plants or in fuel cell-based plants.

Another aspect of the invention relates to the understanding that the addition of biomass, such as functionalized cellulosic or lignocellulosic material, to a mixture comprising a low molecular sugar can facilitate the conversion of the low molecular sugar into a product such as a combustible fuel such as ethanol. The authors of the invention found that the inclusion of biomass in a mixture with a low molecular weight sugar, a solvent or a system of solvents and a microorganism significantly increases the yield of a product obtained by the conversion of sugar, for example, an alcohol such as ethanol, in some cases, without significant conversion or depletion of the biomass itself. The inclusion of biomass can also prevent incomplete, slow or "stuck" product conversion, for example by fermentation.

Biomass may not be converted into a product by itself (such as ethanol), or it may be partially or fully converted into a product together with a low molecular weight sugar.

In cases where the biomass is partially converted, the surface area and porosity of the biomass are increased relative to the surface area and porosity of the original biomass, which can effectively increase the conversion rate of low molecular weight sugar into the product.

In some cases, biomass may be the remains of cellulosic or lignocellulosic material that has undergone saccharification, such as lignin and/or other materials that remain after the conversion of cellulose to sugar.

Thus, in one aspect, the invention relates to a method that includes converting a low molecular weight sugar or a material that includes a low molecular weight sugar, in a mixture with biomass, a microorganism and a solvent or solvent system, such as water or a mixture of water and an organic solvent, into a product, for example, excellent from sugar Examples of solvents or solvent systems include water, hexane, hexadiene, glycerin, chloroform, toluene, ethyl acetate, petroleum ether, liquefied petroleum gas (LPG), ionic liquids, and mixtures thereof. The solvent or solvent system may be in the form of a single phase or two or more phases. Biomass can be, for example, in fibrous form.

In some cases, the presence of biomass material (eg, processed by any method described herein or untreated) present in the production process of a product, such as ethanol, can increase the rate of product production. Without being bound by any particular theory, it is believed that the presence of a solid, such as a solid with a high surface area and/or high porosity, can increase reaction rates by increasing the effective concentration of solutes and providing a substrate on which to flow reaction.

For example, irradiated or non-irradiated biomass material, such as paper fiber, can be added to a fermentation process, such as a corn-ethanol fermentation process or a sugar cane extract fermentation process, to increase the production rate by 10, 15, 20, 30, 40, 50, 75, 10095 or more, for example by 15095. The biomass material may have a large surface area, high porosity and/or low bulk density. In some embodiments, the biomass is present in the mixture in an amount from about 0.5 wt. 95 to about 50 wt. 95, such as from about 1 wt. 95 to about 25 wt. 95 or from about 2 wt. 95 to about 12.5 wt. 95. In other embodiments, the biomass is present in amounts greater than about 0.5 wt. 95, for example more than about 1, 2, 3, 4, 5, 6, 7, 8, 9 wt. 95 or even more than about 10 wt. 95. For example, in some embodiments, an oxidized, sonicated, steam exploded, and/or pyrolyzed biomass material, such as paper or cotton fiber, may be added to the low molecular weight sugar fermentation process, for example, to increase fermentation rate and yield.

Since the biomass material itself is not consumed in the conversion process, the biomass material can be reused in multiple batch processes or it can be used continuously to produce a relatively large volume of product.

Some embodiments include one or more of the following features.

Biomass may contain fibrous material. The conversion may include allowing the microorganism to convert at least a portion of the low molecular weight sugar to ethanol.

For example, conversion may involve fermentation. The microorganism may include a yeast, for example, selected from the group consisting of 5. segemisiae and R. virii, or a bacterium such as 2utotopavz torbiii. Converting can detect 96 performance of at least 14095, in some cases at least 17095.

The method may additionally include irradiating the fibrous material prior to mixing, for example, with ionizing radiation, for example, at a total dose of at least 5 Mrad.

Irradiation can be carried out using a beam of particles. Irradiation can be carried out under conditions chosen to reduce the molecular weight of the biomass.

Biomass may have a bulk density of less than about 0.5 g/cm3. The biomass may have a VET surface area greater than 0.25 m-/g and/or a length-to-diameter ratio of at least 5. The biomass may have a porosity greater than 5095, such as greater than 7095.

The method may further include physical pretreatment of the biomass, for example, by crushing or reducing the size of the biomass by mill grinding, mechanical cutting or tearing, rod mill grinding, or air grinding. Biomass can have internal fibers and can be shredded to the extent that its internal fibers are essentially exposed.

Biomass can be or include cellulosic or lignocellulosic material. For example, the biomass may be selected from the group consisting of paper, paper products, paper waste, wood, compressed wood, sawdust, agricultural waste, sewage, silage, grasses, rice husk, cake, cotton, jute, hemp, flax , bamboo, sisal, abaca, straw, corncob cores, corn straw, millet, alfalfa, hay, coconut fibers, sea grass, algae and their mixtures.

The method may additionally include affecting the biomass by enzymatic hydrolysis and, in some cases, converting the hydrolyzed material into a product.

In another aspect, the invention relates to a method of dissolving cellulosic or lignocellulosic material, which includes mixing the cellulosic or lignocellulosic material with a solvent system containing DMSO and salt.

Solvent systems for cellulosic and lignocellulosic materials include DMSO-salt systems. Such systems include, for example, DMSO in combination with a lithium, magnesium, potassium, sodium or zinc salt. Lithium salts include GiCl, GIVg, Gi, lithium perchlorate and lithium nitrate. Magnesium salts include magnesium nitrate and magnesium chloride. Potassium salts include potassium iodide and potassium nitrate. Examples of sodium salts include iodide and sodium nitrate. Examples of zinc salts include zinc chloride and zinc nitrate. Any salt can be anhydrous or hydrated. Typical loadings of salt in DMSO are from about 1 to about 5095, for example from about 2 to 25, from about 3 to 15, or from about 4 to 12.5 wt. 90.

In other embodiments, the salt may be a fluoride salt, such as tetrabutylammonium fluoride. The method may additionally include irradiation of cellulosic or lignocellulosic material. The cellulosic or lignocellulosic material may be selected from the group consisting of paper, paper products, paper waste, wood, compressed wood, wood sawdust, agricultural waste, sewage, silage, grasses, rice husk, cake, cotton, jute, hemp, flax, bamboo, sisal, abaca, straw, corncob cores, corn straw, millet, alfalfa, hay, coconut fibers, sea grass, algae and their mixtures. In some cases, the cellulosic or lignocellulosic material has a bulk density of less than about 0.5 g/cm (prior to the addition of solvents to the system) and a porosity of at least 5095.

This document describes materials that include a plurality of saccharide units organized in the form of a molecular chain, where from about 1 out of every 2 to about 1 out of every 250 saccharide units include a carboxylic acid group or its complex ester or salt. In another aspect, the materials include a plurality of such molecular chains. For example, approximately 1 of every 8, 1 of every 10, 1 of every 50, or 1 of every 100 saccharide units of each chain may include a carboxylic acid group or its ester or salt. In some embodiments, the saccharide units may include 5 or 6 carbon saccharide units. Such a chain can have from about 10 to about 200 saccharide units, for example from about 10 to about 100 or from about 10 to about 50. For example, each chain can include hemicellulose or cellulose. In some embodiments, each chain also includes saccharide units that include nitroso groups, nitro groups, or nitrile groups.

In some embodiments, the average molecular weight of the materials relative to REO standards can be from about 1,000 to about 1,000,000, such as from 1,500 to 200,000 or from 2,000 to 10,000. For example, the average molecular weight of the materials relative to RES standards can be less than about 10,000.

This document describes methods of changing the molecular and/or supramolecular structure of the biomass feedstock, which include 1) irradiating the biomass feedstock with radiation, such as photons, electrons or ions with energy sufficient to ionize the biomass feedstock, with the formation of the first level of radicals, for example, which can be detected using an electron spin resonance spectrometer; 2) quenching the radicals to such an extent that the radicals are at a second lower level than the first level, such as a level that is no longer detectable by an electron spin resonance spectrometer, such as a level of less than about 1072 spins; and 3) processing of irradiated biomass raw materials to obtain a product. If desired, before irradiation and/or after irradiation, the biomass feedstock can be prepared by reducing one or more sizes of individual fragments of the biomass feedstock.

In some embodiments, the processing step includes obtaining a product, such as a fuel, such as a combusted fuel, such as motor fuel, aviation fuel, or fuel cell fuel, such as for generating electricity, by converting the irradiated biomass feedstock with a microorganism capable of converting at least a part, for example at least about 1 wt. 95, biomass into a product.

In some variants, irradiation is carried out on raw biomass with simultaneous exposure to raw biomass with air, nitrogen, oxygen, helium or argon. In some embodiments, the pretreatment may include pretreatment of the biomass feedstock by steam explosion.

In some embodiments, the method further includes reducing one or more sizes of individual biomass fragments, such as by crushing, wet or dry grinding, cutting, compression, pressing, or a combination of any of these methods. For example, crushing can be done using a cutting device with a rotating knife. Crushing can provide fibers having an average length to diameter ratio greater than 5/1 or at least 5. In some embodiments, the prepared biomass can have a surface area of BET greater than 0.25 mg/g. In some cases, the biomass has internal fibers, and the biomass can be crushed to such an extent that the internal fibers of the biomass are essentially exposed. Biomass can be crushed to such an extent that it has a bulk density of less than about 0.35 g/cm3.

In some embodiments, the method does not include hydrolyzing the biomass with an acid or base.

For example, at least about 70 wt. 95% of the biomass may be non-hydrolyzed, for example at least 95% by weight. 95% of biomass is non-hydrolyzed. In specific embodiments, the biomass is not essentially hydrolyzed.

In some embodiments, the irradiation is carried out on biomass in which less than about 25 wt. 95 biomass is wetted with a liquid such as water. Specifically, in some embodiments, at least one method of pre-treating the biomass is carried out in which the biomass is substantially not wetted with a liquid such as water. Biomass may have, for example, less than about 5 wt. 95 of retained water measured at 25 "C and a relative humidity of 50965.

In some embodiments, the irradiation is carried out on biomass in which less than about 25 wt. 906 of the biomass is in the swollen state, and the swollen state is characterized by the presence of a volume that exceeds by more than about 2.595 the volume in the non-swollen state. In other embodiments, the biomass is mixed with or includes a swelling agent.

Pressure can be applied in any of the ways described in this document. For example, irradiation can be performed on the biomass at a pressure greater than about 2.5 atmospheres (0.25 MPa), such as greater than about 5 or 10 atmospheres (0.5 or 0.1 MPa).

In another aspect, the mixture includes a low molecular weight sugar, a biomass material, and a solvent. In some cases, the mixture also includes a microorganism.

Examples of biomass feedstocks include paper, paper products, waste paper, wood, pressed wood, wood sawdust, agricultural waste, sewage, silage, grasses, rice husk, cake, cotton, jute, hemp, flax, bamboo, sisal, abaca, straw , corn cob kernels, corn straw, millet, alfalfa, hay, coconut fiber, synthetic cellulose, sea grass, algae, or mixtures thereof. Biomass can be or include synthetic material.

Examples of fuel grades include one or more components selected from hydrogen, alcohols, and hydrocarbons. For example, alcohols can be ethanol, n-propanol, isopropanol, n-butanol or their mixtures.

Irradiation, for example, can be performed using ionizing radiation such as gamma rays, electron beams, or ultraviolet C radiation having a wavelength of about 100 nm to about 280 nm. Irradiation can be carried out using a number of methods of applying radiation exposure. Ionizing radiation can include electron beam radiation. For example, radiation may be applied at a total dose of from about 10 Mrad to about 150 Mrad, for example at a dose rate of from about 0.5 to about 10 Mrad/day or from 1 Mrad/s to about 10 Mrad/s. In some embodiments, the implementation of irradiation includes the use of two or more sources of radiation, such as gamma rays and an electron beam.

In some embodiments, the biomass comprises a first cellulose having a first number average molecular weight, and the carbohydrate material comprises a second cellulose having a second number average molecular weight that is lower than the first number average molecular weight. For example, the second number average molecular weight is lower than the first number average molecular weight by more than about a factor. In some embodiments, the first cellulose has a first crystallinity and the second cellulose has a second crystallinity lower than the first crystallinity. For example, the second crystallinity may be lower than the first crystallinity by more than about 1095.

In some embodiments, the first cellulose may have a first oxidation level and the second cellulose may have a second oxidation level greater than the first oxidation level.

The biomass material may further include a buffer such as sodium bicarbonate or ammonium chloride, an electrolyte such as potassium chloride or sodium chloride, a growth factor such as biotin, and/or a base pair such as uracil, a surfactant, a mineral, or a chelating agent. agent.

In some embodiments, the methods include pretreatment with one or more pretreatment methods in addition to irradiation. For example, two or more different pretreatment methods may include radiation exposure and sonication, radiation exposure and oxidation, and radiation exposure and pyrolysis. Optionally, pretreatment of the biomass may include steam explosion.

An enzyme, such as a cellulolytic enzyme, or a chemical reagent, such as sodium hypochlorite, an acid, a base, or a swelling agent may be used to further assist in reducing the molecular weight of the biomass in any manner described herein.

Enzymatic and/or chemical treatment may be performed before, during, or after irradiation or other pretreatment.

If a microorganism is used, it can be a natural microorganism or an engineered microorganism. For example, a microorganism can be a bacterium, such as a cellulolytic bacterium, a fungus, such as a yeast, a plant, or a single-celled organism, such as an algae, a protozoa, or a fungus-like protozoa, such as a slime mold. If the organisms are compatible, mixtures may be used. Generally, different microorganisms can produce a number of useful products, such as fuel, by acting on materials, such as fermentation. For example, alcohols, organic acids, hydrocarbons, hydrogen, proteins, or mixtures of any of these materials can be produced by fermentation or other processes.

Examples of products that can be produced using the methods described herein include mono- and polyfunctional C1-Cb-alkyl alcohols, mono- and polyfunctional carboxylic acids, C1-Cb-hydrocarbons, and combinations thereof. Specific examples of suitable alcohols include methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol, propylene glycol, 1,4-butanediol, glycerin, and combinations thereof. Specific examples of suitable carboxylic acids include formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, palmitic acid, stearic acid, oxalic acid, malonic acid, succinic acid, glutaric acid, oleic acid, linoleic acid, glycolic acid. , lactic acid, γ-hydroxybutyric acid and their combinations. Examples of suitable hydrocarbons include methane, ethane, propane, pentane, n-hexane and combinations thereof. Many of these products can be used as fuel.

Other products are described in US Provisional Application Serial No. 61/139453, the entire contents of which are incorporated herein by reference. Produced products or co-products may be products intended for use as manufactured, or produced products may be intermediate products for any other process described herein or any process described in an application incorporated herein as a reference

Examples of microorganisms that can be used to produce beneficial products include bacteria, yeast, and combinations thereof. For example, a microorganism can be a bacterium, such as a cellulolytic bacterium, a fungus, such as a yeast, a plant, or a single-celled organism, such as an algae, a protozoa, or a fungus-like protozoa, such as a slime mold.

In any of the ways described in this document, radiation can be applied from a device that is in storage.

The term "fibrous material", as used herein, is a material that includes a plurality of loose, discrete and separate fibers. For example, the fibrous material can be obtained from a source of fibers, which is bleached kraft paper, by crushing, for example, using a cutting device with a rotating knife.

The term "sieve", as used herein, means an element capable of sieving material according to size. Examples of sieves include a plate with holes, a cylinder or the like, or a wire sieve, or a cloth.

The term "pyrolysis", as used herein, means the destruction of bonds in a material by the application of thermal energy. Pyrolysis can occur when the material in question is in a vacuum or immersed in a gaseous substance such as an oxidizing gas such as air or oxygen or a reducing gas such as hydrogen.

The oxygen content is determined by elemental analysis by pyrolysis of the sample in a furnace operating at 1300 "C or higher.

The term "biomass" includes any non-fossilized, i.e. renewable, organic matter. Different types of biomass include plant biomass (defined below), microbial biomass, animal biomass (any livestock by-product, livestock waste, etc.) and municipal waste biomass (domestic waste and light industrial waste with recyclable materials such as such as discarded metal and glass). Also, the term "biomass" includes raw or used cellulosic materials, such as rags and towels, made from cotton or cotton blends.

The terms "plant biomass" and "lignocellulosic biomass" refer to virtually any organic material derived from plants (woody or non-woody). Plant biomass may include, but is not limited to, agricultural or food crops (e.g., sugar cane, sugar beet, or corn kernels), or their extract (e.g., sugar from sugar cane and corn starch from corn), crops and agricultural waste, and residues such as corn straw, wheat straw, rice straw, sugarcane bagasse, cotton, etc. In addition, plant biomass includes, but is not limited to, trees, tree energy crops, wood waste and residues such as softwood chips trees, bark waste, wood sawdust, paper and pulp industry waste streams, wood fiber, etc.

In addition, as another source of plant biomass, forage grasses such as millet etc. can potentially be grown on a large scale. For urban areas, the best potential plant biomass feedstocks include horticultural waste (e.g. grass clippings, leaves, tree trimmings and wind ) and vegetable processing waste. "Ligocellulosic feedstock" is any type of plant biomass, such as, but not limited to, non-woody plant biomass, cultivated crops such as, but not limited to, grasses, such as, but not limited to, C4 grasses such such as millet, spartina, weeds, miscant (Chinese cane), dicotyledons (canary) or combinations thereof, or residues from sugar processing such as cake or beet pulp, agricultural residues such as soybean straw, corn straw, rice straw, rice husk , barley straw, corncob core, wheat straw, canola straw, oat straw, oat hulls, corn fiber, recycled wood pulp fiber, wood sawdust, hardwood such as wood and aspen sawdust, softwood, or combinations thereof. In addition, lignocellulosic feedstocks may include cellulosic waste products such as, but not limited to, newsprint, cardboard, wood sawdust, and the like.

The lignocellulosic feedstock may include a single type of fiber or, alternatively, the lignocellulosic feedstock may include a mixture of fibers derived from different lignocellulosic feedstocks. In addition, the lignocellulosic feedstock may contain fresh lignocellulosic feedstock, partially dried lignocellulosic feedstock, fully dried lignocellulosic feedstock, or a combination thereof.

For the purposes of this description, carbohydrates are materials that consist entirely of one or more saccharide units or that include one or more saccharide units. The saccharide units may be functionalized in the ring region with one or more functional groups, such as carboxylic acid groups, amino groups, nitro groups, nitroso groups, or nitrile groups, and still be considered carbohydrates. Carbohydrates can be polymeric (eg, equal to or greater than 10-mer, 100-mer, 1000-mer, 10000-mer, or 100000-mer), oligomeric (eg, equal to 4-mer, 5-mer, b-mer, 7 -mer, 8-mer, 9-mer or 10-mer or exceeding them), trimeric, dimeric or monomeric. If carbohydrates are formed from more than one repeating unit, then all the elements can be the same or different.

Examples of polymeric carbohydrates include cellulose, xylan, pectin, and starch, while examples of dimeric carbohydrates include cellobiose and lactose. Examples of monomeric carbohydrates include glucose and xylose.

Carbohydrates can be part of a supramolecular structure, for example, covalently attached to the structure. Examples of such materials include lignocellulosic materials, such as materials found in wood.

A starch material is a material that is starch or a starch derivative or includes significant amounts of starch or a starch derivative, such as more than about 5 wt. 95 starch or derived starch. For the purposes of this description, starch is a material that includes amylose, amylopectin or their physical and/or chemical mixture, for example a mixture of amylose and pectin, which is 20:80 or 30:70 by weight. 95. For example, rice, corn and their mixtures are starchy materials. Starch derivatives include, for example, maltodextrin, acid-modified starch, basic-modified starch, bleached starch, oxidized starch, acetylated starch, acetylated and oxidized starch, phosphate-modified starch, genetically modified starch, and starch that is resistant to cleavage.

For purposes of this description, a low molecular weight sugar is a carbohydrate or derivative thereof having a formula molecular weight (excluding moisture content) of less than about 2,000, such as less than about 1,800, less than about 1,600, less than about 1,000, less than about 500 , less than about 350 or less than about 250. For example, a low molecular sugar can be a monosaccharide such as glucose or xylose, a disaccharide such as cellobiose or sucrose, or a trisaccharide.

Combustible fuel is a material capable of burning in the presence of oxygen. Examples of combustible fuels include ethanol, n-propanol, n-butanol, hydrogen, and mixtures of any two or more of them.

Swelling agents, as used herein, are materials that cause visible swelling, such as an increase in volume of cellulosic and/or lignocellulosic materials relative to an unswollen state of 2.595 when such materials are used as a solution, such as an aqueous solution . Examples of these include alkalis such as sodium hydroxide, potassium hydroxide, lithium hydroxide and ammonium hydroxides, acidifiers such as mineral acids (such as sulfuric acid, hydrochloric acid and phosphoric acid), salts such as zinc chloride, calcium carbonate, sodium, benzyltrimethylammonium sulfate, and basic organic amines such as ethylenediamine. A "crushed material" as used herein is a material that includes individual fibers in which at least about 5095 individual fibers have a length/diameter ratio (G/0) of at least about 5 and which have an uncompressed bulk density less than about 0.6 g/cm3. Thus, shredded material is different from material that is chopped, chopped, or ground.

Changing the molecular structure of biomass raw materials, as used in this document, means changing the location of chemical bonds, for example, the type and number of functional groups or the conformation of the structure. For example, a change in molecular structure may include a change in the supramolecular structure of the material, oxidation of the material, change in average molecular weight, change in average crystallinity, change in surface area, change in degree of polymerization, change in porosity, change in degree of branching, graft copolymerization with other materials, change in crystal domain size, or resizing the entire domain.

This application incorporates as a reference the full content of international application Me PCT/О52007/022719, filed on October 26, 2007. The complete disclosures of each of the following US patent applications are incorporated herein by reference: prior US applications Serial Nos. 61/049391; 61/049394; 61/049395; 61/049404; 61/049405; 61/049406; 61/049407; 61/049413; 61/049415 and 61/049419, all filed April 2008; US Patent Application Serial Nos. 61/073432; 61/073436; 61/073496; 61/073530; 61/073665 and 61/073674, all filed Jun. 18, 2008; US Provisional Application Serial No. 61/106861, filed Oct. 20, 2008; US Patent Application Serial Nos. 61/139324 and 61/139453, both filed Dec. 19, 2008, and US Patent Application Serial No. 12/417707; 12/417720; 12/417840; 12/417699; 12/417731; 12/417900; 12/417880; 12/417723; 12/417786 and 12/417904, all filed Apr. 2009.

Unless otherwise defined, all technical and scientific terms used in this document have the same meanings that are commonly understood by those skilled in the art to which the present invention belongs.

Although methods and materials similar to or equivalent to the methods or materials described herein may be used in the practice or testing of this invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, this description, including definitions, shall govern. In addition, the materials, methods and examples are illustrative only and are not intended to be limiting.

Other features and advantages of the invention will become apparent from the detailed description and claims presented below.

Description of drawings

In FIG. 1 presents a block diagram illustrating the conversion of biomass into products and by-products.

In FIG. 2 is a block diagram illustrating the conversion of a fiber source into a first and a second fibrous material.

In FIG. C shows a cross-sectional view of a cutting device with a rotating knife.

In FIG. 4 is a block diagram illustrating the conversion of a fiber source into a first, second, and third fibrous material.

In FIG. 5 is a block diagram illustrating the compaction of the material.

In FIG. 6 is a perspective view of a pelletizing press.

In FIG. 7A shows compacted fibrous material in the form of granules.

In FIG. 7B shows a cross-section of hollow granules in which the center of the cavity is on the same line as the center of the granule.

In FIG. 7C shows a cross-section of a hollow pellet in which the center of the cavity is offset relative to the center of the pellet.

In FIG. 70 shows a cross-section of a three-lobed pellet.

In FIG. 8 is a block diagram illustrating the sequence of processing for the processing of raw materials.

In FIG. 9 shows a cross-sectional perspective view of a gamma emitter located in a concrete storage.

In FIG. 10 presents an enlarged perspective view of area E. FIG. 9.

In FIG. 11A presents a block diagram illustrating the sequence of pre-treatment of raw materials by electron beam irradiation.

In FIG. 118 shows a schematic representation of biomass ionized and then oxidized or quenched.

In FIG. 12 presents a schematic view of the system for ultrasound treatment of the technological flow of cellulosic material in a liquid medium.

In FIG. 13 is a schematic view of an ultrasonic treatment device having two transducers attached to a single horn.

In FIG. 14 presents a block diagram illustrating a system for pyrolytic pretreatment of raw materials.

In FIG. 15 is a cross-sectional side view of the pyrolysis chamber.

In FIG. 16 is a cross-sectional side view of the pyrolysis chamber.

In FIG. 17 is a cross-sectional side view of the pyrolyzer, which includes a heating hair.

In FIG. 18 shows a schematic cross-sectional side view of the pyrolyzer by point

Curie.

In FIG. 19 shows a schematic cross-sectional side view of the furnace pyrolyzer.

In FIG. 20 is a schematic cross-sectional top view of a pyrolysis laser device.

In FIG. 21 is a schematic cross-sectional top view of a device for instant pyrolysis with a tungsten hair.

In FIG. 22 is a block diagram illustrating a system for oxidative pretreatment of raw materials.

In FIG. 23 is a block diagram illustrating a general view of the process of converting a fiber source into a product, such as ethanol.

In FIG. 24 shows a schematic cross-sectional view of a steam explosion device.

In FIG. 25 is a schematic cross-sectional side view of a hybrid device for electron beam/ultrasound treatment.

In FIG. 26 presents a picture, obtained with the help of a scanning electron microscope at a magnification of 25X, of a fibrous material obtained from paper with a multilayer coating.

The fibrous material was obtained on a rotary knife cutter using a screen with 1/8 inch (0.32 cm) openings.

In FIG. 27 presents a picture, obtained with the help of a scanning electron microscope at a magnification of 25X, of a fibrous material obtained from bleached kraft cardboard. The fibrous material was obtained on a rotary knife cutter using a screen with 1/8 inch (0.32 cm) openings.

In FIG. 28 presents a picture obtained with the help of a scanning electron microscope at a magnification of 25X of a fibrous material obtained from bleached kraft paperboard. The fibrous material was crushed twice on a rotary knife cutter using a screen with 1/16 inch (0.16 cm) openings for each crushing.

In FIG. 29 presents a picture obtained with the help of a scanning electron microscope at a magnification of 25X of a fibrous material obtained from bleached kraft cardboard. The fibrous material was crushed three times on a cutting device with a rotating knife. A 1/8 inch (0.32 cm) sieve was used during the first crushing; a 1/16 inch (0.16 cm) sieve was used for the second crushing and a 1/32 inch (0.08 cm) sieve was used for the third crushing.

In FIG. 29A-29E present 3-0 Raman spectra of the surface of fibers from samples P132, P132-10,

P132-100, P-1e, P-ZOe and P-100e, respectively.

In FIG. 30 shows a schematic side view of the device for ultrasound treatment, and on

FIG. 31 is a cross-sectional view through the processing cell of FIG. 30.

In FIG. 32 is a scanning electron microscope image at 1000X magnification of fibrous material obtained by crushing millet on a rotary knife cutter and then passing the crushed material through a 1/32 inch (0.08 cm) screen.

In FIG. 33 and 34 show images obtained using a scanning electron microscope at a magnification of 1000X of the fibrous material of FIG. 32 after irradiation with gamma rays at a dose of 10 and 100 Mrad, respectively.

In FIG. 35 presents pictures obtained with the help of a scanning electron microscope at a magnification of 1000X, the fibrous material of FIG. 32 after irradiation with a dose of 10 Mrad and treatment with ultrasound.

In FIG. 36 presents images obtained using a scanning electron microscope at a magnification of 1000X, the fibrous material of FIG. 32 after irradiation with a dose of 100 Mrad and treatment with ultrasound.

In FIG. 37 presents the infrared spectrum of kraft cardboard cut on a cutting device with a rotating knife.

In FIG. 38 presents the infrared spectrum of the kraft paper of FIG. 37 after exposure to gamma radiation at a dose of 100 Mrad.

In FIG. 38A-38I presents the H-NMR spectra for samples P132, P132-10, PI132-100, P-1e, P-5e, P-1be, P-ZOe, P-7Oe and P-100e in example 23. FIG. . 38) presents a comparison of the exchange proton at -16 m.p.h. in FIG. In FIG. 381 -38M are presented "ZS-NMR of sample P-100e with a retention time of 10 seconds. FIG. 38M presents "H-

NMR of sample P-100e at a concentration of 1095 wt./wt.

In FIG. 39 shows a schematic view of the biomass conversion process.

In FIG. 40 presents a schematic view of another biomass conversion process.

Detailed description

Biomass (e.g., plant biomass, such as biomass that is or includes one or more low molecular weight sugars, animal biomass, and municipal waste biomass) can be processed to produce useful products such as fuels, such as fuel for internal combustion engines, jet engines, or raw materials for fuel cells. In addition, using the methods described herein, it is possible to obtain functionalized materials having the desired types and amounts of functional groups, such as carboxylic acid groups, enol groups, aldehyde groups, ketone groups, nitrile groups, nitro groups, or nitroso groups. Such functionalized materials can be, for example, more soluble, more easily utilized by various microorganisms, or they can be more stable over a long time, for example, less prone to oxidation. This document describes systems and processes in which various biomass materials can be used, such as cellulosic materials, lignocellulosic materials, starch materials, or materials that are or include low molecular weight sugars as raw materials.

Such materials are often readily available but may be difficult to process, for example by fermentation, or they may give suboptimal yields at sufficiently low rates.

Raw materials are first physically prepared for processing, often by reducing the size of the raw materials. Physically prepared raw materials can be pretreated or processed using one or more methods selected from radiation exposure, sonication, oxidation, pyrolysis, and steam explosion. Different systems and methods for pretreatment can use combinations of two, three or even four of these technologies.

In some cases, to provide materials that include a carbohydrate, such as cellulose, that can be converted by a microorganism into a number of desired products, such as combustible fuels (eg, ethanol, butanol, or hydrogen), feedstocks that include one or more saccharide units can be process in one or more ways described in this document. Other products and co-products that can be produced include, for example, human food, animal feed, pharmaceuticals and nutraceuticals. A number of examples are presented, ranging from laboratory implementations of individual pretreatment methods to large-scale biomass processing plants.

Types of biomass

In general, any biomass material that is or includes carbohydrates that consist entirely of one or more saccharide units or includes one or more saccharide units can be processed by any of the methods described herein. For example, the biomass material can be cellulosic or lignocellulosic materials, starchy materials such as corn kernels, rice grains or other food products, or materials that are or include one or more low molecular weight sugars such as sucrose or cellobiose.

For example, such materials may include paper, paper products, wood, wood-based materials, pressed wood, grasses, rice husks, cake, cotton, jute, hemp, flax, bamboo, sisal, abaca, straw, corncob cores, coconut fibers, algae, sea grass, synthetic cellulose or mixtures of any of these. Suitable materials include the materials listed in the Summary of the Invention section above.

Fiber sources include cellulosic fiber sources, including paper and paper products (eg, laminated paper and kraft paper), and lignocellulosic fiber sources, including wood and wood-related materials, such as pressed wood. Other suitable fiber sources include natural fiber sources such as grasses, rice husks, rice husks, jute, hemp, flax, bamboo, sisal, abaca, straw, corncob cores, coconut fibers; fiber sources with a high α-cellulose content, such as cotton; and synthetic fiber sources such as extruded yarn (oriented yarn or non-oriented yarn). Natural or synthetic sources of fibers can be obtained from scraps of primary textile materials, such as scraps, or they can be used waste, such as rags. When paper products are used as fiber sources, they can be natural materials, such as pieces of raw materials, or they can be used waste. In addition to raw materials, used product waste, industrial (for example, by-products) waste and processing waste (for example, waste water from paper processing) can also be used as sources of fibers. Also, the source of the fibers can be obtained or formed from human waste (eg sewage), animals or plants. Additional sources of fibers are described in US Patent Nos. 6,448,307, 6,258,876, 6,207,729, 5,973,035, and 5,952,105.

In some embodiments, the carbohydrate is or includes a material that has one or more D-1,4 linkages and has a number average molecular weight of about 3,000 to about 50,000. Such carbohydrate is or includes cellulose (I) that is formed from VD-glucose 1 by condensation of D-(1-4)-glycosidic bonds. This bond is the opposite of a-(1--4)-glycosidic bonds present in starch and other carbohydrates.

no po no on 1 (Fin no ON i xx (o) ; (Fin! 1

Starch materials include starch itself, such as corn starch, wheat starch, potato starch or rice starch, a starch derivative, or a material that includes starch, such as a food or crop. For example, the starchy material may be aracacha, buckwheat, banana, barley, cassava, kudzu, sorrel, sago, sorghum, common house potato, sweet potato, taro, yam (sweet potato), or one or more legumes such as horse beans, lentils or peas Mixtures of these and/or other starchy materials are also considered starchy materials. In specific embodiments, the starch material is obtained from corn. Various corn starches and their derivatives are described in the publication "Sogp eiagsy", Sot Weijpeg5 Avzosiyop (117 Eyyop, 2006), which is attached to this document in the form of appendix A.

Biomass materials that include low molecular weight sugars may contain, for example, at least about 0.5 wt. 96 low molecular weight sugar, for example at least approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 5, 25, 35, 50, 60, 70, 80, 90 wt. 95 or even at least approximately 95 wt. 90 low molecular weight sugar. In some cases, the biomass consists essentially of low molecular sugar, for example, more than 95 wt. 95, for example 96, 97, 98, 99 wt. 95 or essentially 100 wt. 905 low molecular sugar.

Biomass materials that include low molecular sugars can be agricultural or food products, such as sugar cane or sugar beet, or their extract, such as sugar cane or sugar beet juice. Biomass materials that include low molecular weight sugars can be essentially pure extracts, such as unrefined or crystallized table sugar (sucrose). Low molecular weight sugars include sugar derivatives.

For example, low molecular sugars can be oligomeric (eg, equal to or greater than 4-mer, 5-mer, b-mer, 7-mer, 8-mer, 9-mer, or 10-mer), trimeric, dimeric, or monomeric. If carbohydrates are formed by more than one repeating unit, then all the repeating units can be the same or different.

Specific examples of low molecular sugars include cellobiose, lactose, sucrose, glucose and xylose, as well as their derivatives. In some cases, sugar derivatives dissolve more quickly in solution or are utilized by microbes, producing a useful material such as ethanol or butanol. A few of these sugars and sugar derivatives are presented below.

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But sy "TV BZ |. iluvose pu ov me chu. s psa on um naya y l l b; 1-MA5 SHHI g monobasic acid sucrose po on sg-ketoglucanov kielota no

She i sn s wa no t E -chh y m NO ek en y te (y

In he and not, oh ha and still a S a pom oon ok y Y" . i and g y s OH a i! sho ov Glyujhursnovka ul! , acid von / o y and 5-MAx - y-zdnossnonny acid of sucharose also Gah

Fructose no

Sh-ch y za is eh ti B

Kv) oo

Syakhvaroz in Z dn

Mixtures of any of the biomass materials described herein may be used to produce the products described herein, such as ethanol. For example, mixtures of cellulosic materials and starch materials can be used to make any of the products described herein.

Biomass processing systems

In FIG. 1 presents a system for converting biomass 100, in particular biomass with significant amounts of cellulosic and lignocellulosic components and/or starch components, into useful products and by-products. System 100 includes a feedstock preparation subsystem 110, a pretreatment subsystem 114, a primary processing subsystem 118, and a further processing subsystem 122. Feedstock preparation subsystem 110 is fed raw biomass and physically prepared for use as raw materials for further processes (for example, to reduce the size and homogenization of biomass), and biomass is stored both in raw form and in the form of raw materials. Biomass feedstocks with significant amounts of cellulosic and/or lignocellulosic components or starch components may have a high average molecular weight and crystallinity, which may complicate the processing of the feedstock into useful products (for example, fermentation of the feedstock to produce ethanol). For example, in other systems, acids, bases and enzymes are used for the processing of cellulosic, lignocellulosic or starchy raw materials. As described herein, in some embodiments, such treatment is unnecessary or required only in small or catalytic amounts.

The raw material from the raw material preparation subsystem 110 is fed into the pre-processing subsystem 114 and in it the raw material is prepared for use in the main production processes, for example, by reducing the average molecular weight and crystallinity of the raw material. From the pre-processing subsystem 114, the pre-treated feedstock is fed to the main processing subsystem 118 and useful products (eg, ethanol, other alcohols, pharmaceuticals, and/or food products) are produced therein. In some cases, the product exiting the processing subsystem primary processing 118 is suitable directly, but in other cases additional processing is required by the further processing subsystem 122. The further processing subsystem 122 provides further processing of the product stream from the primary processing system 118 that is required for it (eg, distillation and denaturation ethanol), as well as the treatment of waste streams from other subsystems. In some cases, the co-products of the subsystems 114, 118, 122 may also be directly or indirectly suitable as secondary products and/or to increase the overall efficiency of the system 100. For example, the subsystem for further processing 122 may produce treated water for recirculation as technical water in other subsystems and/or may produce combustible waste that can be used as fuel for boilers generating steam and/or electricity.

The optimal size of a biomass conversion plant is influenced by factors including economies of scale and the type and availability of biomass used as feedstock. Increasing plant size tends to increase the economies of scale associated with the processes in the plant However, increasing plant size also tends to increase costs (eg transportation costs) per unit of raw material. Studies analyzing these factors indicate that acceptable plant sizes for biomass conversion can range from 2,000 to 10,000 dry tons per day, depending, at least in part, on the type of feedstock used. The type of feedstock can also affect storage requirements at the facility, where facilities designed primarily to process feedstocks whose availability varies seasonally (eg, corn straw) require more on-site than off-site storage of the feedstock in compared to installations designed for the processing of raw materials, the availability of which is relatively constant (for example, waste paper).

Callisthenics

In some cases, processing methods begin with the physical preparation of raw materials, such as reducing the size of the raw materials, for example, by cutting, grinding, crushing or chopping. In some cases, loose raw materials (such as recycled paper, starch materials or millet) are prepared by crushing or cutting. Sieves and/or magnets can be used to remove very large objects or unwanted objects, such as stones or nails, from the feed stream.

Systems for the preparation of raw materials can be configured to produce streams of raw materials with specific characteristics, such as, for example, specific maximum dimensions, specific ratios of length to width, or specific ratios of surface areas. As part of the raw material preparation, the bulk density of the raw material can be adjusted (for example, increased or decreased).

Size reduction

In some embodiments, the material to be recycled is in the form of a fibrous material that includes fibers obtained by crushing a fiber source. For example, crushing can be done using a cutting device with a rotating knife.

For example, and referring to FIG. 2, the fiber source 210 is crushed, for example in a rotary knife cutter, to produce a first fibrous material 212. The first fibrous material 212 is passed through a first screen 214 having an average hole size of 1.59 mm or less (1/16 inch, 0 .0625 inch), to produce a second fibrous material 216. If desired, the fiber source may be ground prior to crushing, such as by means of a shredder. For example, if paper is used as the fiber source, the paper may first be shredded into strips of, for example, 1/4 to 1/2 inch (0.64 to 1.28 cm) wide using a shredding device, such as a grinding with counter-rotating screws, such as a device manufactured by Mipsop (Shisa, M.M.). As an alternative to shredding, paper can be reduced in size by cutting to the desired size using a guillotine cutting tool. For example, a guillotine cutter can be used to cut paper into sheets that are, for example, 10 inches (25.4 cm) wide and 12 inches (30.5 cm) long.

In some embodiments, crushing the fiber source and passing the resulting first fibrous material through the first sieve are performed simultaneously. Crushing and passing can also be carried out by the method of periodic action.

For example, a cutting device with a rotating knife can be used to simultaneously crush the fiber source and screen the first fibrous material. Referring to FIG. 3, the rotary knife cutting device 220 includes a hopper 222 into which the chopped fiber source 224 obtained by grinding the fiber source can be loaded. The chopped fiber source is crushed between stationary blades 230 and rotating blades 232 to produce a first fibrous material 240. The first fibrous material 240 is passed through a sieve 242, and the resulting second fibrous material 244 is collected in a basket 250. To facilitate collection of the second fibrous material, the basket may be pressurized below nominal atmospheric pressure, such as at least 1095 below nominal atmospheric pressure, such as at least 25956 below nominal atmospheric pressure, at least 5095 below nominal atmospheric pressure, or at least 7595 below nominal atmospheric pressure. In some embodiments, a vacuum source 252 is used to maintain basket pressure below nominal atmospheric pressure.

Crushing can be beneficial for "unfolding" and "stressing" fibrous materials, making the cellulose of the materials more susceptible to chain scission and/or reduced crystallinity. Exposed materials may also be more sensitive to oxidation when exposed to radiation.

The fiber source can be crushed in a dry state, in a hydrated state, for example, with the presence of up to 10 wt. 95 of absorbed water) or in a wet state, for example, with the presence of from about 10 wt.

Fo up to about 75 wt. 95 water. The fiber source can be crushed even when partially or fully immersed in a liquid such as water, ethanol, isopropanol.

The fiber source can also be crushed in a gas atmosphere (such as a flow or atmosphere of a gas other than air), such as oxygen or nitrogen, or steam.

Other methods of obtaining fibrous materials include, for example, mill grinding, mechanical fluffing or tearing, rod mill grinding, or air grinding.

If desired, the fibrous materials can be separated, for example continuously or in batches, into fractions according to their length, width, density, type of material, or some combination of these characteristics.

For example, ferrous metals can be separated from any fibrous material by passing the fibrous material that includes the ferrous metal past a magnet, such as an electromagnet, and then passing the resulting fibrous material through a series of sieves, where each sieve has different sized holes.

Fibrous materials can also be separated, for example, using a high-velocity gas such as air. With this approach, fibrous materials are separated by removing different fractions, which, if desired, can be subjected to photon characterization. Such a separation device is discussed in Hipazeu et al., US patent No. 6,883,667.

The fibrous materials can be irradiated immediately after their preparation, or they can be dried, for example, at about 105 C for 4-18 hours, so that the moisture content before use is, for example, less than about 0.595.

If desired, the lignin can be removed from fibrous materials that include lignin. Also, to facilitate degradation of materials that include cellulose, the material may be treated prior to irradiation with heat, a chemical reagent (eg, a mineral acid, a base, or a strong oxidizing agent such as sodium hypochlorite), and/or an enzyme.

In some embodiments, the average opening size of the first screen is less than 0.79 mm (1/32 inch, 0.03125 inch), such as less than 0.51 mm (1/50 inch, 0.02000 inch), less than 0.40 mm (1/64 in, 0.015625 in), less than 0.23 mm (0.009 in), less than 0.20 mm (1/128 in, 0.0078125 in), less than 0.18 mm (0.007 in), less 0.13 mm (0.005 in) or even less than 0.10 mm (1/256 in, 0.00390625 in). The sieve is made by interweaving monofilament, which has the appropriate diameter to obtain the desired hole size. For example, monofilaments can be made of metal such as stainless steel. As the size of the holes decreases, the structural requirements for the monofilament increase. For example, for aperture sizes less than 0.40 mm, it may be preferable to fabricate screens from monofilaments obtained from a material other than stainless steel, such as titanium, titanium alloys, amorphous metals, nickel, tungsten, rhodium, rhenium, ceramics or glass. In some embodiments, the sieve is made from a plate, such as a metal plate, having holes, for example, cut into the plate using a laser. In some embodiments, the mesh area is less than 5295, such as less than 4195, less than 3695, less than 3195, less than 3095.

In some embodiments, the second fibrous material is crushed and passed through a first sieve or sieve with a different size. In some embodiments, the second fibrous material is passed through a second sieve having an average pore size equal to or less than the pore size of the first sieve.

Referring to FIG. 4, the third fibrous material 220 may be obtained from the second fibrous material 216 by crushing the second fibrous material 216 and passing the resulting material through a second sieve 222 having an average pore size smaller than that of the first sieve 214.

Typically, the fibers of fibrous materials can have a relatively high average length to diameter ratio (eg, greater than 20 to 1), even if they have been subjected to crushing more than once. In addition, the fibers of the fibrous materials described herein may have a relatively narrow length distribution and/or length-to-diameter ratio.

As used herein, the average fiber width (ie diameter) is determined optically by randomly selecting approximately 5000 fibers. Average fiber length is length-weighted adjusted lengths. The surface area of VET (Vgypaceg, Ettei apa TePeg) is a multipoint surface area, and porosity is a quantity determined by mercury porometry.

The average length-to-diameter ratio of the second fibrous material 14 may be, for example, greater than 8/1, such as greater than 10/1, greater than 15/1, greater than 20/1, greater than 25/11, or greater than 50/1 . The average length of the second fibrous material 14 may be, for example, from about 0.5 to 2.5 mm, for example, from about 0.75 to 1.0 mm, and the average width (ie, diameter) of the second fibrous material 14 may be, for example, from about 5 to 50 μm, for example from about 10 to 30 μm.

In some embodiments, the standard deviation of the length of the second fibrous material 14 is less than 6095 from the average length of the second fibrous material 14, such as less than 5090 from the average length, less than 4095 from the average length, less than 2595 from the average length, less than 1095 from the average length, less than 595 from of average length or even less than 195 of average length.

In some embodiments, the VET surface area of the second fibrous material is greater than 0.1 mg/g, such as greater than 0.25 mg/g, greater than 0.5 me/g, greater than 1.0 mg/g, greater than 1.5 m/g , exceeds 1.75 m"/g, exceeds 5.0 m/g, exceeds 10 m"/g, exceeds 25 m"/g, exceeds 35 m"/g, exceeds 50 m-/g, exceeds 60 mg/ g, exceeds 75 mg/g, exceeds 100 mg/g, exceeds 150 mg/g, exceeds 200 mg/g or even exceeds 250 mg/g. The porosity of the second fibrous material 14 may be, for example, greater than 2095, greater than 2595, greater than 3595, greater than 50965, greater than 6095, greater than 7095, for example greater than 8095, greater than 85965, greater than 9095, greater than 9295, greater than 9495, greater than 9595,9595, greater than 9595 9995 or even exceed 99.595.

In some embodiments, the ratio of the average length-to-diameter ratio of the first fibrous material to the average length-to-diameter ratio of the second fibrous material is, for example, less than 1.5, such as less than 1.4, less than 1.25, less than 1.1, less than 1.075, less than 1.05, less than 1.025, or even essentially equal to 1.

In specific embodiments, the second fibrous material is crushed again and the resulting fibrous material is passed through a second sieve having an average pore size smaller than that of the first sieve to produce a third fibrous material. In such cases, the ratio of the average length-to-diameter ratio of the second fibrous material to the average length-to-diameter ratio of the third fibrous material may be, for example, less than 1.5, such as less than 1.4, less than 1.25, or even less than 1.1.

In some embodiments, the third fibrous material is passed through a third sieve to produce a fourth fibrous material. The fourth fibrous material can, for example, be passed through the fourth sieve to obtain the fifth material. Similar sieving processes can be repeated as many times as desired to obtain the desired fibrous material having the desired properties.

Consolidation

Compacted materials may be processed in any of the ways described herein, or any material described herein, such as any fibrous material described herein, may be processed in any one or more ways, described herein and then compacted as described herein.

A material such as a fibrous material having a low bulk density can be compacted into a product having a higher bulk density. For example, a composition of material having a bulk density of 0.05 g/cm" can be compacted by isolating the fibrous material in a relatively gas-tight structure, such as a bag made of polyethylene or a bag made of alternating layers of polyethylene, and nylon, followed by removing the gas contained therein, such as air, from the structure After the air has been removed from the structure, the fibrous material may have, for example, a bulk density greater than 0.3 g/cm, such as 0.5, 0.6, 0.7 g/cm" or more, for example 0.85 g/cm3. After compaction, the product can be processed by any of the methods described in this document, such as irradiation, such as gamma radiation. This can be useful when it is desired to transport the material to another location, such as to a remote manufacturing facility, where the composition of the fibrous material can be added to a solution, for example, to produce ethanol. After puncturing the substantially gas-tight structure, the compacted fibrous material may return to substantially its original bulk density, such as greater than 6095 of its original bulk density, such as 70, 80, 8595 or greater, such as 9595 of its original bulk density. To reduce static electricity in the fibrous material, a static discharge agent may be added to the material.

In some embodiments, the structure, such as a bag, is made of a material that dissolves in a liquid, such as water. For example, the structure can be made of polyvinyl alcohol so that it dissolves when in contact with a water-based system. Such variants of implementation allow adding compacted structures directly to solutions that include a microorganism, without primary release of the contents of the structure, for example, by cutting.

Referring to FIG. 5, the biomass material can be combined with any desired additives and binder and then compacted by applying pressure, for example by passing the material through a certain gap between counter-rotating pressure rollers or by passing the material through a pelletizing press. During the application of pressure, heating may optionally be applied to facilitate compaction of the fibrous material. The compacted material can then be irradiated.

In some embodiments, the material before compaction has a bulk density of less than 0.25 g/cm3, such as 0.20, 0.15, 0.10, 0.05 g/cm3 or less, such as 0.025 g/cm3. Bulk density is determined using AZTM 018958. Briefly, the method includes filling a measuring cylinder with a known volume with a sample and determining the mass of the sample. The bulk density is calculated by dividing the mass of the sample in grams by the known volume of the cylinder in cubic centimeters.

Preferred binders include binders that are water-soluble, water-swellable, or have a glass transition temperature of less than 25°C, as determined by differential scanning calorimetry. By water-soluble binders are binders having a solubility in water of at least about 0.05 wt 95. By water-swellable binders is meant binders whose volume increases by more than 0.595 when exposed to water.

In some embodiments, binders that are water-soluble or water-swellable include functional groups that are capable of forming a bond, such as a hydrogen bond, with the fibers of a fibrous material, such as a cellulose fibrous material. For example, a functional group can be a carboxylic acid group, a carboxylate group, a carbonyl group, such as an aldehyde or a ketone, a sulfonic acid group, a sulfonate group, a phosphoric acid group, a phosphate group, an amide group, an amino group, a hydroxyl group, such as an alcohol, and combinations thereof. groups, for example, a carboxylic acid group and a hydroxyl group. Specific examples of monomers include glycerol, glyoxal, ascorbic acid, urea, glycine, pentaerythritol, monosaccharide or disaccharide, citric acid and tartaric acid. Suitable saccharides include glucose, sucrose, lactose, ribose, fructose, mannose, arabinose and erythrose. Examples of polymers include polyglycols, polyethylene oxide, polycarboxylic acids, polyamides, polyamines and polysulfonic acids, polysulfonates. Specific examples of polymers include polypropylene glycol (PPO), polyethylene glycol (PES), polyethylene oxide, such as ROIMOHF, copolymers of ethylene oxide and propylene oxide, polyacrylic acid (PAA), polyacrylamide, polypeptides, polyethyleneimine, polyvinylpyridine, poly(sodium-4-styrenesulfonate) and poly(2 -acrylamidomethyl-1-propanesulfonic acid).

In some embodiments, the binder comprises a polymer that has a glass transition temperature of less than 25°C. Examples of such polymers include thermoplastic elastomers (TPEs). Examples of TPEs include polyester block amides such as polyester block amides, available under the trade name REVAHF, polyester elastomers such as polyester elastomers available under the trade name NUTKEY F, and styrenic block copolymers such as block copolymers available under the trade name KKATOMF).Other suitable polymers having a glass transition temperature state less than 25 "C, include copolymer of ethylene and vinyl acetate (EMA), polyolefins, such as polyethylene, polypropylene, copolymers of ethylene-propylene, and copolymers of ethylene and alpha-olefins, such as 1-octene, such as copolymers available under the trade name EMOSACEF) . In some embodiments, for example, when the material is pulped paper with a multilayer coating, the material is compacted without adding a special polymer with a low glass transition temperature.

In a specific embodiment, the binder is lignin, for example, natural or synthetically modified lignin.

A suitable amount of binder added to the material, calculated on a dry weight basis, is, for example, from about 0.0195 to about 5095, for example 0.03, 0.05, 0.1, 0.25, 0.5, 1.0, 5, 1095 or more, for example 2595, based on the total weight of the compacted material. The binder can be added to the material in the form of an undiluted pure liquid, in the form of a liquid in which the binder is dissolved, in the form of a dry powder of the binder, or in the form of granules of the binder.

Compacted fibrous material can be made in a pelletizing press. Referring to

FIG. 6, the pelletizing press 300 has a loading hopper 301 for holding the uncompacted material 310, which includes a carbohydrate-containing material such as cellulose. The loading hopper is connected to an auger 312 which is driven by a variable speed motor 314 so that the uncompacted material can be transported to the mixing device 320, in which the uncompacted material is mixed by blades 322 which are rotated by the motor 330 of the mixing device. Other ingredients, such as any of the additives and/or fillers described herein, may be added via inlet 332. If desired, heat may be added while the fibrous material is in the mixing device. After mixing, the material is removed from the mixing device through the sleeve 340 to another auger 342. The sleeve, controlled by the drive mechanism 344, allows the material to flow smoothly from the mixing device to the auger. The screw rotates under the action of motor 346 and controls the supply of fibrous material to the die assembly with rollers 350. Specifically, the material is fed into a hollow cylindrical die 352 that rotates about a horizontal axis and has radial holes 250. The die 352 rotates about an axis under the action of motor 360, which includes a power meter that indicates the total energy consumed by the engine. Compacted material 370, for example, in the form of pellets, falls from the chute 372 and is collected and processed, for example, by irradiation.

It is convenient that after compaction the material is in the form of granules or chips that take different forms. Then the pellets can be irradiated. In some embodiments, the granules or chips are cylindrical in shape, for example having a maximum transverse dimension of, for example, 1 mm or more, for example 2, 3, 5, 8, 10, 15 mm or more, for example 25 mm. Other convenient forms include pellets or chips that are plate-shaped, for example having a thickness of 1 mm or more, for example 2, 3, 5, 8, 10 mm or more, for example 25 mm; width, for example, 5 mm or more, for example 10, 15, 25, 30 mm or more, for example 50 mm; and a length of 5 mm or more, for example 10, 15, 25, 30 mm or more, for example 50 mm.

Next, referring to FIG. 7A-7O, the pellets can be made to have a cavity inside. As shown, the cavity can be located mainly in line with the center of the pellet (FIG. 7B) or it can be offset from the center of the pellet (FIG. 7C). Making the pellet hollow inside can increase the dissolution rate in the liquid after irradiation.

Next, referring to FIG. 7O, the granule may have, for example, a cross-sectional shape that is multilobed, such as three-lobed as shown, or four-lobed, five-lobed, six-lobed or ten-lobed. Making granules with this transverse shape can also increase the dissolution rate in the solution after irradiation.

Alternatively, the compacted material may have any other desired shape, for example the compacted material may be in the form of a plate, cylinder or briquette.

Examples

In one example, half-gallon (1.9 L) juice cartons made from white kraft paperboard having a bulk density of 20 lb/ft may be used as raw materials. (0.32 g/cm3). The paperboard can be folded flat and then fed into a shredder to produce a confetti-like material that is 0.1 inch (0.25 cm) to 0.5 inch (1.27 cm) wide, 0 .25 inch (0.63 cm) to 1 inch (2.54 cm) and a thickness equivalent to the thickness of the parent material (approximately 0.075 inch (0.19 cm)). The confetti-like material can be fed into a cutting device with a rotating knife that crushes the confetti-like fragments, breaking the fragments and releasing the fibrous material.

In some cases, multiple shredder-shredder systems can be arranged with sequential production. In one embodiment, two shredder-shredder systems may be arranged in series, where the product of the first shredder is fed as input to the second shredder. In another embodiment, the three shredder-shredder systems can be arranged in series, where the product of the first shredder is fed as input to the second shredder, and the product of the second shredder is fed as input to the third shredder. It is expected that multiple passes through crusher-crusher systems can reduce particle size and increase the total surface area in the feed stream.

In another example, the fibrous material obtained by shredding and shredding juice cartons can be processed to increase its bulk density. In some cases, the fibrous material may be sprayed with water or diluted mother liquor

ROG MOH"M MUK M10 (polyethylene oxide) prepared in water. The soaked fibrous material can then be processed in a granulation press operating at room temperature. The granulation press can increase the volume density of the source material flow by more than one order of magnitude.

Preliminary processing

Physically prepared raw materials can be pre-processed for use in main production methods, for example, by reducing the average molecular weight and crystallinity of the raw materials and/or increasing the surface area and/or porosity of the raw materials. In some embodiments, the cellulosic and/or lignocellulosic material includes a first cellulose having a first number average molecular weight, and the resulting carbohydrate includes a second cellulose having a second number average molecular weight that is lower than the first number average molecular weight.

For example, the second number average molecular weight is lower than the first number average molecular weight by more than about 2595, for example, it can be 2x, 3x, 5x, 7x, 10x, 25x, and even 100x less.

In some embodiments, the first cellulose has a first crystallinity and the second cellulose has a second crystallinity that is lower than the first crystallinity, e.g., by about 2, 3, 5, 10, 15, or 2590.

In some embodiments, the first cellulose has a first oxidation level and the second cellulose has a second oxidation level that is higher than the first oxidation level, such as by 2, 3, 4, 5, 10, or 2590.

Pretreatment methods may include one or more methods selected from irradiation, sonication, oxidation, pyrolysis, and steam explosion. Different pretreatment systems can be used, combining two, three or even four of these technologies.

Combined pretreatment

In some embodiments, the biomass can be processed using two or more of any of the methods described herein, such as two, three, four or more methods selected from radiation exposure, sonication, oxidation, pyrolysis, and steam explosion, or to , or in between or after preparation of raw materials as described in this document. The methods can be applied to biomass in any order or simultaneously. For example, a carbohydrate can be produced by applying radiation, sonication, oxidation, pyrolysis and, optionally, steam explosion to cellulosic and/or lignocellulosic material (in any order or simultaneously). The resulting carbohydrate material can then be converted by one or more microorganisms, such as bacteria, yeast or mixtures of yeast and bacteria, into a number of desired products as described herein. Complex methods can provide materials that can be more easily utilized by various microorganisms due to their lower molecular weight, lower crystallinity and/or higher solubility. Complex methods can provide synergy and can reduce the total required energy costs compared to any single method.

For example, in some embodiments, a feedstock is provided that includes a carbohydrate obtained by a process that includes irradiation and sonication, irradiation and oxidation, irradiation and pyrolysis, or irradiation and steam explosion (in any order or simultaneously) of cellulosic and/or lignocellulosic material. . Then the provided raw material can be brought into contact with a microorganism having the ability to convert at least a part, for example at least about 1 wt. 95, raw materials into a product, such as burned fuel.

Pre-processing conditions

In some embodiments, the method does not include hydrolysis of cellulosic and/or lignocellulosic material, in particular, with an acid or a base, for example, a mineral acid such as hydrochloric or sulfuric acid. If desired, a portion of the raw material may or may not include hydrolyzed material. For example, in some embodiments, at least about 70 wt. 95 percent of the raw material is unhydrolyzed material, for example, at least 95 wt. 95% of the raw material is non-hydrolyzed material. In some embodiments, substantially all of the raw material is non-hydrolyzed material.

Any raw material or any reactor or fermenter loaded with the raw material may include a buffer such as sodium bicarbonate, ammonium chloride, or Ttg5; an electrolyte such as potassium chloride, sodium chloride or calcium chloride; a growth factor such as biotin and/or a base pair such as uracil or its equivalent; a surfactant such as TmeepF or polyethylene glycol; a mineral such as calcium, chromium, copper, iodine, iron, selenium or zinc; or a chelating agent such as ethylenediamine, ethylenediaminetetraacetic acid (EDTO) (or its salt form, such as sodium or potassium EDTO) or dimercaprol.

When radiation exposure is used, it can be applied to any sample that is dry or wet, or even dispersed in a liquid such as water. For example, irradiation can be carried out on cellulosic and/or lignocellulosic material in which less than about 25 wt. A cellulosic and/or lignocellulosic material has a surface wetted by a liquid such as water. In some embodiments, the irradiation is performed on a cellulosic and/or lignocellulosic material in which the cellulosic and/or lignocellulosic material is substantially unwetted by a liquid such as water.

In some embodiments, any processing described herein is performed after the cellulosic and/or lignocellulosic material remains dry as received or after it has been dried, for example, using heat and/or reduced pressure. For example, in some embodiments, the cellulosic and/or lignocellulosic material has less than about 5 wt. 95 of retained water, measured at 25 "C and at a relative humidity of 50905.

If desired, a swelling agent as defined herein may be used in any method described herein. In some embodiments, when cellulosic and/or lignocellulosic material is processed using radiation exposure, less than about 25 wt. 95 cellulosic and/or lignocellulosic material is in the swollen state, the swollen state being characterized as having a volume greater than 2.595 times the volume in the unswollen state, such as greater than 5.0, 7.5, 10, or 1595 exceeds the volume in the unswollen state. In some embodiments, when radiation exposure is applied to the cellulosic mAbo lignocellulosic material, the cellulosic and/or lignocellulosic material is essentially not in a swollen state. In specific embodiments, when radiation exposure is used, the cellulosic and/or lignocellulosic material includes a swelling agent, and the swollen cellulosic and/or lignocellulosic material receives a dose of less than about 10 Mrad.

When radiation exposure is used in any method, it can be applied by simultaneously exposing the cellulosic and/or lignocellulosic material to air, oxygen-enriched air, or even oxygen itself, or it can be under an inert gas such as nitrogen, argon, or helium. If maximum oxidation is desired, an oxidizing atmosphere such as air or oxygen may be used.

When radiation exposure is used, it can be applied to biomass, such as cellulosic and/or lignocellulosic material, at pressures greater than about 2.5 atmospheres (253 kPa), such as greater than 5 (506 kPa), 10 (1012 kPa), 15 (1518 kPa), 20 (2036 kPa), or even more than about 50 atmospheres (5060 kPa).

Radiation treatment

For the processing of raw materials from a wide variety of different sources, one or more processing sequences can be used for the purpose of extracting useful substances from the raw materials, as well as for the purpose of obtaining partially degraded organic material that performs the function of an input stream in further stages and/or processing sequences. Irradiation can reduce the molecular weight and/or crystallinity of raw materials. In some embodiments, materials are irradiated using energy stored in the material, which releases an electron from its atomic orbital. Radiation exposure can be provided by 1) heavy charged particles such as alpha particles or protons, 2) electrons produced, for example, in beta decay or in electron beam accelerators, or 3) electromagnetic radiation such as gamma rays, X-rays or ultraviolet rays. In one approach, radiation generated by radioactive substances can be used to irradiate raw materials. In some embodiments, any combination of (1)-(3) can be used in any order or simultaneously. In another approach, electromagnetic radiation (for example, generated using electron beam sources) can be used to irradiate raw materials. The doses used depend on the desired effect and the specific raw material. For example, high doses of radiation exposure can destroy chemical bonds in the components of the raw material, and low doses of radiation exposure can increase the formation of chemical bonds (eg, cross-linking) in the components of the raw material. In some cases where chain scission is desired and/or polymer chain functionalization is desired, heavier than electron particles such as protons, helium group nuclei, argon ions, silicon ions, neon ions, carbon ions, phosphorus ions, oxygen or nitrogen ions. When ring-opening chain cleavage is desired, positively charged particles can be used as Lewis acid properties to enhance ring-opening chain cleavage. For example, when oxygen-containing functional groups are desired, irradiation in the presence of oxygen or even irradiation with oxygen ions can be carried out. For example, when nitrogen-containing functional groups are desired, irradiation in the presence of nitrogen or even irradiation with nitrogen ions can be performed.

Referring to FIG. 8, in one method, the first material 2, which is or includes cellulose having a first number average molecular weight ("Mm:") is irradiated, for example by treatment with ionizing radiation (for example, in the form of gamma radiation, X-ray radiation, ultraviolet (UV) light from 100 to 280 nm, a beam of electrons or other charged particles), to obtain a second material 3, which includes cellulose having a second number average molecular weight ("Mkg"), which is lower than the first number average molecular weight. The second material (or the first and second materials) can be mixed with a microorganism (e.g., bacteria or yeast) that can utilize the second and/or the first material, producing a fuel 5 that is or includes hydrogen, an alcohol (e.g., ethanol or butanol, such as n-, sec-, or tert-butanol), an organic acid, a hydrocarbon, or a mixture of any of these.

Since the second material C has cellulose having a reduced molecular weight relative to the first material and, in some cases, also reduced crystallinity, the second material is generally more capable of dispersing, swelling and/or dissolving in the solution containing the microorganism. These properties make the second material 3 more susceptible to chemical, enzymatic and/or biological action relative to the first material 2, which can significantly increase the production rate and/or production level of the desired product, such as ethanol. Radiation exposure can also sterilize materials.

In some embodiments, the second number average molecular weight ("Micg") is lower than the first number average molecular weight ("Mmi") by more than about 1095, such as by 15, 20, 25, 30, 35, 40, 50, 6095, or even more than by about 7,595.

In some cases, the second material contains cellulose that has a crystallinity ("Cg) that is lower than the crystallinity (C) of the cellulose of the first material. For example, the ("Cg) may be lower than (Si) by more than about 1095, such as 15, 20, 25, 30, 35, 4095 or even more than about 5095.

In some embodiments, the initial crystallinity index (before irradiation) is from about 4095 to about 87.595, such as from about 5095 to about 7595 or from about 6095 to about 7095, and the crystallinity index after irradiation is from about 1095 to about 5095, such as from about 1595 to about 4595 or from about 2095 to about 4095. However, in some embodiments, such as after extensive irradiation, the crystallinity index may be less than 595. In some embodiments, the material after irradiation is essentially amorphous.

In some embodiments, the starting number average molecular weight (before irradiation) is from about 200,000 to about 3,200,000, such as from about 250,000 to about 1,000,000 or from about 250,000 to about 700,000, and the number average molecular weight after irradiation is from about 50,000 to about 200,000, e.g. from about 60,000 to about 150,000 or from about 70,000 to about 125,000. However, in some embodiments, such as after extensive irradiation, the number average molecular weight may be less than about 10,000 or even less than about 5,000.

In some embodiments, the second material may have an oxidation level (702) greater than the oxidation level (70) of the first material. A higher level of oxidation of a material can contribute to its ability to disperse, swell, and/or dissolve, further increasing the susceptibility of materials to chemical, enzymatic, or biological attack. In some embodiments, to increase the oxidation level of the second material relative to the first material, the irradiation is carried out in an oxidizing atmosphere, such as air or oxygen, producing a second material that is more oxidized than the first material. For example, the second material may have more hydroxyl groups, aldehyde groups, ketone groups, ester groups, or carboxylic acid groups that may increase its hydrophilicity.

Ionizing radiation

Each form of radiation ionizes biomass through specific interactions determined by the energy of the radiation. Heavy charged particles mainly ionize matter through Coulomb scattering; moreover, these interactions generate energetic electrons that can further ionize matter. Alpha particles are identical to the nucleus of a helium atom and are produced by the alpha decay of various radioactive nuclei, such as isotopes of bismuth, polonium, astatine, radon, francium, radium, some actinoids such as actinium, thorium, uranium, neptunium, curium, californium, americium and plutonium.

When particles are used, they can be neutral (uncharged), positively charged, or negatively charged. When charged, charged particles can carry a single positive or negative charge, or multiple charges, such as one, two, three, or even four or more charges. In cases where chain cleavage is desired, positively charged particles may be desirable, in part due to their acidic nature. When particles are used, the particles may have the mass of the rest electron or a mass greater than, for example, 500, 1000, 1500, or 2000 or more times the mass of the rest electron. For example, the particles may have a mass of from about 1 atomic unit to about 150 atomic units, such as from about 1 atomic unit to about 50 atomic units, or from about 1 to about 25, such as 1, 2, 3, 4, 5, 10, 12 or 15, atomic units. Accelerators used to accelerate particles can be OS-electrostatic, OS-electrodynamic, PE-linear, linear with magnetic induction or continuous. For example, cyclotron accelerators are available from IVA, Veidiit, such as the KpodoihopF system), and OS-type accelerators are available from KOI, currently

IVA Ipdysvigia!, such as OupatygopF). Ions and ion accelerators are discussed in the publications of Ipigodisiogu Mysieag

RPuzis5, Keppeii 5. Kgape, Chopp UmMieu 5 5op5, Ips. (1988), Kgeo Rgeies, RI2ICA V 6 (1997) 4, 177-206, a copy of which is attached as an appendix V, Spy, Umiyat T., "Omegmiezhm oi Gidpe-Iop Veat Tnegaru",

Soishtryv-ONio, ISVO-IAEA Meeiiipo, 18-20 Magsp 2006, a copy of which is attached to this document in the form of appendix C, maga, MU. ei ai., "AKegpaipd-Rpaze-BROsizei 1N-OTI og Neamu-ip Medvdisai!

Assei|egayugv", Rgoseedipd5 oi ERAS 2006, Edipriguy, Zsoyapa, a copy of which is attached to this document in the form of an appendix O, and Geipeg, SM.

Rgoseedipd5 oi ERAS 2000, Mieppa, A!izvigia, a copy of which is attached to this document in the form of an appendix

IS.

Electrons interact by means of Coulomb scattering and inhibition of radiation caused by changes in the speed of electrons. Electrons can be generated by radioactive nuclei that undergo beta decay, such as isotopes of iodine, cesium, technetium, and iridium.

Alternatively, an electron gun can be used as a source of electrons based on thermionic emission.

Electromagnetic radiation acts through the following three processes: photoelectric absorption, Compton scattering, and vapor formation. The predominant effect is determined by the energy of the incident radiation and the atomic number of the material. The sum of the interactions leading to the absorption of radiation in the cellulosic material can be expressed using the mass absorption coefficient.

Electromagnetic radiation is divided into gamma rays, X-rays, ultraviolet rays, infrared rays, microwaves or radio waves, depending on the wavelength.

For example, gamma radiation can be used to irradiate materials.

Referring to FIG. 9 and 10 (enlarged view of area K), the gamma emitter 10 includes sources of gamma radiation 408, such as 90Co tablets, a work table 14 for holding the materials to be irradiated, and a storage device 16, for example, made of a plurality of iron plates, all of which are in a concrete-shielded chamber (storage) 20, which includes a labyrinthine entrance 22 behind a leaded door 26. The accumulator 16 includes a plurality of channels 30, such as sixteen or more channels, that allow the gamma radiation sources to pass through the accumulator near desktop

In the process of work, the sample to be irradiated is placed on the work table. The irradiator is configured to deliver the desired dose level and control equipment is connected to the experimental unit 31. The operator then leaves the protective chamber, passing through the entrance in the form of a labyrinth and through a leaded door. The operator serves the control panel 32, instructing the computer 33 to bring the radiation sources 12 into the working position using the cylinder 36 connected to the hydraulic pump 40.

Gamma radiation has the advantage of a significant depth of penetration into various sample materials.

Sources of gamma rays include radioactive nuclei such as isotopes of cobalt, calcium, technetium, chromium, gallium, indium, iodine, iron, krypton, samarium, selenium, sodium, thallium, and xenon.

X-ray sources include collisions of the electron beam with metal targets such as tungsten or molybdenum or alloys, or compact light sources such as commercially produced I upseap light sources.

Sources of ultraviolet radiation include deuterium or cadmium lamps.

Infrared radiation sources include ceramic lamps with a sapphire, zinc, or selenide window.

Microwave sources include klystrons, CE-type biemip sources, or atomic beam sources using hydrogen, oxygen, or nitrogen gas.

Various other irradiation devices may be used in the methods described herein, including field ionization sources, electrostatic ion separators, field ionization generators, thermionic emission sources, ultrahigh frequency discharge ion sources, recirculation or static accelerators, dynamic linear accelerators, accelerators

Van de Graafois and bent tandem accelerators. Such devices are disclosed, for example, in the previous application

US Serial No. 61/073665, the contents of which are incorporated herein by reference in their entirety.

Electronic beam

In some embodiments, an electron beam is used as a source of radiation. The electron beam has the advantage of high dose levels (eg 1, 5 or even 10 Mrad per second), high performance, less protective insulation and less isolation equipment.

Electrons can also be more efficient at providing chain cleavage. In addition, electrons having an energy of 4-10 MeV can have a penetration depth of 5 to 30 mm or more, for example 40 mm.

Electron beams can be generated, for example, using electrostatic generators, cascade generators, transformer generators, low-energy scanning accelerators, low-energy linear cathode accelerators, linear accelerators, and pulse accelerators. Electrons may be suitable as a source of ionizing radiation, for example, for relatively thin stacks of material, such as less than 0.5 inch (1.27 cm), such as less than 0.4 inch (1.02 cm), 0.3 inch ( 0.76 cm), 0.2 in (0.51 cm) or less than 0.1 in (0.25). In some embodiments, the energy of each electron in the electron beam is from about 0.3 MeV to about 2.0 MeV (megaelectronvolts), such as from about 0.5 MeV to about 1.5 MeV or from about 0.7 MeV to about 1 .25 MeV.

In FIG. 11A presents a basic technological scheme 3000, which includes various stages in the sequence of preliminary processing of raw materials with an electron beam. In the first stage, 3010 dry raw materials are supplied from the feedstock source. As discussed above, the dry feedstock from the feedstock source may be preprocessed prior to delivery to the electron beam devices. For example, if the raw material is derived from plant sources, certain parts of the plant material may be removed prior to harvesting the plant material and/or prior to delivering the plant material using a raw material transport device.

Alternatively or additionally, as reflected in optional stage 3020, the biomass raw material can be subjected to mechanical processing (eg, to reduce the average length of fibers in the raw material) before delivery to devices for electron beam irradiation.

At step 3030, the dry raw material is moved into the raw material transport device (eg, on a conveyor belt) and is distributed along the transverse length of the raw material transport device approximately uniformly in volume. This can be done, for example, manually or by inducing a localized vibrational movement at some point of the device for transporting raw materials before processing by irradiation with an electron beam.

In some embodiments, the mixing system supplies a chemical reagent 3045 to the raw material in an optional process 3040 in which a slurry is formed. The combination of water with processed raw materials in the mixing stage 3040 leads to an aqueous suspension of raw materials that can be transported, for example, through a system of pipes, rather than using, for example, a conveyor belt.

The next stage 3050 is a cycle that includes exposure to the raw material (in dry form or in the form of a suspension) of electron beam irradiation from one or more (for example, M) devices for electron beam irradiation. The suspension of feedstock is moved through each of the M "streams" of electron beams at stage 3052. Movement through and between the streams may occur at a constant rate, or there may be a pause during passage through each stream, followed by rapid movement to the next stream. At step 3053, a small portion of the feedstock slurry is exposed to each stream for some predetermined exposure time.

Electron beam devices can be purchased commercially from Op Veat

Arriisayopv, I oimaiip-Ia-Metsme, VeYidisht or She Tyap Sogrogayop, Zap Oiedo, SA. A typical electron energy can be 1, 2, 4.5, 7.5 or 10 MeV. A typical power of an electron beam irradiation device can be 1, 5, 10, 20, 50, 100, 250 or 500 kW. The efficiency of depolymerization of the raw material suspension depends on the used electron energy and the applied dose, while the exposure time depends on the power and dose. Typical doses can be 1, 5, 10, 20, 50, 100 or 200 kGy.

When choosing the optimal power characteristics of the device for electron beam irradiation, labor costs, capital costs, depreciation costs and the location of the device are taken into account.

When choosing the optimal levels of the exposure dose of electron beam irradiation, energy consumption and issues of ecology, safety and health are taken into account (E5N). When choosing the optimal electron energy, energy consumption is taken into account; in this case, a lower electron energy may be advantageous from the point of view of promoting the depolymerization of a certain feed stock suspension (see, for example, Voysyaga, et al.,

Sei Ishiose (2006) 13: 601-610). As a rule, generators are placed in storage, for example, made of lead or concrete, or leaded concrete.

To ensure a more efficient depolymerization process, it may be useful to perform double-pass electron beam irradiation. For example, a raw material transport device can direct the raw material (in dry form or in slurry form) down and in a reverse direction relative to its original direction of transport. Dual-pass systems can handle thicker stock slurries and can provide more uniform depolymerization through the thickness of the stock slurry.

An electron beam irradiation device can generate either a fixed beam or a scanning beam. A scanning beam with long scan sweep lengths and high scan speeds may be preferred, as this can effectively replace a large fixed beam width. In addition, sweep lengths of 0.5, 1, 2 m or more are available.

After a portion of the raw material suspension has been transported through M devices for electron beam irradiation, in some embodiments it may be necessary, as in stage 3060, to mechanically separate the liquid and solid components of the raw material suspension. In these embodiments, residual solid particles are filtered from the liquid part of the raw material suspension and returned to the slurry preparation stage 3040. Then, the solid part of the raw material suspension is moved to the next processing stage 3070 using a raw material transport device. In other embodiments, the raw material is maintained in the form of a suspension for additional processing.

Electromagnetic radiation

In embodiments in which exposure is performed using electromagnetic radiation, the electromagnetic radiation may have, for example, an energy per photon (in electron volts) greater than 102 eV, such as greater than 103, 102, 105, 105 eV, or even greater than 107 eV. In some embodiments, the electromagnetic radiation has an energy per photon of 107 to 107 eV, such as 105 to 105 eV. Electromagnetic radiation can have a frequency, for example, greater than 1016 Hu, greater than 1017, 1018, 1019, 1020 Hu, or even greater than 102! Gu. In some embodiments, the electromagnetic radiation has a frequency between 1078 and 1022 Hz, such as between 10792 and 102! Hz.

Doses

In some embodiments, exposure (with any radiation source or combination of sources) is conducted until the material receives a dose of at least 0.25 Mrad, such as at least 1.0 Mrad, at least 2.5 Mrad, at least 5.0 Mrad, or at least 10.0 Mrad. In some embodiments, the irradiation is carried out until the material receives a dose of 1.0 to 6.0 Mrad, for example, 1.5 to 4.0 Mrad.

In some embodiments, the irradiation is carried out at a dose level of 5.0 to 1500.0 kilorads/hour, for example, from 10.0 to 750.0 kilorads/hour or from 50.0 to 350.0 kilorads/hour.

In some embodiments, the implementation uses two or more sources of radiation, such as two or more sources of ionizing radiation. For example, samples can be treated, in any order, with an electron beam, followed by gamma radiation and UV radiation having wavelengths from about 100 nm to about 280 nm. In some embodiments, samples are treated using three sources of ionizing radiation, such as an electron beam, gamma radiation, and energetic UV radiation.

Alternatively, in another example, the fibrous material, which includes cellulosic and/or lignocellulosic material, is irradiated and optionally treated with sound energy, such as ultrasound.

One example uses half-gallon (1.9 L) juice cartons made from white kraft paperboard having a bulk density of 20 lb/ft3 (0.32 g/cm3) as raw material. The paperboard can be folded flat and then fed into a sequence of three shredder-shredder systems arranged in series, where the product of the first shredder is fed as input to the second shredder and the product of the second shredder is fed as input material to the third device for grinding.

The fibrous material obtained in the shredder-shredder system can be sprayed with water and processed using a pellet press operating at room temperature. Compacted pellets can be placed in a glass ampoule, which is evacuated under high vacuum and then refilled with argon gas. The ampoule is sealed in an argon atmosphere. Alternatively, in another example, the ampoule is sealed in an air atmosphere. The granules in the ampoule are irradiated with gamma radiation for about 3 hours at a dose level of about 1 Mrad per hour to produce irradiated material in which the cellulose has a lower molecular weight than the starting material.

Additives to enhance molecular weight reduction during irradiation

In some embodiments, various materials, such as solids or liquids, may be added to the biomass prior to irradiation to enhance molecular weight reduction. In cases where a liquid is used, the liquid can be in contact with the external surfaces of the biomass or the liquid can be inside the biomass, for example it can be poured into the biomass.

For example, the material may be a neutral weak base such as alanine, ammonia, an ammonia/water mixture, such as 25 wt. 95 ammonia in water, water, methylamine, dimethylamine, trimethylamine, pyridine, or an anionic base such as an acetic acid salt (eg, sodium acetate), sodium carbonate, sodium bicarbonate, or a hydrogen sulfide ion salt (eg, sodium hydrosulfide).

Alternatively, the material may be a neutral weak acid such as formic acid, acetic acid, trichloroacetic acid, hydrogen, hydrogen sulfide, or a cationic acid such as an ammonium salt.

Extinction and controlled functionalization of biomass

After treatment with one or more types of ionizing radiation, such as photon radiation (such as X-rays or gamma rays), electron beam irradiation, or particles heavier than electrons that are positively or negatively charged (such as protons or carbon ions), any of hydrocarbon-containing materials or mixtures described in this document become ionized, that is, they include radicals at levels that are detectable with an electron spin resonance spectrometer. The current practical limit of radical detection is approximately 1077 spins at room temperature. After ionization, any biomass material,

which has been ionized, can be quenched to reduce the level of radicals in the ionized biomass, for example, so that the radicals are no longer detectable with an electron spin resonance spectrometer. For example, radicals can be quenched by applying sufficient pressure to the biomass and/or using a fluid medium in contact with the ionized biomass, such as a gas or liquid, which reacts (quenches) with the radicals. The use of a gas or liquid to at least aid radical quenching also allows the operator to control the functionalization of the ionized biomass with the desired amount and type of functional groups, such as carboxylic acid groups, enol groups, aldehyde groups, nitro groups, nitrile groups, amino groups, alkylamino groups, alkyl groups, chloroalkyl groups, or chlorofluoroalkyl groups. In some cases, such quenching can increase the stability of some of the ionized biomass materials. For example, quenching can increase the resistance of biomass to oxidation.

Functionalization by quenching may also increase the solubility of any biomass described herein, may increase its thermal stability, which may be important in the manufacture of composites and paperboards described herein, and may improve utilization of the material by various microorganisms. For example, the functional groups that are provided to the biomass material as a result of quenching can act as receptor sites for binding by microorganisms, for example to enhance the hydrolysis of cellulose by various microorganisms.

In FIG. 118 illustrates changing the molecular and/or supramolecular structure of the biomass feedstock by pre-treating the biomass feedstock with ionizing radiation, such as electrons or ions with energy sufficient to ionize the biomass feedstock, providing a first level of radicals. As shown in FIG. 11B, if the ionized biomass remains in the atmosphere, it is oxidized, for example, to such an extent that carboxylic acid groups are formed due to the reaction with atmospheric oxygen. In some cases, for some materials, such oxidation is desirable, since it can contribute to further lowering the molecular weight of carbohydrate-containing biomass, and oxidizing groups, for example, carboxylic acid groups, can in some cases be useful for solubility and utilization by a microorganism. However, since the radicals can "live" for some time after exposure, for example, more than 1 day, 5 days, 3 days, 3 months, 1 months, or even more than 1 year, the properties of the material can continue to change over time, which, in some cases, , may be undesirable. The detection of radicals in irradiated samples by means of electron spin resonance spectroscopy and the lifetime of radicals in such samples are considered in the publications of Vagiotsa ei aiY., Rpuvzis5 ip Medisipe apa Vioidodu, 46 (2001), 461-471 and Vagpoiona ei ai.,

Vadiayop Rgoiesip Oovziteigu, Moi. 84, Language 1-4, pp. 293-296 (1999), which are attached to this document as Appendix E and Appendix C, respectively. As shown in FIG. 118V, the ionized biomass can be quenched to functionalize and/or stabilize the ionized biomass. At any point in time, such as when the material is "live", "partially live" or fully decomposed, the pre-treated biomass can be converted into a product such as a fuel, food or composite.

In some embodiments, quenching includes applying pressure to the biomass, for example, by mechanically deforming the biomass, such as direct mechanical compression of the biomass in one, two, or three dimensions, or by applying pressure to a fluid medium in which the biomass is immersed, such as isostatic pressing. In such cases, deformation of the material itself yields radicals that are often trapped in crystalline domains close enough for the radicals to recombine or react with another group. In some cases, the pressure is applied together with the application of heating, such as an amount of heat sufficient to raise the temperature of the biomass to a level above the melting point or to the softening temperature of a biomass component such as lignin, cellulose, or hemicellulose. Heating can increase molecular mobility in the polymer material, which can contribute to radical quenching. If pressure is used for quenching, it can be higher than about 1000 psi. in. (6.9 MPa), such as above about 1250 psi. in. (8.6 MPa), 1450 lb/sq. in. (10 MPa), 3625 lb/sq. in. (25.2 MPa), 5075 lb/sq. in. (35 MPa), 7250 lb/sq. in. (50 MPa), 10,000 psi. in. (69 MPa) or even higher than 15,000 psi. inch (103.4 MPa).

In some embodiments, quenching includes contacting the biomass with a fluid medium, such as a liquid or gas, such as a gas capable of reacting with radicals, such as acetylene or a mixture of acetylene in nitrogen, ethylene, chlorinated ethylenes or chlorofluoroethylenes, propylene, or mixtures of these gases. In other specific embodiments, quenching includes contacting the biomass with a liquid, such as a liquid soluble in the biomass or at least capable of penetrating the biomass and reacting with radicals such as a diene such as 1,5-cyclooctadiene. In some specific embodiments, quenching includes contacting the biomass with an antioxidant such as vitamin E. If desired, the biomass feedstock may include an antioxidant dispersed therein, and the quenching may occur as a result of the antioxidant dispersed in the biomass feedstock contacting radicals .

Other extinguishing methods are possible. For example, to quench any ionized biomass material described herein, any method of quenching radicals in polymeric materials described in Mygagodii et al., US Patent Application Publication No. 2008/0067724 and Mygagodii et al., US Pat. Mo. 7166650, which are attached as Appendix H and Appendix I, respectively. Additionally, any quenching agent (described as a "sensitizing agent" in the Mygaiodia disclosures above) and/or any antioxidant described in any Mygaiodia reference can be used to quench any ionized biomass material.

Functionalization can be enhanced using heavy charged ions, such as any of the heavier ions described herein. For example, if it is desired to increase oxidation, charged oxygen ions can be used for irradiation. If nitrogen functional groups are desired, nitrogen ions or ions that include nitrogen can be used. Similarly, if sulfur or phosphorus groups are desired, then sulfur or phosphorus ions can be used during irradiation.

In some embodiments, after quenching, any of the quenched materials described herein may be further treated by one or more methods selected from radiation exposure, such as ionizing or non-ionizing radiation, sonication, pyrolysis, and oxidation to further alter the molecular and/or or supramolecular structure.

In specific embodiments, the functionalized materials described herein are treated with an acid, base, nucleophile, or Lewis acid to further alter the molecular and/or supramolecular structure, such as further lowering the molecular weight.

Examples of acids include organic acids such as acetic acid and mineral acids such as hydrochloric, sulfuric and/or nitric acid. Examples of bases include strong mineral bases such as hydroxide ion source, basic ions such as fluoride ions, or weaker organic bases such as amines. Even water and sodium bicarbonate, for example, when dissolved in water, can provide a change in molecular and/or supramolecular structure, such as an additional decrease in molecular weight.

Particle beam irradiation in fluid media

In some cases, cellulosic or lignocellulosic materials can be exposed to particle beam irradiation in the presence of one or more additional fluid media (eg, gases and/or liquids). The effect on the material of a beam of particles in the presence of one or more additional fluid media can increase the efficiency of processing.

In some embodiments, the material is irradiated with a beam of particles in the presence of a fluid medium, such as air. Particles accelerated in one or more types of accelerators described in this document (or in another type of accelerator) exit the accelerator through an outlet (eg, a thin membrane such as a metal foil), pass through a volume of space occupied by the fluid environment, and then fall on the material. In addition to the direct processing of the material, some of the particles form additional chemical particles by interaction with the particles of the fluid medium (for example, ions and/or radicals generated by various components of the air, such as ozone and nitrogen oxides). These formed chemical particles can also interact with the material and can act as initiators of various chemical bond breaking reactions in the material. For example, any oxidizing agent formed can oxidize the material, which can lead to a decrease in molecular weight.

In certain embodiments, additional fluids can be selectively applied to the path of the particle beam before it hits the material. As discussed above, as a result of reactions between the particles of the beam and the particles of the supplied fluids, additional chemical particles can be formed, which react with the material and can contribute to the functionalization of the material and/or otherwise selectively change certain properties of the material. One or more additional fluids can be directed into the path of the beam, for example, from a downpipe. The direction and flow rate of the fluid(s) being fed can be selected according to the desired power and/or direction of exposure to control overall treatment efficiency, including both particle treatment effects and interaction effects of dynamically formed particles from the given fluid medium with the material. In addition to air, typical media that can be supplied to the ion beam include oxygen, nitrogen, one or more noble gases, one or more halogens, and hydrogen.

Irradiation of biomass materials with low bulk density and cooling of irradiated biomass

In the process of treating biomass materials with ionizing radiation, especially at high dose levels, such as levels greater than 0.15 Mrad per second, for example 0.25, 0.35, 0.5, 0.75 Mrad/s or even more than 1 Mrad/ c, biomass materials can store significant amounts of heat, so that the temperature of biomass materials increases. While, in some embodiments, elevated temperatures may be preferred, for example, when a higher reaction rate is desired, it is useful to control the heating of the biomass to maintain control over chemical reactions initiated by ionizing radiation, such as cross-linking, chain cleavage, and/or or graft copolymerization, for example, to maintain process control. Low bulk density materials, such as materials having a bulk density of less than about 0.4 g/cm3, such as less than about 0.35, 0.25 g/cm3, or less than about 0.15 g /cm3, especially when combined with materials that have thin cross-sections, such as fibers that have small cross-sectional dimensions, are generally easier to cool. In addition, photons and particles can primarily penetrate deeper into or through materials of relatively low bulk density, which may allow processing of larger volumes of materials at higher speeds and may enable applications of photons and particles , which have lower energies, such as 0.25, 0.5, 0.75 or 1.0 MeV, which can reduce shielding requirements for safety.

For example, in one method of altering the molecular and/or supramolecular structure of the biomass feedstock, the biomass is pretreated at a first temperature with ionizing radiation, such as photons, electrons, or ions (eg, singly or multiply charged cations or anions), for a sufficient time and/or at a sufficient a dose for raising the temperature of the biomass raw material to a second temperature that exceeds the first temperature. The pretreated biomass is then cooled to a third temperature, which is lower than the second temperature. Finally, if desired, the cooled biomass can be treated one or more times with radiation, such as ionizing radiation. If desired, the biomass can be cooled after and/or during each irradiation treatment.

The biomass feedstock can be physically prepared as discussed above, for example, by reducing the size of one or more individual fragments of the biomass feedstock so that the feedstock can be processed more efficiently, such as being more easily cooled and/or more permeable to ionizing radiation.

In some embodiments, ionizing radiation is used in a total dose of less than 25

Mrad or less than 10 Mrad, such as less than 5 Mrad or less than 2.5 Mrad, and at a rate greater than 0.25

Mrad per second, for example greater than 0.5, 0.75 or greater than 1.0 Mrad/s, before cooling the biomass.

Pre-treatment of biomass raw materials with ionizing radiation can be carried out by pneumatically transporting the biomass raw materials into a fluid medium, such as a gas such as nitrogen or air. To facilitate molecular weight reduction and/or functionalization of materials, the gas may be saturated with any swelling agent described herein and/or steam. For example, you can use acidic steam. To facilitate molecular weight reduction, water can be acidified with an organic acid such as formic or acetic acid or a mineral acid such as sulfuric or hydrochloric acid.

Pre-treatment of biomass raw materials with ionizing radiation can be carried out as the biomass raw materials fall under the influence of gravity. This process can effectively reduce the bulk density of the biomass feedstock as it is processed, and it can help cool the feedstock.

For example, biomass can be transported from a first belt at a first height above ground level, and then it can enter a second belt at a second level above ground level, which is lower than the first level. For example, in some embodiments, the trailing edge of the first strip and the leading edge of the second strip form a gap. Preferably, ionizing radiation, such as a beam of electrons, protons, or other ions, may be applied to the gap region to prevent damage to the biomass transport system.

Cooling the biomass may include contacting the biomass with a fluid medium, such as a gas, at a temperature below the first or second temperature, such as nitrogen gas at or about 77K (-196 "C). Even water, such as water at below the nominal room temperature (for example, 25 "7C).

It is often preferred that the biomass feedstock has internal fibers and prior to exposure to ionizing radiation, the biomass feedstock is shredded to such an extent that its internal fibers are essentially exposed. This crushing can provide material with a low bulk density, which has small cross-sectional dimensions, which can contribute to the reduction of molecular weight and/or functionalization of the biomass. For example, in some embodiments, the biomass is or includes individual fibers and/or particles having a maximum size of no more than about 0.5 mm, such as no more than about 0.25 mm, no more than about 0.1 mm, or no more than about 0.05 mm.

In some embodiments, the biomass feedstock to which ionizing radiation is applied has a bulk density of less than about 0.35 g/cm3, such as less than about 0.3, 0.25, 0.20 g/cm3, or less than about 0.15 g/cm3, during the application of ionizing radiation. In such embodiments, the biomass feedstock may be cooled, and then ionizing radiation may be applied to the cooled biomass. In some preferred embodiments, the biomass feedstock is or includes individual fibers and/or particles having a maximum size of no more than about 0.5 mm, such as no more than about 0.25 mm, no more than about 0.1 mm, no more than about 0.05 mm or no more than about 0.025 mm.

Ultrasound treatment

One or more sonication series can be used to process raw materials from a wide variety of different sources to extract useful substances from the raw materials, as well as to provide partially degraded organic material that serves as an input stream for further stages and/or processing sequences. Ultrasound treatment can reduce the molecular weight and/or crystallinity of raw materials.

Referring again to FIG. 8, in one method, a first material 2 that includes cellulose having a first number average molecular weight ("Mmi") is dispersed in a medium such as water and sonicated or otherwise cavitated to produce a second material 3 that includes cellulose, having a second number average molecular weight ("Mmg") that is lower than the first number average molecular weight. The second material (or, in certain embodiments, the first and second materials) can be mixed with a microorganism (e.g., bacteria or yeast) that can utilize the second and/or first material to produce a fuel 5 that includes hydrogen, an alcohol, an organic acid, hydrocarbons or mixtures of any of them.

Since the second material contains cellulose having a reduced molecular weight relative to the first material and, in some cases, also reduced crystallinity, the second material is generally more capable of dispersing, swelling and/or dissolving in a solution containing a microorganism, e.g. in a concentration of more than 105 microorganisms/ml. These properties make the second material C more susceptible to chemical, enzymatic and/or microbial attack relative to the first material 2, which can significantly increase the production rate and/or production level of the desired product, such as ethanol. Ultrasonication can also sterilize materials, but should not be used as long as the microorganisms are expected to be alive.

In some embodiments, the second number average molecular weight ("Mg") is lower than the first number average molecular weight ("Mm:") by more than about 1095, such as 15, 20, 25, 30, 35, 40, 50, 6095, or even more than about 7,595.

In some cases, the second material contains cellulose having a crystallinity (Cg) that is lower than the crystallinity (Si) of the cellulose of the first material. For example, (Cg) may be lower than (7Ci) by more than about 1095, such as by 15, 20, 25, 30, 35, 4095, or even more than about 5095.

In some embodiments, the initial crystallinity index (before sonication) is from about 4095 to about 87.595, such as from about 5095 to about 7595 or from about 6095 to about 7095, and the crystallinity index after sonication is from about 1095 to about 5095, e.g. from about 1595 to about 4595 or from about 2095 to about 4095. However, in certain embodiments, such as after extensive sonication, the crystallinity index may be less than 595. In some embodiments, the sonicated material is essentially amorphous.

In some embodiments, the starting number average molecular weight (before sonication) is from about 200,000 to about 3,200,000, such as from about 250,000 to about 1,000,000 or from about 250,000 to about 700,000, and the number average molecular weight after sonication is from about 50,000 to about 200,000 , such as from about 60,000 to about 150,000 or from about 70,000 to about 125,000.

However, in some embodiments, such as after extensive sonication, the number average molecular weight may be less than about 10,000 or even less than about 5,000.

In some embodiments, the second material may have an oxidation level (702) greater than the oxidation level (70) of the first material. A higher level of oxidation of a material can contribute to its ability to disperse, swell and/or dissolve, further increasing the susceptibility of materials to chemical, enzymatic or microbial attack. In some embodiments, to increase the oxidation level of the second material relative to the first material, the sonication is performed in an oxidizing atmosphere, producing a second material that is more oxidized than the first material. For example, the second material may have more hydroxyl groups, aldehyde groups, ketone groups, ester groups, or carboxylic acid groups that may increase its hydrophilicity.

In some embodiments, the sonication medium is an aqueous medium. If desired, the medium may include an oxidizing agent such as a peroxide (eg, hydrogen peroxide), a dispersing agent, and/or a buffer. Examples of dispersing agents include ionic dispersing agents, such as sodium lauryl sulfate, and nonionic dispersing agents, such as poly(ethylene glycol).

In other embodiments, the sonication medium is non-aqueous. For example, sonication can be carried out in a hydrocarbon such as toluene or heptane, in an ether such as diethyl ether or tetrahydrofuran, or even in a liquefied gas such as argon, xenon or nitrogen.

Without being bound by any particular theory, it is believed that sonication destroys the bonds in the cellulose by forming bubbles in the medium containing the cellulose, which grow and then rapidly collapse. In the process of bubble collapse, which can take place in less than a nanosecond, the implosive force raises the local temperature in the bubble to about 5100K (-4827 "C) (in some cases even higher; see, for example, Zyviisk ei aY, Maishge 434, 52- 55) and produces pressures from a few hundred atmospheres to over 1000 atmospheres or more. It is these high temperatures and pressures that break the bonds. Also, without being tied to any particular theory, the reduced crystallinity is believed to be a consequence, at least in part , extremely high cooling rates during bubble collapse, which can exceed about 10"K/second. High cooling rates generally prevent the cellulose from organizing and crystallizing, resulting in materials that have reduced crystallinity. Ultrasonic Systems and Ultrasonic Chemistry considered, for example, in the publications of Oiyi ei ai., US patent Mo 5766764; Kobregi5, US patent Mo 5828156; Mazop, Spetivigu m/y OPgavotspa, Eivemiyi, Ohio, (199 0); ZyviiskK (vayog), Oigazotspa: yv

SNnetisa)i, Rnuzisai! apa Viioodisa! Enesiv, MSN, M/vipneit, (1988); Rgise, "Sitepi Ttepavz ip Zopospetivighu"

Vouai bosieiu oi Spetivigu, Satbgidde, (1992); ZiviisK ei a!., App. Wow Maieg. 5 29, 295, (1999); BiviiskK ei ai., Mayte 353, 414 (1991); NiPeg eyi a!., Rpuv. German And ek. 69, 1182 (1992); Vagbeg vy a!., Maishige, 352, 414 (1991); BiviisK eyi ai., U. At. Spent 5os, 108, 5641 (1986); Tapo eyi aI., Spet. Sott., 2119 (2000); Mapd ei a)., Adumapsed Maieg., 12, 1137 (2000); And updats her ai., u. oi Saia|uvziv, 201, 22 (2001); ReiKaz ei ai!., Sing.

Sott., 988 (2001); Mikpepko eyi ai., Apdem/. Spent Ipiveg. Ed. (Ossetbeg 2001); zpNaii iei a!., U. Rnu5. Snet

In 103, 3358 (1999); Amimi ei ai!., U. Ateg. Spent 5 people 121, 4196 (1999); and Amimi ei ai., U. Ateg. Spent Goiter. 122, 4331 (2000).

Systems for ultrasound treatment

In FIG. 12 shows a general system in which a flow of cellulosic material 1210 mixes with a flow of water 1212 in a vessel 1214 to form a process stream 1216. A first pump 1218 removes the process stream 1216 from the vessel 1214 towards a flow cell 1224. An ultrasonic transducer 1226 transmits ultrasonic energy to the process stream 1216 as the process flow passes through the flow cell 1224. The second pump 1230 removes the process flow 1216 from the flow cell 1224 in the direction of further processing.

The container 1214 includes a first receiver 1232 and a second receiver 1234 in hydraulic communication with the volume 1236. A conveyor (not shown) delivers a stream of cellulosic material 1210 into the container 1214 through the first receiver 1232. The water stream 1212 enters the container 1214 through the second receiver 1234. in some embodiments, the water flow 1212 enters the volume 1236 along a tangential line, which provides a vortex-creating flow in the volume 1236. In certain embodiments, the cellulosic material flow 1210 and the water flow 1212 may enter the volume 1236 along opposite axes to improve mixing in the volume.

Valve 1238 controls the flow of water stream 1212 through second receiver 1232 to obtain a desired ratio of cellulosic material to water (eg, approximately 1095 cellulosic material, weight to volume). For example, 2,000 tons/day of cellulosic material can be combined with 1 million to 1.5 million gallons/day (3,800 to 5,700 m3/day) of water, such as 1.25 million gallons/day (4,700 m3/day).

The mixing of cellulosic material and water in the container 1214 is controlled by the size of the volume 1236 and the flow rates of the cellulosic material and water into the volume. In some embodiments, the volume 1236 is sized to provide minimal residence time for the cellulosic material and water during mixing. For example, when 2,000 tons/day of cellulosic material and 1.25 million gallons/day (4,700 m3/day) of water flow through vessel 1214, the volume 1236 may be approximately 32,000 gallons (120 m3) to achieve the minimum mixing residence time. which is about 15 minutes.

The container 1214 includes a mixer 1240 in hydraulic communication with the volume 1236. The mixer 1240 mixes the contents of the volume 1236, completely dispersing the cellulosic material in the water of the volume. For example, mixer 1240 may be a rotating blade located in container 1214. In some embodiments, mixer 1240 disperses cellulosic material substantially homogeneously in water.

In addition, the vessel 1214 includes an outlet 1242 in hydraulic communication with the volume 1236 and the process flow 1216. The mixture of cellulosic material and water in the volume 1236 flows out of the vessel 1214 through the outlet 1242. The outlet 1242 is located near the bottom of the vessel 1214 to provide ejection under by gravity of the mixture of cellulosic material and water from the container 1214 into the technological stream 1216.

A first pump 1218 (e.g., any of several vortex impeller pumps manufactured by Ez5so Rytr5 4 Sopigoii5, Go5 Apdeie5, SaIogpia) moves the contents of the process stream 1216 toward the flow cell 1224. In some embodiments, the first pump 1218 agitates the contents of the process stream 1216 so that the mixture of cellulosic material and water becomes substantially homogeneous at the inlet 1220 of the flow cell 1224. For example, the first pump 1218 agitates the process stream 1216, creating a turbulent flow along the process stream between the first pump and the inlet 1220 of the flow cell 1224.

The flow cell 1224 includes a reactor volume 1244 in hydraulic communication with an inlet 1220 and an outlet 1222. In some embodiments, the reactor volume 1244 is a stainless steel tube capable of withstanding elevated pressure (eg, 10 bar). Additionally or alternatively, the reactor volume 1244 includes a rectangular cross-section.

The flow cell 1224 further includes a heat exchanger 1246 in thermal contact with at least a portion of the reactor volume 1244. A cooling fluid 1248 (e.g., water) flows into the heat exchanger 1246 and absorbs the heat generated during the sonication of the process stream 1216 in the reactor volume tank 1244. In some embodiments, the flow rate and temperature of the cooling fluid 1248 in the heat exchanger 1246 are controlled to maintain an approximately constant temperature in the reactor volume 1244. In some embodiments, the temperature of the reactor volume 1244 is maintained at 20-50 C, for example at 25, 30, 35, 40 or 45 "C. Additionally or alternatively, heat transferred to the cooling fluid 1248 from the reactor volume 1244 may be used in other parts of the entire process.

The transition compartment 1226 provides hydraulic communication between the reactor volume 1244 and the booster 1250 coupled (eg, mechanically coupled using a flange) to the ultrasonic transducer 1226. For example, the transition compartment 1226 may include a flange and a block of sealing rings arranged to create a tight seal with the connection between the reactor volume 1244 and the booster 1250. In some embodiments, the ultrasonic transducer 1226 is a high-power ultrasonic transducer manufactured by Neiijespeg OKgasopisw oi Tepom, Septap.

In operation, generator 1252 supplies electricity to ultrasonic transducer 1226 .

The ultrasonic transducer 1226 includes a piezoelectric element that converts electrical energy into sound in the ultrasonic range. In some embodiments, materials are sonicated using sound having a frequency of from about 16 kHz to about 110 kHz, such as from about 18 kHz to about 75 kHz or from about 20 kHz to about 40 kHz (e.g., sound having a frequency of 20 to 40 kHz). In some examples, the sonication is performed at a frequency of from about 15 kHz to about 25 kHz, such as from about 18 to 22 kHz. In specific embodiments, sonication can be performed using a horn of 1 kW or more, such as 2, 3, 4, 5 kW, or even 10 kW.

Then the ultrasonic energy is delivered to the working environment through the booster 1248.

Ultrasonic energy passing through the booster 1248 in the reactor volume 1244 creates a series of compressions and rarefiations of the process stream 1216 with an intensity sufficient to cause cavitation in the process stream 1216. The cavitation disaggregates the cellulosic material dispersed in the process stream 1216. Cavitation also results in production of free radicals in the water of process stream 1216. These free radicals act to further destroy cellulosic material in process stream 1216.

Typically, an ultrasonic energy of between 5 and 4000 MJ/m3, eg 10, 25, 50, 100 , 250, 500, 750, 1000, 2000 or 3000 MJ/m3. After being exposed to ultrasonic energy in reactor volume 1244, process stream 1216 exits flow cell 1224 through outlet channel 1222. A second pump 1230 moves process stream 1216 for further processing (for example, any of several vortex impeller pumps manufactured by E55so Rytrz » 8

Sopigo!v, I o5 Apdeiez, Saiyogpia).

Although certain embodiments have been described, other embodiments are possible.

As an example, although process flow 1216 has been described as a single flow path, other schemes are possible. For example, in some embodiments, process flow 1216 includes a plurality of parallel flow paths (eg, flow at a rate of 10 gallons per minute (38 L/min)). Additionally or alternatively, a plurality of parallel flow paths of process flow 1216 flow in separate flow cells and are sonicated in parallel (eg, using a plurality of 16 kW ultrasonic transducers).

As another example, although one ultrasonic transducer 1226 has been described as being coupled to the flow cell 1224, other schemes are possible. In some embodiments, a plurality of ultrasonic transducers 1226 are located in the flow cell 1224 (eg, ten ultrasonic transducers may be located in the flow cell 1224). In some embodiments, the sound waves generated by each of the plurality of ultrasonic transducers 1226 are time-adjusted (eg, synchronized with each other in phase) to enhance the cavitation effect on the process stream 1216 .

As another example, although one flow cell 1224 has been described, other designs are possible. In some embodiments, the second pump 1230 moves the process stream into a second flow cell, where the second booster and ultrasonic transducer further sonicate the process stream 1216 .

As another example, although reactor volume 1244 has been described as a closed volume, in certain embodiments, reactor volume 1244 is open to ambient conditions. In such embodiments, the ultrasonic pretreatment can be performed substantially simultaneously with other pretreatment methods. For example, ultrasonic energy can be applied to the process flow 1216 in the reactor volume 1244 simultaneously with the supply of the electron beam to the process flow 1216.

As another example, although a streaming method is described, other schemes are possible. In some embodiments, sonication can be performed intermittently. For example, the volume can be filled with a 1095 (weight by volume) mixture of cellulosic material in water and exposed to sound at an intensity of from about 50 W/cm2 to about 600 W/cm-, for example from about 75 W/cm2 to about 300 W/cmg or from about 95 W/cmg to about 200 W/cm-. Additionally or alternatively, the bulk mixture may be sonicated for from about 1 hour to about 24 hours, such as from about 1.5 hours to about 12 hours or from about 2 hours to about 10 hours. In certain embodiments, the material is sonicated for a predetermined period of time and then allowed to settle for a second predetermined period of time before being sonicated again.

Now referring to FiG. 13, in some embodiments, two electroacoustic transducers are mechanically coupled to one horn. As shown, a pair of piezoelectric transducers 60 and 62 are connected to the slotted rectangular horn 64 by respective intermediate connecting horns 70 and 72, the latter of which are also known as auxiliary horns. The mechanical vibration provided by the transducers responding to the high-frequency electrical energy applied to them is transmitted to the corresponding coupling horns, which can be designed to provide mechanical amplification, for example, with a ratio of 1 to 1.2. Horns are provided with appropriate mounting flanges 74 and 76 to support the transducer and horn system in a stationary housing.

The vibration transmitted from the transducers through the connecting or auxiliary horns is transmitted to the input surface 78 of the horn and is transmitted through the horn to the opposite output surface 80, which, in operation, is in forced contact with the object (not shown) that processed, to which vibration is applied.

High-frequency electrical energy provided by the power source 82 is supplied to each of the converters electrically connected in parallel through the compensating transformer 84 and corresponding series-connected capacitors 86 and 90, with one capacitor connected in series by electrical connection to each of the converters. The compensating transformer is also known as "Baiishp", which means "balancing device". The compensating transformer includes a magnetic core 92 and a pair of identical coils 94 and 96, also called the primary coil and the secondary coil, respectively.

In some embodiments, the transducers include commercially available piezoelectric transducers such as models 105 or 502, each designed to operate at 20 kHz and a maximum power of 3 kW. The potential difference when turning on the power to ensure the maximum dynamic amplitude on the output surface of the converter is 930 rms volts. The electrical current through the converter can vary from zero to 3.5 amps depending on the load resistance. At 930 rms volts, the output swing is about 20 micrometers. The maximum deviation of the final voltage for the same dynamic amplitude can therefore be 186 volts. Such a deviation in voltage can lead to large stray currents between the converters. Balancing device 430 ensures a balanced condition by providing equal current through the converters, thus eliminating the possibility of stray currents. The wire size in the coil must be selected for the maximum load current specified above, and the maximum potential difference occurring at the input of the coil is 93 volts.

As an alternative to the application of ultrasonic energy, high-frequency rotor-stator devices can be used. This type of device generates high shear microcavitation forces that can disintegrate biomass upon contact with such forces. Two types of commercially available high-frequency rotor-stator dispersing devices are Zyrgaip'm devices, which are manufactured by Kgyrr Ipdyhigiesesppic OtnN and supplied to the Rogt-OiYiimeg Ueschivspiapa Strnoi Soppesistia market, and Oizrach'"M devices, which are manufactured and supplied to the Ica- M/oikv5, Ips. oh

Sipsippaiii, Oiio. The operation of such a microcavitation device is considered by 5bitsagi, US patent No. 5370999.

Although the ultrasonic transducer 1226 is described as including one or more piezoelectric active elements for generating ultrasonic energy, other schemes are possible. In some embodiments, the implementation of the ultrasonic transducer 1226 includes active elements made of other types of magnetostrictive material (eg, ferrous metals). The design and operation of such an ultrasonic transducer with high power is considered by Napzep ei AI., patent

US Mo 6624539. In some embodiments, the ultrasonic energy is transferred to the process stream 16 using an electro-hydraulic system.

Although the ultrasonic transducer 1226 is described as using the electromagnetic response of magnetostrictive materials to generate ultrasonic energy, other schemes are possible. In some embodiments, acoustic energy in the form of an intense shock wave using an underwater discharge can be applied to the process flow 16. In some embodiments, ultrasonic energy is transferred to the process stream 16 through a thermodynamic system.

For example, high energy density acoustic waves can be generated by applying a potential across an enclosed volume of electrolyte, thereby heating the enclosed volume and causing a pressure increase that is further transmitted through the sound propagating medium (eg, process flow 1216). The scheme and operation of such a thermo-hydraulic converter is considered by Nagitapp ei ai., patent

USA Mo 6383152.

Pyrolysis

One or more pyrolysis processing sequences can be used to process raw materials from a wide variety of different sources to extract useful substances from the raw materials, as well as to obtain partially degraded organic material that serves as an input stream for further stages and/or processing sequences.

Referring again to FIG. 8, in one method, a first material 2 that includes cellulose having a first number average molecular weight ("Mmi") is pyrolyzed, for example, by heating the first material in a tube furnace, to produce a second material 3 that includes cellulose having a second number average molecular weight molecular weight ("Mmg"), which is lower than the first number average molecular weight. The second material (or, in certain embodiments, the first and second materials) is mixed with a microorganism (e.g., bacteria or yeast) that can utilize the second material or the first material, producing a fuel 5 that includes hydrogen, an alcohol (e.g., ethanol or butanol, such as n-, sec-, or tert-butanol), an organic acid, a hydrocarbon, or a mixture of any of these.

Since the second material contains cellulose having a reduced molecular weight relative to the first material and, in some cases, also reduced crystallinity, the second material is generally more capable of dispersing, swelling and/or dissolving in a solution containing a microorganism, e.g. in a concentration of more than 105 microorganisms/ml. These properties make the second material C more susceptible to chemical, enzymatic and/or microbial attack relative to the first material 2, which can significantly increase the production rate and/or production level of the desired product, such as ethanol. The first and second materials can also be sterilized by pyrolysis.

In some embodiments, the second number average molecular weight ("Mg") is lower than the first number average molecular weight ("Mmi) by more than about 1095, such as 15, 20, 25, 30, 35, 40, 50, 6095, or even more than by about 7,595.

In some cases, the second material contains cellulose having a crystallinity (Cg) that is lower than the crystallinity (C) of the cellulose of the first material. For example, (Cg) may be lower than (Ci) by more than about 1095, such as 15, 20, 25, 30, 35, 4095, or even more than about 5095.

In some embodiments, the initial crystallinity (before pyrolysis) is from about 4095 to about 87.595, such as from about 5095 to about 7595 or from about 6095 to about 7095, and the crystallinity index after pyrolysis is from about 1095 to about 5095, such as from about 1595 to about 4595 or from about 2095 to about 4095. However, in certain embodiments, such as after extensive pyrolysis, the crystallinity index may be less than 595. In some embodiments, the material after pyrolysis is essentially amorphous.

In some embodiments, the starting number average molecular weight (before pyrolysis) is from about 200,000 to about 3,200,000, such as from about 250,000 to about 1,000,000 or from about 250,000 to about 700,000, and the number average molecular weight after pyrolysis is from about 50,000 to about 200,000, e.g. from about 60,000 to about 150,000 or from about 70,000 to about 125,000. However, in some embodiments, such as after extensive pyrolysis, the number average molecular weight may be less than about 10,000 or even less than about 5,000.

In some embodiments, the second material may have an oxidation level (702) greater than the oxidation level (70) of the first material. A higher level of oxidation of a material can contribute to its ability to disperse, swell and/or dissolve, further increasing the susceptibility of materials to chemical, enzymatic or microbial attack. In some embodiments, to increase the level of oxidation of the second material relative to the first material, pyrolysis is carried out in an oxidizing atmosphere, producing a second material that is more oxidized than the first material.

For example, the second material may have more hydroxyl groups, aldehyde groups, ketone groups, ester groups, or carboxylic acid groups that may increase its hydrophilicity.

In some embodiments, the pyrolysis of materials is continuous. In other embodiments, the material is pyrolyzed for a predetermined period of time and then allowed to cool for a second predetermined period of time before pyrolysis again.

Systems for pyrolysis

In FIG. 14 presents a flow chart 6000, which includes various stages of a pyrolytic system for pre-treatment of raw materials. In the first stage 6010, dry raw material is supplied from the source of raw material.

As described above, the dry raw material from the feedstock source may be preprocessed prior to delivery to the pyrolysis chamber. For example, if the raw material is obtained from plant sources, then certain parts of the plant material may be removed before harvesting the plant material and/or before delivering the plant material to the raw material transport device.

Alternatively or additionally, the biomass feedstock can be subjected to mechanical processing 6020 (eg, to reduce the average fiber length in the feedstock) prior to delivery to the pyrolysis chamber.

After mechanical processing, the raw material undergoes the moisture correction stage 6030. The nature of the moisture correction stage depends on the moisture content in the mechanically processed raw material. Typically, feedstock pyrolysis occurs most efficiently when the moisture content of the feedstock is from about 1095 to about 3095 (eg, 15 to 2595) by weight of the feedstock. If the moisture content in the raw material exceeds approximately 40 wt. 95, then the excessive heat load provided by the water content in the raw material increases the energy consumption in the subsequent stages of pyrolysis.

In some embodiments, if the raw material has a moisture content that exceeds approximately 30 wt. 95, then drier raw material 6220, which has a low moisture content, can be mixed with it, obtaining a mixed raw material at stage 6030 with an average moisture content that is within the limits indicated above. In certain embodiments, raw materials with a high moisture content can simply be dried by distributing the raw material on a moving conveyor that cycles the raw material through an in-line heating element. The heating element evaporates part of the water present in the raw material.

In some embodiments, if the feedstock from stage 6020 has a moisture content that is very low (eg, lower than about 10 wt. 95), the mechanically processed feedstock can be combined with feedstock 6230 having a higher moisture content, such as sewage sludge.

Alternatively or additionally, water 6240 can be added to the dry raw material of stage 6020 to increase the moisture content.

At step 6040, the raw material, now with its moisture content adjusted to be within acceptable limits, may be preheated in an optional preheat step 6040.

The preheating stage 6040 can be used to raise the temperature of the raw material to 75-1507C in preparation for further pyrolysis of the raw material. Depending on the nature of the feedstock and the specific design of the pyrolysis chamber, preheating the feedstock can ensure that the heat distribution in the feedstock remains more uniform during pyrolysis and can reduce the heat load on the pyrolysis chamber.

The feedstock is then transported to a pyrolysis chamber to undergo pyrolysis in step 6050. In some embodiments, feedstock transport is aided by the addition of one or more compressed gases 6210 to the feedstock stream. The gases create a pressure gradient in the feedstock transport channel, moving the feedstock into the pyrolysis chamber ( and even through a pyrolysis chamber). In certain variants, the implementation of transportation of raw materials occurs mechanically; namely, the raw material is transported to the pyrolysis chamber by a transportation system that includes a conveyor, such as a screw conveyor.

It is also possible to add other gases 6210 to the raw material before the pyrolysis chamber. In some embodiments, for example, one or more catalytic gases may be added to the feedstock to aid in the decomposition of the feedstock in the pyrolysis process. In certain embodiments, one or more absorbers can be added to the raw material to trap volatile substances released during pyrolysis. For example, various sulfur-based compounds such as sulfides may be released during pyrolysis, and an agent such as hydrogen gas may be added to the feedstock to ensure desulfurization of the pyrolysis products. Hydrogen combines with sulfides to form gaseous hydrogen sulfide, which can be removed from pyrolyzed raw materials.

Pyrolysis of the feedstock in the chamber may involve heating the feedstock to relatively high temperatures that cause partial decomposition of the feedstock. Typically, the feedstock is heated to a temperature in the range of 150 to 1100°C. The temperature to which the feedstock is heated depends on a number of factors, including feedstock composition, average feedstock particle size, moisture content, and desired pyrolysis products. For many types of biomass feedstocks use, for example, pyrolysis temperatures from 300 to 550 "С.

The residence time of the raw material in the pyrolysis chamber, as a rule, depends on a number of factors, including the temperature of the pyrolysis, the composition of the raw material, the average particle size of the raw material, the moisture content and the desired pyrolysis products. In some embodiments, the raw materials are subjected to pyrolysis at a temperature slightly above the decomposition temperature of the material in an inert atmosphere, such as from about 2°C above to about 10°C above the decomposition temperature or from about 3°C above to about 7°C above the temperature decomposition. In such embodiments, the material is typically held at this temperature for more than 0.5 hours, such as more than 1.0 hours or more than about 2.0 hours. In other embodiments, the materials are pyrolyzed at a temperature significantly above the decomposition temperature of the material in an inert atmosphere, such as from about 75°C above to about 175°C above the decomposition temperature or from about 85°C above to about 150°C above the decomposition temperature . In such embodiments, the material is typically held at this temperature for less than 0.5 hour, such as less than 20 minutes, less than 10 minutes, less than 5 minutes, or less than 2 minutes. In other embodiments, the materials are subjected to pyrolysis at an extreme temperature, such as from about 200 "C above to about 500 "C above the decomposition temperature of the material in an inert environment or from about 250 "C above to about 400 "C above the decomposition temperature. In such embodiments, the material is typically held at this temperature for less than 1 minute, such as less than 30 seconds, 15 seconds, 10 seconds, 5 seconds, 1 second, or less than 500 ms.

Such implementation options are usually called instant pyrolysis.

In some embodiments, the feedstock in the chamber is heated relatively quickly to the selected pyrolysis temperature. For example, the chamber can be designed to heat the raw material at a rate of 500 to 11000 "S/s, for example, from 500 to 1000 "S/s.

Turbulent flow of raw material in the pyrolysis chamber is usually preferred because it provides relatively efficient heat transfer to the raw material from the heating subsystem. Turbulent flow can be achieved, for example, by driving the raw material through the chamber using one or more carrier gases 6210 that are pumped. As a rule, carrier gases are relatively inert to the raw material, even at high temperatures in the pyrolysis chamber. Illustrative carrier gases include, for example, nitrogen, argon, methane, carbon monoxide and carbon dioxide. Alternatively or additionally, mechanical transport systems, such as screw conveyors, can transport the raw material and circulate it in the pyrolysis chamber, creating a turbulent flow of the raw material.

In some variants, pyrolysis of raw materials takes place essentially in the absence of oxygen and other reactive gases. Oxygen can be removed from the pyrolysis chamber by periodically purging the chamber with nitrogen under high pressure (eg, nitrogen pressure of 2 bar or more). After purging the chamber, the gas mixture present in the chamber for pyrolysis (for example, in the process of pyrolysis of raw materials) may include less than 4 mol. 95 oxygen (for example, less than 1 mol. 95 oxygen and even less than 0.5 mol. 95 oxygen).

The absence of oxygen ensures the absence of ignition of raw materials at elevated pyrolysis temperatures.

In certain embodiments, relatively small amounts of oxygen can be supplied to the raw material and be present in the pyrolysis process. This method is called oxidative pyrolysis. As a rule, oxidative pyrolysis occurs in several stages of heating. For example, in the first stage of heating, the raw material is heated in the presence of oxygen to ensure partial oxidation of the raw material. At this stage, the available oxygen in the pyrolysis chamber is used up. Then, in the subsequent stages of heating, the temperature of the raw material is additionally increased. However, as a result of the consumption of all the oxygen in the chamber, the ignition of the raw material does not occur, but the pyrolytic decomposition of the raw material occurs without ignition (for example, with the formation of hydrocarbon products). As a rule, the process of heating raw materials in a pyrolysis chamber to initiate decomposition is endothermic. However, during oxidative pyrolysis, the formation of carbon dioxide by the oxidation of raw materials is an exothermic process. The heat released during the formation of carbon dioxide can contribute to the subsequent heating stages during pyrolysis, thereby reducing the heat load provided by the raw material.

In some embodiments, pyrolysis takes place in an inert environment, for example, when raw materials are surrounded by argon or gaseous nitrogen. In certain embodiments, pyrolysis may occur in an oxidizing environment, such as air or argon enriched air. In some embodiments, pyrolysis can occur in reducing ambient conditions, for example, when raw materials are surrounded by gaseous hydrogen. Various chemicals such as oxidizing agents, reducing agents, acids, or bases can be added to the material prior to or during the pyrolysis process to promote pyrolysis. For example, sulfuric acid can be added, or a peroxide (eg, benzoyl peroxide) can be added.

As discussed above, a number of different processing conditions can be used depending on factors such as feedstock composition and desired pyrolysis products. For example, for a cellulosic feedstock, relatively mild pyrolysis conditions can be used, including flash pyrolysis temperatures of 375 to 450°C and residence times of less than 1 second. As another example, for an organic solid waste material such as sewage sludge of water, usually use instant pyrolysis temperatures from 500 to 650 "C with a residence time of 0.5 to 3 seconds. As a rule, many parameters of the pyrolysis process, including residence time, pyrolysis temperature, raw material turbulence, moisture content, composition of raw materials, composition of pyrolysis products, and total composition of gases, can be regulated automatically by a system of regulators and an automated control system.

After the pyrolysis stage 6050, the pyrolysis products pass through the quenching stage 6250 to reduce the temperature of the products before further processing. Typically, the quench stage 6250 includes spraying pyrolysis products with streams of cooling water 6260. The cooling water also forms a slurry that includes solid undissolved product and various dissolved products. Also, the product stream contains a mixture that includes various gases, including gaseous products, carrier gases, and other types of process gases.

The flow of products is transported through the gas line installed in the line to the gas separator, which performs the gas separation stage 6060, in which gaseous products and other gases are separated from the suspension formed during quenching of pyrolysis products. The separated mixture of gases is optionally sent to the fan 6130, which increases the pressure of the gas by blowing the mixture with air. The gas mixture may be subjected to a filtration step 6140 in which the gas mixture is passed through one or more filters (eg, activated carbon filters) to remove particles and other impurities. In the next stage 6150, the filtered gas can be compressed and stored until use.

Alternatively, the filtered gas may be subjected to further processing steps 6160 .

For example, in some embodiments, the filtered gas may be subjected to condensation to separate various gaseous compounds from the gas mixture. Various compounds may include, for example, various hydrocarbon products (eg, alcohols, alkanes, alkenes, alkynes, ethers) formed in the pyrolysis process. In certain embodiments, filtered gas containing a mixture of hydrocarbon components may be combined with vapor gas 6170 (eg, a mixture of water vapor and oxygen) and subjected to a cracking process to reduce the molecular weight of the hydrocarbon components.

In some embodiments, the pyrolysis chamber includes heat sources that burn hydrocarbon gases such as methane, propane, and/or butane, heating the feedstock. A portion of the separated gases 6270 may be recirculated into the pyrolysis chamber for combustion, generating process heat to support the pyrolysis process.

In certain embodiments, process heat can be supplied to the pyrolysis chamber, which can be used to increase the temperature of the raw materials. For example, when raw materials are irradiated with radiation (for example, gamma radiation, electron beam irradiation, or other types of radiation exposure), raw materials can be heated to relatively high temperatures. Heated raw materials can be cooled by a heat exchange system that removes part of the excess heat from the irradiated raw materials. The heat exchange system can be configured to transport part of the thermal energy into the pyrolysis chamber in order to heat (or preheat) the raw material, thereby reducing the energy consumption of the pyrolysis process.

A suspension containing liquid and solid pyrolysis products may be subjected to an optional dehydration step 6070, in which excess water may be removed from the suspension by processes such as mechanical compression and evaporation. Excess water 6280 may be filtered and then recycled for further use in quenching pyrolysis decomposition products in stage 6250.

The dewatered slurry then passes through a mechanical separation stage 6080, in which the solid product material 6110 is separated from the liquid product material 6090 by means of a series of fine filters with increasing degrees of purification. In step 6100, the liquid product material 6090 may be further condensed (eg, by evaporation) to remove waste water 6190 and purified by methods such as extraction. Extraction may include the addition of one or more organic solvents 6180, for example, to separate products such as oils from products such as alcohols. Suitable organic solvents include, for example, various hydrocarbons and halogenated hydrocarbons. The purified liquid products 6200 can then be subjected to further processing steps. Waste water 6190 may be filtered if necessary and recycled for further use in quenching decomposition products by pyrolysis in stage 6250.

After separation in step 6080, the solid product material 6110 is optionally subjected to a drying step 6120, which may include evaporation of water. The solid material 6110 can then be stored for further use or subjected to further processing steps, depending on the situation.

The parameters of the pyrolysis process discussed above are illustrative. As a rule, the values of these parameters can vary widely depending on the nature of the raw materials and desired products. Moreover, a wide variety of pyrolysis methods can be used, including the use of heat sources such as hydrocarbon flames and/or furnaces, infrared lasers, microwave heaters, induction heaters, resistive heaters, and other heating devices and configurations.

A wide variety of different pyrolysis chambers can be used to decompose raw materials. In some embodiments, for example, pyrolysis of the feedstock may include heating the material using a resistive heating element, such as a metal hair or metal tape. Heating can occur through direct contact between the resistive heating element and the material.

In certain embodiments, the pyrolysis may include heating the material by induction, for example, using a Curie point pyrolyzer. In some embodiments, the pyrolysis may include heating the material using radiation, such as infrared radiation. Radiation can be generated by a laser, such as an infrared laser.

In certain embodiments, pyrolysis may include heating the material with convective heat. Convective heat can be generated by a flowing stream of heated gas. The heated gas may be maintained at a temperature of less than about 1200 "C, such as less than about 1000 "C, less than 750 "C, less than 600 "C, less than 400 "C, or even less than 300 "C. The heated gas may be maintained at a temperature greater than about 250° C. Convective heat may be generated by a heated body surrounding the first material, such as in a furnace.

In some embodiments, the pyrolysis may include heating the material with steam at a temperature greater than about 250°C.

The embodiment of the chamber for pyrolysis is shown in FIG. 15. The chamber 6500 includes an insulated chamber wall 6510 with a vent 6600 for exhaust gases, a plurality of furnaces 6520 that generate heat for the pyrolysis process, a transport channel 6530 for transporting raw materials through the chamber 6500, a screw conveyor 6590 for transporting raw materials through the channel 6530 in a turbulent flow and a quenching system 6540, which includes a screw conveyor 6610 for transporting pyrolysis products, water nozzles 6550 for spraying the pyrolysis products with cooling water, and a gas separator for separating gaseous products 6580 from a slurry 6570 containing solid and liquid products.

Another embodiment of the pyrolysis chamber is shown in FIG. 16. The chamber 6700 includes an insulated chamber wall 6710, a feed channel 6720, an inclined inner wall of the chamber 6730, furnaces 6740 that generate heat for the pyrolysis process, a vent 6750 for waste gases, and a gas separator 6760 for separating gaseous products 6770 from liquid and solids 6780. Chamber 6700 is configured to rotate in the direction shown by arrow 6790 to ensure proper mixing and turbulent flow of raw materials within the chamber.

The next embodiment of the pyrolysis chamber is shown in FIG. 17. Filament pyrolyzer 1712 includes a sample holder 1713 with a resistive heating element 1714 in the form of a wire wound around an open space defined by the sample holder 1713. Optionally, the heated element can rotate about an axis 1715 (as indicated by arrow 1716) to turn the material, which includes the cellulosic material, in the sample holder 1713. The space 1718, defined by the enclosure 1719, is maintained at a temperature above room temperature, for example from 200 to 250 "C. In a typical application, a carrier gas, such as an inert gas, or an oxidizing or reducing gas, moves transversely through the sample holder 1713 while the resistive heating element is rotated and heated to the desired temperature, for example 325°C. After an appropriate period of time, such as 5 to 10 minutes, the pyrolyzed material is removed from the sample holder.

The system shown in FIG. 17, can be scaled and made continuous. For example, instead of a wire, a screw conveyor can be used as a heating element. Material can continuously enter the sample holder, coming into contact with the heated screw, which pyrolyzes the material.

At the same time, the screw can push pyrolyzed material out of the sample holder, allowing new non-pyrolyzed material to enter.

Another embodiment of the pyrolysis chamber is shown in FIG. 18, which shows a Curie point pyrolyzer 1820 that includes a sample chamber 1821 containing a ferromagnetic foil 1822.

The sample chamber 1821 is surrounded by a radio frequency coil 1823. The space 1824 defined by the enclosure 1825 is maintained at a temperature above room temperature, for example 200 to 250 °C. In a typical application, the carrier gas moves in a transverse direction through the sample holder 1821, while as foil 1822 is subjected to inductive heating by an applied radio frequency field to pyrolyze the material at the desired temperature.

Another embodiment of the pyrolysis chamber is shown in FIG. 19. Furnace pyrolyzer 130 includes a movable sample holder 131 and a furnace 132. In normal use, the sample is lowered (as indicated by arrow 137) into the heating zone 135 of the furnace 132, while the carrier gas fills the jacket 136 and moves transversely through the sample holder 131. The sample is heated to a desired temperature for a desired period of time to obtain a pyrolyzed product. The pyrolyzed product is removed from the pyrolyzer by lifting the sample holder (as indicated by arrow 134).

In certain embodiments, as shown in FIG. 20, the cellulosic target 140 can be pyrolyzed by treating the target contained in the vacuum chamber 141 with laser radiation, such as radiation having a wavelength of about 225 nm to about 1500 nm.

For example, the target can be destroyed at 266 nm using the fourth harmonic wave of the laser

Ma-mAsS (Zresiga RPuzis5, SK170, Zap duoze, Saiii). The optical configuration shown allows the practically monochromatic light 143 generated by the laser 142 to be directed using mirrors 144 and 145 to the target after passing through the lens 146 in the vacuum chamber 141. As a rule, the pressure in the vacuum chamber is maintained at a level of less than 105 mm Hg. In some embodiments, the implementation uses infrared radiation, for example, radiation from a Ma-zhAs laser, which is 1.06 micrometers. In such embodiments, an infrared-sensitive dye can be combined with the cellulosic material to produce a cellulosic target.

The infrared dye can increase the heating of the cellulosic material. Laser destruction is described by Viapspei-Ripsopeg eii a. in US patent Mo 5942649.

Referring to FIG. 21, in some embodiments, the cellulosic material may be subjected to flash pyrolysis by coating a tungsten filament 150, such as a 5 to 25 mm tungsten filament, with the desired cellulosic material while the material is contained in a vacuum chamber 151. To ensure pyrolysis, the filament is passed current that causes rapid heating of the hair for a desired period of time. As a rule, heating is continued for seconds, and then the hair is allowed to cool. In some embodiments, the heating is carried out several times to ensure the desired degree of pyrolysis.

In certain embodiments, the carbohydrate-containing biomass material can be heated in the absence of oxygen in a fluidized bed reactor. If desired, the carbohydrate-containing biomass may have relatively thin cross-sections and may include any of the fibrous materials described herein for efficient heat transfer. The material can be heated by heat exchange from hot metal or ceramic, such as glass, pellets or sand in the reactor, and the resulting pyrolyzed liquid or oil can be transported to a central treatment plant to produce combustible fuels or other useful products.

Oxidation

One or more oxidative processing sequences can be used to process raw materials from a wide variety of different sources in order to extract useful substances from the raw materials and obtain partially degraded organic material that serves as an input stream for further stages and/or processing sequences.

Referring again to FIG. 8, the first material 2, which includes cellulose having a first number average molecular weight ("Mm:") and having a first oxygen content (70), is subjected to oxidation, for example, by heating the first material in a tube furnace in a stream of air or oxygen-enriched air, with obtaining the second material 3, which includes cellulose that has a second number average molecular weight ("Mkg) and has a second oxygen content (702) that is lower than the first oxygen content (70).

The second material (or, in certain embodiments, the first and second materials) can, for example, be mixed with a resin, such as a molten thermoplastic resin, or with a microorganism to produce a composite 4 having the desired mechanical properties, or a fuel 5.

Such materials can also be combined with a solid and/or liquid. For example, a liquid can be in the form of a solution, and a solid can be in the form of particles. The liquid and/or solid may include a microorganism, such as a bacterium, and/or an enzyme. For example, a bacterium or enzyme can act on a cellulosic or lignocellulosic material to produce a fuel such as ethanol or a co-product such as protein. Fuel and co-products are described in R IVKOSV5 MATEMKIAII 5 AMO

SOMROBITE5, OM 11/453951, submitted on June 15, 2006. The entire content of each of the above applications is incorporated herein by reference.

In some embodiments, the second number average molecular weight is no more than 9795 below the first number average molecular weight, such as no more than 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 30, 20, 12.5, 10.0, 7.5, 5.0, 4.0, 3.0, 2.5, 2.095 or not more than 1.095 below the first number average molecular weight. The amount of molecular weight reduction depends on the application.

In some embodiments, in which the materials are used to produce a fuel or coproduct, the starting number average molecular weight (before oxidation) is from about 200,000 to about 3,200,000, such as from about 250,000 to about 1,000,000 or from about 250,000 to about 700,000, and the number average molecular weight after oxidation is from about 50,000 to about 200,000, such as from about 60,000 to about 150,000 or from about 70,000 to about 125,000. However, in some embodiments, such as after extensive oxidation, the number average molecular weight may be less than about 10,000 or even less than about 5000.

In some embodiments, the second oxygen content is at least about 595 greater than the first oxygen content, such as greater than 7.5, 10.0, 12.5, 15.0, or 17.595. In some preferred embodiments, the second oxygen content is at least about 20.095 greater than the oxygen content of the first material. Oxygen content is measured by elemental analysis by pyrolysis of a sample in a furnace operating at 1300 "C or more. A suitable device for elemental analysis is an analyzer

GESO SNM5-932 with MTE-900 high-temperature pyrolysis furnace.

In some embodiments, the oxidation of the first material 200 does not significantly change the crystallinity of the cellulose. However, in some cases, such as after excessive oxidation, the second material has a cellulose having a crystallinity ("C2g") that is lower than the crystallinity (C) of the cellulose of the first material. For example, (Cg) may be lower than (7C1) by about more than 595 , for example by 10, 15, 2095 or even 2595. This may be desirable to increase the solubility of materials in a liquid, such as a liquid that includes a bacterium and/or an enzyme.

In some embodiments, the initial crystallinity index (before oxidation) is from about 4095 to about 87.595, such as from about 5095 to about 7595 or from about 6095 to about 70905, and the crystallinity index after oxidation is from about 3090 to about 75.095, such as from about 35.095 to about 70.095 or from about 37.595 to about 65.095. However, in certain embodiments, such as after extensive oxidation, the crystallinity index may be less than 595. In some embodiments, the material after oxidation is essentially amorphous.

Without being bound by any particular theory, oxidation is believed to increase the number of hydrogen-bonding groups in the cellulose, such as hydroxyl groups, aldehyde groups, ketone groups, carboxylic acid groups, or anhydride groups, which can increase its ability to dispersion and/or its solubility (for example, in liquid). To further enhance the resin's dispersibility, the resin may include a component that contains hydrogen-bonding groups, such as one or more anhydride groups, carboxylic acid groups, hydroxyl groups, amide groups, amino groups, or mixtures of any of these groups. In some preferred embodiments, the component includes a polymer that is copolymerized with maleic anhydride and/or has grafted maleic anhydride. Such materials are available from Europe under the trade name EGOBAVOMOF)

As a rule, the oxidation of the first material 200 occurs in an oxidizing environment.

For example, oxidation can be accomplished or accelerated by pyrolysis in an oxidizing medium such as air or argon enriched air. Various chemicals, such as oxidizing agents, acids, or bases, may be added to the material prior to or during oxidation to promote oxidation. For example, a peroxide (for example, benzoyl peroxide) can be added before oxidation.

Systems for oxidation

In FIG. 22 presents a technological scheme 5000, which includes various stages in the system of oxidative pretreatment of raw materials. In the first stage 5010, the dry raw material is supplied from the raw material source. The feedstock source may include, for example, a platform or storage container that is connected to an in-line oxidation reactor via a conveyor belt or other feedstock transport device.

As described above, the dry feedstock from the feedstock source may be preprocessed prior to delivery to the oxidation reactor. For example, if the raw material is obtained from plant sources, then certain parts of the plant material may be removed before harvesting the plant material and/or before delivering the plant material to the raw material transport device.

Alternatively or additionally, the biomass feedstock can be subjected to mechanical processing (for example, to reduce the average fiber length in the feedstock) before delivery to the reactor for oxidation.

After mechanical processing 5020, raw material 5030 is transported to a mixing system that supplies water 5150 to the raw material during mechanical mixing. The combination of water with processed raw materials at the stage of mixing 5040 leads to an aqueous suspension 5050 of raw materials, which can then be treated with one or more oxidizers.

As a rule, one liter of water is added to the mixture for every 0.02-1.0 kg of dry raw materials. The ratio of raw materials and water in the mixture depends on the source of raw materials and specific oxidizers used further in the process in general. For example, in conventional industrial lignocellulosic biomass processing sequences, aqueous suspension 5050 feedstock includes from about 0.5 kg to about 1.0 kg of dry biomass per liter of water.

In some embodiments, one or more fiber-protecting additives 5170 may also be added to the feedstock slurry at the feedstock mixing stage 5040. Fiber-protecting additives help reduce the degradation of certain types of biomass fibers (eg, cellulosic fibers) during feedstock oxidation. Fiber-protecting additives can be used, for example, if the desired product of the processing of lignocellulosic raw materials includes cellulose fibers. Typical fiber-protecting additives include magnesium compounds such as magnesium hydroxide. Concentrations of fiber-protecting additives in suspension 5050 raw materials can be, for example, from 0.1 to 0.495 dry weight of biomass raw materials.

In certain embodiments, the aqueous suspension 5050 of the raw material can be subjected to optional extraction 5180 with an organic solvent to remove water-insoluble substances from the suspension.

For example, extraction of slurry 5050 with one or more organic solvents results in a purified slurry and organic waste stream 5210, which includes water-insoluble substances such as fats, oils and other non-polar hydrocarbon-based substances. Suitable solvents for carrying out the extraction of suspension 5050 include, for example, various alcohols, hydrocarbons and halogenated hydrocarbons.

In some embodiments, the aqueous suspension 5050 of the raw material can be subjected to an optional heat treatment 5190 to further prepare the raw material for oxidation. An example of heat treatment includes heating a suspension of raw materials in the presence of compressed steam. In fibrous biomass raw materials, the compressed steam provides swelling of the fibers, giving a large part of the fiber surfaces to the aqueous solvent and oxidants, which are fed in the further stages of processing.

In certain embodiments, the aqueous suspension of feedstock 5050 can be optionally treated with basic substances 5200. Treatment with one or more basic substances can facilitate the separation of lignin from cellulose in the lignocellulosic biomass feedstock, thereby improving subsequent oxidation of the feedstock. Typical basic substances include hydroxides of alkali and alkaline earth metals, such as sodium hydroxide, potassium hydroxide and calcium hydroxide. Generally, a plurality of basic substances can be used, usually in concentrations of from about 0.0195 to about 0.595 of the dry weight of the raw material.

Aqueous slurry 5050 of feedstock is transported (eg, through an in-line piping system) to a chamber, which may be a pre-oxidative processing chamber or an oxidation reactor. At the stage of preliminary oxidative processing 5060, one or more oxidizing agents 5160 are added to the liquid suspension 5050 of the raw material to form an oxidizing medium. In some embodiments, for example, oxidizing agents 5160 may include hydrogen peroxide.

Hydrogen peroxide can be added to suspension 5050 in the form of an aqueous solution and in ratios ranging from 3 to 30-35 wt. 95 suspension 5050. Hydrogen peroxide has a number of advantages as an oxidizer.

For example, an aqueous solution of hydrogen peroxide is relatively inexpensive, relatively chemically stable, and not particularly harmful relative to other oxidizing agents (and thus does not require burdensome storage procedures and expensive protective equipment). Moreover, hydrogen peroxide decomposes with the formation of water in the process of oxidation of raw materials, so that the treatment of the waste stream is relatively simple and inexpensive.

In certain embodiments, the oxidizing agents 5160 may include oxygen (eg, oxygen gas) either alone or in combination with hydrogen peroxide. Gaseous oxygen can be used to bubble suspension 5050 in ratios ranging from 0.5 to 10 wt. 95 slurry 5050. Alternatively or additionally, oxygen gas can also be fed into the aqueous phase in equilibrium with the slurry 5050 (eg, a steam header over the slurry 5050). Oxygen gas can be supplied to either the pre-oxidative processing chamber or the oxidation reactor (or both), depending on the configuration of the oxidative processing system. Of course, for example, the partial pressure of oxygen in the vapor above the suspension 5050 exceeds the atmospheric pressure of oxygen and is in the range from 0.5 to 35 bar depending on the nature of the raw material.

Oxygen gas can be supplied in its pure form or it can be mixed with one or more carrier gases. For example, in some embodiments, high-pressure air provides oxygen in the water vapor. In certain embodiments, gaseous oxygen can be continuously supplied in the vapor phase to ensure that during the processing of raw materials, the concentration of oxygen in the vapor remains within certain predetermined limits. In some embodiments, gaseous oxygen may first be introduced in sufficient concentration to oxidize the feedstock, and then the feedstock may be transported to a closed, pressurized vessel (eg, an oxidation reactor) for processing.

In certain embodiments, the oxidants 5160 may include released oxygen (eg, oxygen radicals). As a rule, the released oxygen (atomic) is formed, if necessary, in the oxidation reactor or in a chamber in hydraulic communication with the oxidation reactor, as a result of one or more decomposition reactions. For example, in some embodiments, atomic oxygen can be formed as a result of the reaction between MO and O»5 in a gas mixture or in solution. In certain embodiments, atomic oxygen may be formed upon decomposition of NOSI in solution. Other ways in which atomic oxygen can be generated include electrochemical generation, such as in an electrolyte solution.

As a rule, the oxygen released is an effective oxidant due to the relatively high reactivity of oxygen radicals. However, the evolved oxygen can also be a relatively selective oxidant. For example, when lignocellulosic raw materials are treated with released oxygen, the selective oxidation of lignin with respect to other components of the raw material, such as cellulose, takes place. As a result, the oxidation of raw materials with released oxygen provides a method of selective removal of the lignin fraction from certain types of raw materials. Of course, in order to achieve effective oxidation, concentrations of released oxygen from about 0.5 to 595 of the dry weight of the raw material are used.

Without regard to theory, it is believed that the released oxygen reacts with the lignocellulosic raw material by at least two different mechanisms. According to the first mechanism, the released oxygen undergoes a reaction of attachment to lignin, leading to partial oxidation of lignin, which solubilizes lignin in an aqueous solution. As a result, solubilized lignin can be removed from the rest of the raw material by washing. According to the second mechanism, the released oxygen destroys butane cross-links or opens aromatic rings connected through butane cross-links. As a result, the solubility of the aqueous solution of lignin increases, contributing to the separation of the lignin fraction from the rest of the raw material by washing.

In some embodiments, the oxidizing agents 5160 include ozone (Oz). The use of ozone can introduce some conditions for the handling of chemical reagents in the sequence of oxidative processing.

If heated excessively, the aqueous ozone solution can rapidly decompose with potentially adverse consequences for both the people operating the system and the system equipment. Thus, ozone is usually formed in a thermally insulated container with thickened walls, separate from the container containing the suspension of raw materials, and transported to it at the appropriate stage of the method.

Without wishing to be bound by theory, it is believed that ozone decomposes into oxygen and oxygen radicals, and that oxygen radicals (eg, atomic oxygen) are responsible for the oxidizing properties of ozone as described above. Ozone tends to preferentially oxidize the lignin fraction in lignocellulosic materials, leaving the cellulose fraction relatively unaffected.

Conditions for ozone-based oxidation of biomass raw materials usually depend on the nature of the biomass.

For example, for cellulosic and/or lignocellulosic raw materials, ozone concentrations from 0.1 to 20 g/m3 of dry raw materials ensure effective oxidation of raw materials. As a rule, the water content in the 5050 suspension is from 10 to 80 wt. 95 (for example, from 40 to 60 wt. 905). In the ozone-based oxidation process, the temperature of the 5050 suspension can be maintained in the range of 0 to 100 C to avoid intensive ozone decomposition.

In some embodiments, the feed stock slurry 5050 may be treated with an aqueous alkaline solution that includes one or more alkali or alkaline earth metal hydroxides, such as sodium hydroxide, potassium hydroxide, and calcium hydroxide, and then treated with an ozone-containing gas in an oxidation reactor. It was found that this process significantly increases the decomposition of biomass in suspension 5050. As a rule, for example, the concentration of hydroxide ions in an alkaline solution is from 0.001 to 10 wt. 95 of suspension 5050. After wetting the raw material by contact with an alkaline solution, a gas containing ozone is supplied to the reactor for oxidation, where it contacts the raw material and oxidizes it.

Oxidizers 5160 may also include other substances. In some embodiments, for example, halogen-based oxidants such as chlorine and chlorine oxygen compounds (eg, hypochlorite) can be added to slurry 5050. In certain embodiments, nitrogen-containing oxidants can be added to the suspension 5050. Illustrative nitrogen-containing oxidizing agents include, for example, MO and

MO". Nitrogen-containing substances can also combine with oxygen in suspension 5050, creating additional oxidants. For example, both MO and MO' combine with oxygen in the 5050 slurry to form nitrate compounds that are effective oxidizing agents for biomass feedstocks. Halogen- and nitrogen-based oxidants can, in some embodiments, provide bleaching of the biomass feedstock, depending on the nature of the feedstock. Bleaching may be desirable for certain biomass-derived products that are extracted in further processing stages.

Other oxidizing agents may include, for example, various peroxyacids, peroxyacetic acids, persulfates, percarbonates, permanganates, osmium tetroxide, and chromium oxides.

After the stage of preliminary oxidation processing 5060, the suspension 5050 of the raw material is oxidized in the stage 5070. If the oxidizing agents 5160 are added to the suspension 5050 in the reactor for oxidation, then the oxidation proceeds in the same reactor. Alternatively, if oxidizers 5160 are added to slurry 5050 in a pretreatment chamber, slurry 5050 is transported to the reactor for oxidation through an in-line piping system. After entering the oxidation reactor, the oxidation of the biomass feedstock continues under a controlled set of ambient conditions.

Of course, for example, an oxidation reactor is a cylindrical container that is closed from the external environment and is under pressure. Both cyclic and continuous operation are possible, although environmental conditions are generally easier to control in flow-through cyclic processing operations.

Oxidation of suspension 5050 raw materials usually takes place in an oxidation reactor at elevated temperatures. For example, the temperature of the 5050 suspension in the oxidation reactor is usually maintained above 100 °C, in the range from 120 to 240 °C. For many types of biomass feedstocks, oxidation is particularly effective if the temperature of the 5050 slurry is maintained in the range of 150 to 220 "C. The 5050 slurry can be heated using a variety of heat transfer devices.

For example, in some embodiments, the oxidation reactor contacts a heating bath that includes oil or molten salts. In certain embodiments, a series of heat exchange tubes surround and contact the oxidation reactor, and circulating hot fluid in the tubes heats the slurry 5050 in the reactor. Other heating devices that can be used to heat the suspension 5050 include, for example, resistive heating elements, induction heaters, and microwave sources.

The residence time of the suspension 5050 raw material in the oxidation reactor can be varied as desired for processing the raw material. As a rule, the 5050 suspension is in the reactor during oxidation from 1 minute to 60 minutes. For a relatively soft biomass material such as lignocellulosic material, the residence time in the reactor for oxidation can be from 5 minutes to 30 minutes, for example, at a reactor oxygen pressure of 3 to 12 bar and a slurry temperature of 160 to 210 °C However, for other types of raw materials, the residence time in the reactor for oxidation can be longer, for example up to 48 hours.

To determine the appropriate residence time of slurry 5050 in the reactor for oxidation, aliquots of the slurry can be withdrawn from the reactor at certain intervals and analyzed to determine the concentrations of specific products of interest, such as complex saccharides. Information on the increase in concentrations of certain products in suspension 5050 as a function of time can be used to determine the residence time for specific classes of raw material.

In some embodiments, in the process of oxidation of the suspension of 5050 raw materials, it is possible to correct the pH of the suspension by adding one or more chemicals to the oxidation reactor. For example, in certain embodiments, oxidation occurs more efficiently at a pH in the range of approximately 9-11. To maintain pH in this range, substances such as hydroxides of alkali and alkaline earth metals, carbonates, ammonia and buffer solutions can be introduced into the reactor for oxidation.

Circulation of the slurry 5050 may be important in the oxidation process to ensure sufficient contact between the oxidizers 5160 and the feedstock. Circulation of the suspension can be achieved using various methods. For example, in some embodiments, the oxidation reactor may be equipped with a mechanical stirring device that includes impeller blades or a bladed wheel. In certain embodiments, the oxidation reactor may be a loop reactor in which the aqueous solvent in which the feedstock is suspended is simultaneously drained from the bottom of the reactor and recirculated to the top of the reactor by pumping, thereby ensuring that the slurry is continuously mixed multiple times and does not stagnate in reactors

After the oxidation of the raw material is complete, the suspension is transported to the separation device where the mechanical separation stage 5080 takes place. Typically, the mechanical separation stage 5080 includes one or more stages of fine filtration with an increasing degree of filtration of the suspension for mechanical separation of solid and liquid components.

The liquid phase 5090 is separated from the solid phase 5100, after which the two phases are recycled independently. The solid phase 5100 may optionally be subjected to a drying step 5120, for example, in a drying device. The drying step 5120 may include, for example, mechanically distributing the solid material on the surface to dry and evaporate water from the solid phase 5100 by gently heating the solid material. After the drying step 5120 (or, alternatively, without the drying step 5120 ), the solid phase 5100 is transported for further processing steps 5140 .

The liquid phase 5090 may optionally be subjected to a drying step 5110 to reduce the concentration of water in the liquid phase. In some embodiments, for example, the drying step 5110 may include evaporation and/or distillation, and/or extraction of water from the liquid phase 5090 by gently heating the liquid. Alternatively or additionally, one or more chemical dewatering reagents may be used to remove water from the liquid phase of 5090. After the drying step 5110 (or, alternatively, without the drying step 5110), the liquid phase 5090 is transported for further processing steps 5130, which may include various chemical and biological processing steps, such as chemical and/or enzymatic hydrolysis.

The drying stage 5110 produces a waste stream 5220, an aqueous solution that includes dissolved chemicals such as acids and bases in relatively low concentrations. Treatment of the waste stream 5220 may include, for example, pH neutralization with one or more mineral acids or bases. Depending on the concentration of dissolved salts in the waste stream 5220, the solution may undergo partial deionization (for example, by passing the waste stream through an ion exchange system). The waste stream, which includes primarily water, may then be recirculated into the overall process (eg, as water 5150 ), may be diverted to another process, or disposed of.

Typically, in the case of lignocellulosic biomass feedstock, after the separation step 5070, the liquid phase 5090 includes a plurality of soluble poly- and oligosaccharides, which can then be separated and/or reduced to shorter chain length saccharides by further processing steps. The solid phase 5100 usually includes mainly cellulose, for example, with smaller amounts of hemicellulose and lignin products.

In some embodiments, the oxidation can be carried out at an elevated temperature in a reactor, such as a pyrolysis chamber. For example, referring again to FIG. 17, raw materials may be oxidized in a filament pyrolyzer 1712. In a typical application, an oxidizing carrier gas, such as air or an air/argon mixture, is moved transversely through the sample holder 1713 while the resistive heating element is rotated and heated to the desired temperature , for example 325 °C. After a suitable period of time, for example from 5 to 10 minutes, the oxidized material is removed from the sample holder. The system shown in FIG. 17 can be scaled and made continuous. For example, instead of a wire, a screw can be used as the heating element screw conveyor. Material can continuously enter the sample holder, impinging on the heated screw, which pyrolyzes the material. At the same time, the screw can push the oxidized material out of the sample holder, providing an opportunity for new unoxidized material to be introduced.

Raw materials can also be oxidized in any of the systems for pyrolysis presented at

FIG. 18-20 and described above in the section "Systems for pyrolysis".

Referring again to FIG. 21, raw materials can be rapidly oxidized by coating a tungsten filament 150, along with an oxidizing agent such as peroxide, with a desired cellulosic material while the material is in a vacuum chamber 151. To provide oxidation, a current is passed through the filament which causes the filament to rapidly heat up. during the desired period of time. Usually, heating is continued for several seconds, and then the hair is allowed to cool. In some embodiments, the heating is carried out several times to ensure the desired degree of oxidation.

Referring again to FIG. 12, in some embodiments, raw materials may be oxidized by sound and/or cavitation. Typically, to achieve oxidation, materials are sonicated in an oxidizing medium, such as water saturated with oxygen or another chemical oxidizer, such as hydrogen peroxide.

Referring again to FIG. 9 and 10, in certain embodiments, ionizing radiation is used to promote the oxidation of raw materials. As a rule, to achieve oxidation, materials are irradiated in an oxidizing medium, such as air or oxygen.

For example, gamma radiation and/or electron beam irradiation can be used to irradiate materials.

Other ways

Steam explosion can be used alone without any of the methods described in this document or in combination with any of the methods described in this document.

In FIG. 23 shows a general representation of the entire process of converting a fiber source 400 into a product 450, such as ethanol, in a manner that includes crushing and steam explosion to produce a fibrous material 401, which is then hydrolyzed and converted, such as fermented, to produce a product. The fiber source can be converted into fibrous material 401 in a number of possible ways, including at least one crushing method and at least one steam explosion method.

For example, one embodiment includes comminuting the fiber source followed by an optional screening step(s) and an optional additional comminution step(s) to produce a comminuted fiber source 402, which may then be steam exploded to form a fibrous material 401. After the process steam explosion is optionally followed by a fiber extraction process to remove liquids or "solution" 404 formed in the steam explosion process. The material obtained after the steam explosion of the crushed fiber source can be further crushed in an optional additional crushing stage(s) and/or an optional sieving stage(s).

In another method, the fibrous material 401 is first steam-exploded to form a steam-exploded fiber source 410. The resulting steam-exploded fiber source is then subjected to an optional fiber extraction process to remove liquids or solution.

The resulting steam-exploded fiber source can then be crushed to form a fibrous material. The steam-exploded fiber source may also be subjected to one or more additional sieving steps and/or one or more optional additional crushing steps. The method of crushing and steam explosion of the fiber source to obtain crushed and steam-exploded fibrous material is further discussed below.

Prior to crushing or steam explosion, the fiber source may be cut into fragments or strips of confetti-like material. Crushing processes are carried out on the material in a dry state (for example, having less than 0.25 wt. 95 of absorbed water), in a hydrated state, or even when the material is partially or completely immersed in a liquid such as water or isopropanol. Also, the method may optionally include stages of drying the output stream after steam explosion or crushing to provide additional stages of dry crushing or steam explosion. The stages of crushing, sieving and steam explosion can be carried out in the presence or absence of various chemical solutions.

In the steam explosion method, the fiber source or the fragmented fiber source is contacted with steam under high pressure, and the steam diffuses into the structures of the fiber source (eg, lignocellulosic structures). The steam is then condensed under high pressure, thereby "wetting" the fiber source.

Moisture in the fiber source can hydrolyze any acetyl groups in the fiber source (eg, acetyl groups in hemicellulose fractions) to form organic acids such as acetic and uronic acids. Acids, in turn, can catalyze the depolymerization of hemicellulose, the release of xylan and limited amounts of glucan. Then, when the pressure is released, the "soaked" fiber source (or crushed fiber source, etc.) "explodes". Condensed moisture is instantly evaporated by the sudden pressure drop, and the expansion of the water vapor exerts a shear force on the fiber source (or shredded fiber source, etc.). Sufficient shear force leads to mechanical destruction of the internal structures (for example, lignocellulosic structures) of the fiber source.

The shredded and steam-exploded fibrous material is then converted into a useful product such as ethanol. In some embodiments, the fibrous material is converted to fuel. One way to convert fibrous material into fuel is through hydrolysis to produce fermentable sugars 412, which are then fermented to produce a product. Other known or unknown ways of converting fibrous materials into fuel can also be used.

In some embodiments, prior to mixing with the microorganism, the shredded and steam-exploded fibrous material 401 is sterilized to kill any competing microorganisms that may be present on the fibrous material. For example, the fibrous material can be sterilized by exposing the fibrous material to radiation such as infrared radiation, ultraviolet radiation, or ionizing radiation such as gamma radiation. Microorganisms can also be killed using chemical sterilants such as bleach (eg, sodium hypochlorite), chlorhexidine, or ethylene oxide.

One of the methods of hydrolysis of shredded and steam-exploded fibrous material is the use of cellulases. Cellulases are a group of enzymes that act synergistically to hydrolyze cellulose. It is also possible to use the commercially available enzyme complex AssayYegazeF 1000, which contains a complex of enzymes that ensure the conversion of lignocellulosic biomass into fermentable sugars.

According to current understanding, cellulase components include endoglucanases, exoglucanases (cellobiohydrolases) and β-glucosidases (cellobiases). Synergism between cellulase components exists when hydrolysis by a combination of two or more components exceeds the sum of the types of activity exhibited by individual components. The generally accepted mechanism of action of the cellulase system (in particular, the system of T. Iopdibgaspiasht) on crystalline cellulose is that endoglucanase hydrolyzes the internal B-1,4-glycosidic bonds of amorphous regions, thereby increasing the number of exposed non-reducing ends. Then, exoglucanases cleave cellobiose units from the non-reducing ends, which, in turn, are hydrolyzed by p-glucosidases to individual glucose units. There are several configurations of both endo- and exoglucanases that differ in stereospecificity.

As a rule, optimal hydrolysis of cellulose requires a synergistic action of components in different configurations. However, cellulases are more prone to hydrolyze the amorphous regions of cellulose. There is a linear relationship between crystallinity and hydrolysis rates, with higher crystallinity indices corresponding to slower enzyme hydrolysis rates. Amorphous regions of cellulose are hydrolyzed at twice the rate of crystalline regions. Hydrolysis of shredded and steam-exploded fibrous material can be carried out by any method of biomass hydrolysis.

The steam explosion of biomass sometimes causes the formation of by-products, such as toxic substances, which are inhibitory to the activity of microbes and enzymes. Thus, the method of converting shredded and steam-exploded fibrous material into fuel may optionally include a liming stage prior to fermentation to precipitate some of the toxic substances.

For example, the pH value of crushed and steam-exploded fibrous material can be increased so that it exceeds 10 by adding calcium hydroxide (Ca(OH)g), followed by a step of lowering the pH to about 5 by adding Na25Ox. The calcined fibrous material can then be used as is without removing the precipitate. As shown in FIG. 23, an optional liming stage is carried out immediately before the hydrolysis stage of the shredded and steam-exploded fibrous material, but it is also envisaged to carry out the liming stage after the hydrolysis stage and before the fermentation stage.

In FIG. 24 shows an example of a steam explosion device 460. The steam explosion device 460 includes a reaction chamber 462 into which a fiber source and/or fibrous material is placed through a fiber source inlet 464. Closing the fiber source inlet valve 465 closes the reaction chamber. The reaction chamber further includes a compressed vapor inlet 466 that includes a steam valve 467. The reaction chamber further includes an explosion pressure relief outlet 468 that includes an outlet valve 469 connected to the cyclone 470 via a connection pipe 472. When the reaction chamber contains the fiber source and/or the crushed fiber source and is closed by the shut-off valves 465, 467 and 469, steam is delivered to the reaction chamber 462 by opening the steam inlet valve 467, allowing the steam to escape through the steam inlet 466. After the reaction chamber reaches the desired temperature, which may take approximately 20-60 seconds, the holding time begins. The reaction chamber is held at the desired temperature for the desired holding time, which is typically about 10 seconds to 5 minutes. At the end of the holding period, the outlet valve opens, allowing the blast pressure to be released. The depressurization process moves the contents of the reaction chamber 462 from the depressurization outlet 468, through the connecting pipe 472, into the cyclone 470. The steam-exploded fiber source or fibrous material then exits the cyclone in slurry form into a collection basket 474, in while most of the residual steam exits the cyclone to the atmosphere through vent 476. The steam explosion device further includes a purge outlet 478 with a purge outlet valve 479 connected to a connecting pipe 472. The purge outlet valve 479 closed in the process of using the steam explosion device 460 in the steam explosion, but open in the process of washing the reaction chamber 462.

The set temperature of the reaction chamber 462 is preferably from 180 to 240 °C or from 200 to 220 °C. The holding time is preferably from 10 seconds to 30 minutes, or from 30 seconds to 10 minutes, or from 1 minute to 5 minutes.

Since the steam explosion process results in a deposit in the form of a steam-exploded fibrous material, the steam-exploded fibrous material may not necessarily be subjected to a fiber drawing process where a "solution" is separated from the steam-exploded fibrous material. This step of fiber extraction is desirable because it enables further crushing and/or screening processes and can provide the possibility of converting the fibrous material into fuel. The fiber separation process is carried out using a mesh fabric to separate the fibers from the solution. In addition, drying processes may also be included to obtain a fibrous material or a steam-exploded fiber source for further processing.

Combination of devices for irradiation, pyrolysis, ultrasound treatment and/or oxidation

In some embodiments, it may be useful to combine two or more separate devices for irradiation, sonication, pyrolysis and/or oxidation into a single hybrid device. With the use of such a hybrid device, several processes can be carried out sequentially or even simultaneously, which gives the advantage of increasing the productivity of pre-treatment and potential cost savings.

For example, the processes of electron beam irradiation and ultrasound treatment are considered.

Each individual process is effective in reducing the average molecular weight of the cellulosic material by an order of magnitude or more, and several orders of magnitude when performed sequentially.

Both irradiation and sonication processes can be applied using a hybrid electron beam/ultrasound device, as illustrated in FIG. 25.

A hybrid electron beam/ultrasound treatment device 2500 is shown above an empty bath (3-5 cm deep) for suspending cellulosic material 2550 dispersed in an aqueous oxidizing medium such as hydrogen peroxide or carbamide peroxide. The hybrid device 2500 has a power source 2510 that powers both the electron beam emitter 2540 and the sonication horns 2530 .

Electron beam emitter 2540 generates an electron beam that passes through electron beam targeting device 2545 to impinge on slurry 2550 containing cellulosic material. The electron beam aiming device may be a scanner that transmits the beam over a range up to about 6 feet (183 cm) in a direction approximately parallel to the surface of the slurry 2550 .

On either side of the electron beam emitter 2540 are sonication horns 2530 that deliver ultrasonic wave energy to the slurry 2550. The sonication horns 2530 terminate in a removable nozzle 2535 that contacts the slurry 2550.

The 2530 sonicator horns are at risk of damage due to prolonged residual exposure to electron beam exposure. Thus, the horns can be protected by a standard shield 2520, for example, made of lead or an alloy containing a heavy metal, such as Lipovich metal, which is impermeable to electron beam radiation. However, precautions must be taken to ensure that the presence of the screen does not affect the ultrasonic energy. The removable tips 2535, which are constructed of the same material as the horns 2530 and attached to the horns 2530, come into contact with the cellulosic material 2550 during the processing process and are expected to be damaged. As such, the 2535's removable tips are designed to be easily replaceable.

Another advantage of this simultaneous electron beam and ultrasound treatment process is that these two processes have complementary results. In the case of a separate electron beam irradiation, an insufficient dose can lead to cross-linking of some of the polymers in the cellulosic material, which reduces the efficiency of the depolymerization process in general. To achieve a degree of depolymerization similar to that achieved using electron beam irradiation and ultrasound treatment separately, lower doses of electron beam irradiation and/or ultrasound irradiation can also be used. The electron beam device can also be combined with one or more of the high-frequency rotor-stator devices that can be used as an alternative to sonication devices.

Other device combinations are also possible. For example, an ionizing radiation treatment device that generates gamma radiation emitted, for example, by 90Co tablets,

can be combined with an electron beam source and/or an ultrasonic wave source. In this case, the shielding requirements may be more stringent.

The radiation devices for biomass pretreatment discussed above can also be combined with one or more devices that perform one or more pyrolysis processing sequences. Such a combination can also have the advantage of high performance. However, caution is necessary, as there may be conflicting requirements between some radiation exposure methods and pyrolysis. For example, when using devices for ultrasound irradiation, it may be necessary to immerse raw materials in a liquid oxidizing medium. On the other hand, as discussed above, it may be preferable for a sample of raw material to be pyrolyzed to have a specific moisture content. In this case, new systems automatically measure and monitor specific moisture content and regulate it. In addition, some or all of the above devices, especially the pyrolysis device, can be combined with an oxidation device as discussed above.

The main methods

Fermentation

Generally, different microorganisms can produce a number of useful products, such as fuel, by acting on pre-treated biomass material, for example by fermenting it.

For example, alcohols, organic acids, hydrocarbons, hydrogen, proteins, or mixtures of any of these materials can be produced by fermentation or other methods.

A microorganism can be a natural microorganism or an engineered microorganism. For example, a microorganism can be a bacterium, such as a cellulolytic bacterium, a fungus, such as yeast, a plant, or a single-celled organism such as algae, a protozoan, or a fungus-like single-celled organism, such as a slime mold. When organisms are compatible, mixtures of organisms can be used.

One or more enzymes, such as a cellulolytic enzyme, can be used to aid in the breakdown of cellulose-containing materials. In some embodiments, materials comprising cellulose are first treated with an enzyme, for example, by combining the material and the enzyme in an aqueous solution. This material can then be combined with a microorganism. In other embodiments, materials including cellulose, one or more enzymes, and a microorganism are combined simultaneously, for example, by combining in an aqueous solution.

Also, to promote the destruction of materials that include cellulose, the materials after irradiation can be treated with heat, a chemical reagent (eg, a mineral acid, a base, or a strong oxidizer such as sodium hypochlorite), and/or an enzyme.

In the fermentation process, the sugars released at the stage of cellulolytic hydrolysis or saccharification are fermented, for example, into ethanol by a fermenting microorganism such as yeast. Suitable fermenting microorganisms are capable of converting carbohydrates such as glucose, xylose, arabinose, mannose, galactose, oligosaccharides or polysaccharides into fermentation products.

Fermenting microorganisms include strains of the genus Zasspagotusev5 5yr. for example Zzasspagotuse5 segemiziae (baker's yeast), sasspagotusev aiiaysiv, sasspagotuse5 imagyt; genus Kisumegotusev, for example species Kinumegotuse5 taghiapi5, Kipumegotusev5 Ppadiiye; genus Sapaiida, for example Sapaiida rzecydoihorisaii5 and Sapaiida Bgazvzisae, Rispia 5Iiriyiz (relative of Sapaida 5Ppepagae), genus Siamizroga, for example species Siamizroga Is5yapiae and Siamizroga oripiae, genus Raspuzoiep, for example species of Raspuzoiep TapporpiyYiv, genus Vgegappotuse5 (e.g. R., 1996, Seyisho5ze riosopmegviop yesppoiodu, ip Naparook op VioyeiNnapo!: Rgodisiop apa Shi2ainop, UUutap, S.E., ed., Tauog v Egapsiv, Mavzpipdiop, OS, 179-212).

Commercially available yeasts include, for example, Keya 5iagv/I ezaigte Evapoi Kei (available from

Kea iag/ ezaite, BOTH), EACIF (available from RIeizsptapp'5 Ueabi, branch of Vygp5 RpPiyir Rosa Ips.,

BA), BOREKZTAKTYE (available from AIZhesi), SECT ZTKAMOF (available from Segi Zigapa AV, Zmedep) and

EECMOI F (available from OBM 5resianiev).

Bacteria that can ferment biomass into ethanol and other products include, for example, 7utotopa5 tobii5 and SiIovigidiit (PpegtoseiPysht (RPyirridih, 1996, above). 1155-1160) isolated anaerobic, mesophilic, cellulolytic bacteria from the forest soil, Siovigidicht rpujeheptepiap5 5r. pom., which will convert cellulose into ethanol.

Fermentation of biomass into ethanol and other products can be carried out using certain types of thermophilic or genetically engineered microorganisms, such as species

Tpeptoapaegobasieg, including T. taiyNgapii, and yeast species such as Rispia species. An example of a strain of T. taiNgapia is AZMA4, described in the publications of Zoppe-Napzep et al. (Arriiea Mistoriiodu apa ViotesppoIodu 1993, 38, 537-541) or AnHipd ei a. (Agop. Misgobio!. 1997, 168, 114-119).

For fermentation or conversion, you can use yeast and bacteria Khupotopav. The optimum pH for yeast is about pH 4 to pH 5, while the optimum pH for 7utopahs is about pH 5 to pH 6. A typical fermentation time is about

24 to 96 hours at temperatures ranging from 26 to 40 "C, but thermophilic microorganisms prefer higher temperatures.

Enzymes that break down biomass such as cellulose into lower molecular weight carbohydrate materials such as glucose in the process of saccharification are called cellulolytic enzymes or cellulases. These enzymes can be a complex of enzymes that act synergistically, destroying crystalline cellulose. Examples of cellulolytic enzymes include: endoglucanases, cellobiohydrolases, and cellobiases (B-glucosidases). The cellulosic substrate is first hydrolyzed by endoglucanases in random regions, producing oligomeric intermediates.

These intermediates then become substrates for exocleaving glucanases such as cellobiohydrolase to form cellobiose, which is cleaved from the ends of the cellulose polymer. Cellobiose is a water-soluble dimer of glucose linked by a DV-1,4 bond. Finally, cellobiase breaks down cellobiose to form glucose.

Cellulase is able to destroy biomass and can be of fungal or bacterial origin. Suitable enzymes include cellulases from the genera Vasiy5, Rehepdotopav5, Nitisoia, Eizagisht, TpyeYiamia,

Asgetopisht, Spguzozrogisht and Tgispodegip, and also include species of Nitisoia, Sorgipi5, Tpiviamia,

Eizagist, Museiorpipoga, Asgetopist, Serpaiozorigist, Zsuiaiidiit, Repisiiist or Azregoii5 (see, e.g., EP 458162), especially cellulases produced by strains selected from the species of Nitisoia ipzoiliep5 (reclassified as Zsuiaidiit (pegtorpist, see, e.g., US Pat. No. 30,4435 ),

Soriipye sipegeiv5, Ryvapyst ohuzrogyt, Museiorpiipoga Sheptorpiia, Megiriishiv didapiev5, Tniviamia

Ieiteviyv5, Astetopiit 5r., Astetopiit regzisipit, Astetopisht astetopisht, Astetopisht Brgaspurepiit,

Asgtetopisht aisptotovrogyt, Astetopisht obBrsiamaisht, Astetopisht ripKepyopiae, Astetopiyt gozeodgizeit, Astetopiyt ipsoYogaisht ii Asgetopisht iigaisht; mainly from the species Nitisoia ipzhoiYep5 О5М 1800, Ryzapyit ohuzrogyt О5M 2672, Museiiorpiypoga Ipegtorpia SV5 117.65, Serpaiozorogyst 5r. NUM-202, Astetopisht 5r. SV5 478.94, Astetopisht v5r. SV5 265.95, Astetopisht regsicipit SV5 169.65,

Asgetopisht Asgetopisht ANA 9519, SerNnaIozrogisht v5r. SV 535.71, Astetopisht Brgaspurepit SV5 866.73, Astetopisht aisptotovzrogyt SV5 683.73, Asgetopisht oBbsiamaisht SV 311.74, Astetopisht ripKkepopiaee SV5 157.10, Astetopisht gozeodgizveit SV5 134.56, Astetopisht ipsoiis SV5 64.56.

Asgetopisht iigaisht SV5 299.70N. Cellulolytic enzymes can also be obtained from

Spguzozrogisht, mainly strain SPpguzozrogisht IshsKpomzhepze. In addition, you can use

Typispodepta (in particular Thispodegta migide, Thispodegta geezei and Thispodegta Copipadia), alkalophilic

Vasiyi5 (see, for example, US Patent No. 3,844,890 and EP 458,162) and Zigeriotusez (see, for example, EP 458,162). The bacterium, sasspagorpadiz adedgadap5, produces a mixture of enzymes capable of breaking down a number of cellulosic materials, and it can also be used in this process.

Anaerobic cellulolytic bacteria, such as new cellulolytic species, were also isolated from the soil

Siozygiditis, Siozygidicht rpusotegtepiapz 5 years. d. (see I ezopipe ei. ai., Ipegpaiopai doigpai oi Zuzietaiiys apa EmoIshiopagu Misgorioiodu (2002), 52, 1155-1160).

It is also possible to use cellulolytic enzymes obtained using recombinant technology (see, for example, UMO 2007/071818 and UMO 2006/110891).

The cellulolytic enzymes used can be obtained by fermentation of the indicated strains of microorganisms on a nutrient medium containing suitable sources of carbon and nitrogen and inorganic salts, using methods known in the field (see, for example, Veppei, 9. MU. apa

I abige, G.. (ea5z.), Moge Sepe Mapirshiaiop5 ip Rypdi, Asadetis Rge55, SA 1991). Suitable media are available from commercial suppliers or can be obtained according to published compositions (for example, in Ategisap Toure Siyige SoPesiop catalogs). Temperature ranges and other conditions suitable for cellulase growth and production are known in the art (see, e.g., Vaieu,

U.E., apa Ov, O.E., Viospetisa! Epaipeegipd Eipdatepia!5, MsStam/-NIIII VooK Sotrapu, MU, 1986).

Processing of cellulose with cellulase is usually carried out at temperatures from 30 to 65 "С. Cellulases are active in the pH range from approximately 3 to 7. The duration of the saccharification stage can be up to 120 hours. The dosage of the cellulase enzyme ensures a fairly high level of cellulose conversion.

For example, a suitable cellulase dosage is typically 5.0 to 50 filter paper units (EPO or ME) per gram of cellulose. ERI is a standard measure and is defined and measured according to Spoze (1987, Ryge apa Arri. Spect. 59:257-268).

Portable fermenters can be used as described in US Patent Application Serial No. 60/832735, currently published International Application Ser. No. MO 2008/011598.

Gasification

In addition to using pyrolysis to pretreat raw materials, pyrolysis can also be used to process pretreated raw materials to extract useful materials. In particular, a form of pyrolysis known as gasification can be used to produce fuel gases along with various other gaseous, liquid and solid products. For gasification, pretreated feedstock is fed into a pyrolysis chamber and heated to a high temperature, typically 700°C or more. The temperature used depends on a number of factors, including the nature of the feedstock and the desired products.

Some amounts of oxygen (eg, pure gaseous oxygen and/or air) and steam (eg, superheated steam) are also added to the pyrolysis chamber to aid gasification. These compounds react with carbon-containing feedstock in a multistage reaction to form a gas mixture called syngas (or "syngas"). Basically, during gasification, a limited amount of oxygen is supplied to the pyrolysis chamber to ensure the combustion of part of the raw material with the formation of carbon monoxide and the generation of process heat. The process heat can then be used to start a second reaction that converts the additional feedstock into hydrogen and carbon monoxide.

In the first stage of the general reaction, heating the raw material leads to carbonaceous residues, which can include a wide variety of different hydrocarbon-based compounds. Certain volatile substances (for example, certain gaseous hydrocarbon materials) can be formed, which leads to a decrease in the total weight of the raw material. Then, in the second stage of the reaction, some of the volatile material formed in the first stage reacts with oxygen in a combustion reaction to produce both carbon monoxide and carbon dioxide. During this combustion reaction, heat is released, which triggers the third stage of the reaction. In the third stage, carbon dioxide and steam (for example, water) react with carbonaceous residues formed in the first stage, with the formation of carbon monoxide and hydrogen gas.

Carbon monoxide can also react with steam, according to the water-gas reaction of changes (uageg da5 5pPPiUyu, with the formation of carbon dioxide and additional gaseous hydrogen.

Gasification can be used as the main process for obtaining products directly from pre-treated raw materials, for example, for subsequent transportation and/or sale.

Alternatively or additionally, gasification can be used as an auxiliary fueling process for the overall processing system. The hydrogen-rich syngas produced in the gasification process can be burned, for example, to generate electricity and/or process heat, which can be directed for use in other parts of the processing system. As a result, the overall recycling system can be at least partially self-sufficient. In the process and/or after gasification, a number of other products can also be obtained, including pyrolysis oils and hydrocarbon-based gaseous substances; they can be separated and stored or transported if desired.

A number of different pyrolysis chambers are suitable for gasification of pretreated feedstocks, including the pyrolysis chambers described herein. In particular, fluidized bed reactor systems, in which pretreated raw materials are subjected to fluidization in steam and oxygen/air, provide relatively high productivity and direct separation of products. The solid carbonaceous residues remaining after gasification in the fluidized bed system (or other pyrolysis chambers) can be burned to generate additional process heat used to start subsequent gasification reactions.

Next processing

Distillation

After fermentation, the obtained fluids can be subjected to distillation using, for example, a "fermentation column" to separate ethanol and other alcohols from most of the water and residual solids. The steam coming out of the fermentation column can be, for example, mass. 95 ethanol and it can be fed to the distillation column. A mixture of substantially azeotropic (92.595) ethanol and water from the distillation column can be purified to pure (99.595) ethanol using vapor phase molecular sieves. The sludge from the fermentation columns can be sent to the first stage of a three-stage evaporator. The reflux condenser can provide the heat for this first stage. After the first stage, the solids can be separated using a centrifuge and dried in a rotary dryer. A portion (2595) of the product from the centrifuge can be reused for fermentation, and the remainder can be sent to the second and third stages of the evaporator. Much of the evaporator condensate can be returned to the process as fairly clean condensate, where a small portion is allocated to waste water treatment to prevent the formation of low-boiling compounds.

Treatment of waste water

Waste water treatment is used to minimize the need for additional water for the water treatment plant for reuse in the plant. Wastewater treatment can also provide fuels (such as sludge and biogas) that can be used to increase the overall efficiency of the ethanol production process. For example, as described in more detail below, sludge and biogas can be used to generate steam and electricity that can be used in various manufacturing processes.

The waste water is first pumped through a sieve (for example, a grate) to remove large particles that collect in the waste container. In some embodiments, large particles are sent to landfills. Additionally or alternatively, the large particles are burned to generate steam and/or electricity, as detailed below. Typically, grid spacing is between 1/4 inch (0.6 cm) and 1 inch (2.5 cm) (eg 1/2 inch (1.3 cm)).

The waste water then flows into an equilibration tank, where the concentration of organic compounds in the waste water is balanced during the settling time. As a rule, the settling time is from 8 hours to 3b hours (for example, 24 hours). A mixer for mixing the contents of the container is located in the container. In some embodiments, mixers located throughout the container are used to mix the contents of the container. In certain embodiments, the mixer essentially mixes the contents of the equilibration container so that the conditions (eg, concentration and temperature of the waste water) throughout the container are the same.

The first pump pumps water from the balancing tank through the liquid-liquid heat exchanger. The heat exchanger is regulated (for example, by adjusting the flow rate of the fluid through the heat exchanger) so that the waste water leaving the heat exchanger has the desired temperature for anaerobic treatment. For example, the desired temperature for anaerobic treatment can be from 40 to 60 "С.

After leaving the heat exchanger, the waste water enters one or more anaerobic reactors. In some embodiments, the concentration of sludge in each anaerobic reactor is the same as the total concentration of sludge in the waste water. In other embodiments, the sludge in the anaerobic reactor has a higher concentration than the total concentration of the sludge in the waste water.

A nutrient solution containing nitrogen and phosphorus is measured into each anaerobic reactor containing waste water. The feed solution reacts with the bottom in an anaerobic reactor to form biogas, which can contain 5,095 methane and has a calorific value of approximately 12,000 British thermal units, or Vis, per pound (28,000 kJ/kg). Biogas exits each anaerobic reactor through a vent and enters a collector, where multiple biogas streams are combined into a single stream.

The compressor pumps the biogas stream into a steam boiler or internal combustion engine, as described in more detail below. In some embodiments, the compressor also pumps a single stream of biogas through the desulfurization catalyst. Additionally or alternatively, the compressor may pump a single stream of biogas through a sedimentation trap.

A second pump pumps the anaerobic output stream from the anaerobic reactors to one or more aerobic reactors (eg, activated sludge reactors). Each aerobic reactor is equipped with an aerator for mixing the anaerobic effluent, bottoms, and oxygen (for example, oxygen contained in air). In each aerobic reactor, the oxidation of cellular material in the anaerobic effluent leads to the production of carbon dioxide, water, and ammonia.

The aerobic outlet stream is moved (for example, by gravity) to a separator, where the sludge is separated from the treated water. A portion of the bottoms is returned to one or more aerobic reactors to create an increased concentration of bottoms in the aerobic reactors, thereby promoting the aerobic destruction of cellular material in the waste water. The conveyor removes excess sludge from the separator. As described in more detail below, excess bottoms are used as fuel to generate steam and/or electricity.

Treated water is pumped from the separator into a sump. Solids dispersed in the treated water settle to the bottom of the sump and are subsequently removed. After the settling period, the treated water is pumped out of the settling tank through a fine filter to remove any additional solids remaining in the water. In some embodiments, chlorine is added to the treated water to kill pathogenic bacteria. In some variants of implementation, one or more methods of physical and chemical separation are used for additional purification of treated water. For example, treated water can be pumped through a coal absorption reactor. As another example, treated water can be pumped through a reverse osmosis reactor.

In the processes described in this document, when water is used in any of the processes, it may be domestic wastewater, such as municipal wastewater or sewage. In some embodiments, wastewater or fecal water is sterilized prior to use.

Sterilization can be carried out by any desired method, for example by irradiation, steam or chemical sterilization.

Incineration of waste

Alcohol production from biomass can lead to various by-product streams suitable for generating steam and electricity for use in other parts of the plant.

For example, steam generated by burning by-product streams can be used in the distillation process. As another example, electricity generated by burning by-product streams can be used to power electron beam generators and ultrasonic transducers used for pretreatment.

Byproducts used to generate steam and electricity are generated from multiple sources throughout the process. For example, anaerobic digestion of waste water produces biogas with a high methane content and a small amount of waste biomass (scum). As another example, post-distillation solids (eg, unconverted lignin, cellulose and hemicellulose remaining after pretreatment and core processes) can be used as fuel.

The biogas is fed to an internal combustion engine connected to an electric generator to generate electricity. For example, biogas can be used as a fuel source in a natural gas engine with electric ignition. As another example, biogas can be used as a fuel source in a direct injection natural gas engine. As another example, biogas can be used as a fuel source for an internal combustion turbine. Additionally or alternatively, the internal combustion engine can be designed in a configuration for combined electrical and thermal energy production. For example, exhaust heat from internal combustion engines can be used to provide hot water or steam throughout production.

The residue and solids after distillation are burned to heat the water flowing through the heat exchanger. In some embodiments, the water flowing through the heat exchanger evaporates and is superheated to form steam. In certain embodiments, the steam is used in the reactor for pretreatment and during heat exchange in the processes of distillation and evaporation. Additionally or alternatively, the steam is expanded, powering a multi-stage steam turbine coupled to an electric generator. The steam leaving the steam turbine is condensed with cooling water and returned to the heat exchanger to be reheated to steam. In some embodiments, the rate of flow of water through the heat exchanger is regulated to provide a given production of electricity from a steam turbine coupled to an electric generator. For example, water may be added to the heat exchanger to allow the steam turbine to operate above the limiting conditions (for example, the turbine spins fast enough to spin an electric generator).

Although some embodiments have been described, other embodiments are possible.

As an example, while the biogas is described as being fed to an internal combustion engine coupled to an electric generator, in certain embodiments the biogas may be passed through a fuel reformer to produce hydrogen. The hydrogen is then converted into electricity using a fuel cell.

As another example, while the biogas is described as being burned separately from the bottoms and solids after distillation, in certain embodiments, all waste by-products may be burned together with steam.

Products/co-products

Alcohols

The produced alcohol can be a monohydric alcohol, such as ethanol, or a polyhydric alcohol, such as ethylene glycol or glycerin. Examples of alcohols that can be produced include methanol, ethanol, propanol, isopropanol, butanol, such as n-, sec-, or tert-butanol, ethylene glycol, propylene glycol, 1,4-butanediol, glycerin, or mixtures of these alcohols.

Each of the alcohols produced at the plant has a commercial value as an industrial raw material.

For example, ethanol can be used to make varnishes and perfumes. As another example, methanol can be used as a solvent used as a component in a windshield wiper fluid. As another example, butanol can be used in plasticizers, resins, glazes, and brake fluids.

The bioethanol produced at the plant is valuable as an ingredient used in the food and beverage industry. For example, the ethanol produced at the plant can be purified to the level of food grade alcohol and used as the main ingredient in alcoholic beverages.

The bioethanol produced at the plant also has commercial value as a transportation fuel.

The use of ethanol as a transportation fuel can be accomplished with relatively little capital investment from manufacturers and owners of electric ignition engines (eg, changing the injection control, fuel-air ratio, and fuel injection system components). Many car manufacturers now offer flex-fuel cars capable of running on ethanol/gasoline blends containing up to 85 vol. 95 ethanol (for example, a standard screw-up on Spema Taioe 4x4).

The bioethanol produced at this facility can be used as engine fuel to improve environmental and economic conditions outside the facility. For example, ethanol produced at this facility and used as a fuel can reduce greenhouse gas emissions from anthropogenic sources (eg, transportation sources). As another example, the ethanol produced at this facility and used as an engine fuel can also replace the gasoline consumed, derived from petroleum and refined.

Bioethanol has a higher octane number than conventional gasoline and thus can be used to improve the performance (for example, to provide higher compression ratios) of spark-ignition engines. For example, small amounts (such as 10 vol. 95) of ethanol can be blended with gasoline to act as an octane-enhancing additive to the fuel used in spark-ignition engines. As another example, higher amounts (eg, 85 vol. 95) of ethanol can be mixed with gasoline to further increase the octane number and replace large volumes of gasoline.

Bioethanol strategies are considered, for example, OiRagdo u doita! ой ОшиооК ог Viota55 ЕШТапой

Rhodisiip apa Oetapa (EIIA Eogesabviv), 2002; Zpeepap in ViotesppoYod Rgodgez5, 15:8179, 1999; Mapip in

Epigute Mistore5 TesppoiIoadu, 31:274, 2002; Steeg in VioSusie, 61-65, Argii 2005; And upa in Misgorioiodu apa

Moiesshiag Vioiodu Nemiemv, 66:3, 506-577, 2002; Tsypdaani ei aiI., in US patent Me 4292406; and VeMPatu, in US Pat. No. 4,094,742.

Organic acids

The organic acids produced may include monocarboxylic acids or polycarboxylic acids.

Examples of organic acids include formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, palmitic acid, stearic acid, oxalic acid, malonic acid, succinic acid, glutaric acid, oleic acid, linoleic acid, glycolic acid, lactic acid acid, γ-hydroxybutyric acid or mixtures of these ee

Food

In some embodiments, all or part of the fermentation process can be interrupted before the cellulosic material is completely converted to ethanol. Fermentation intermediates include high concentrations of sugars and carbohydrates. These fermentation intermediates can be used to prepare foods for human or animal consumption. In some embodiments, pre-treatment of cellulosic material with irradiation ensures sterility of fermentation intermediates (eg, suitable for human consumption). In some embodiments, the fermentation intermediates require further processing before being used as food products. For example, a dryer can be used to remove moisture from fermentation intermediates in order to facilitate storage, processing and increase shelf life. Additionally or alternatively, the fermentation intermediates can be ground to fine particles in a stainless steel laboratory mill to produce a flour-like substance.

Animal feed

Bard and soluble substances can be converted into a valuable by-product of the distillation-dehydration process. After the distillation-dehydration process, the bard and solubles can be dried to improve the storage or handling of the material. The resulting dry bard and solubles (0005) are low in starch, high in fat, high in protein, high in fiber, and high in phosphorus. Thus, for example, WHO may be valuable as a feed source for animals (eg as a feed source for dairy cattle). OOS5 can be further combined with nutritional supplements to meet the specific nutritional needs of specific categories of animals (for example, the balance of digestible lysine and phosphorus for pig diets).

Pharmaceutical drugs

The pretreatment processes discussed above can be applied to plants with medicinal properties. In some embodiments, sonication can stimulate biological activity and/or bioavailability of medicinal plant components with medicinal properties.

Additionally or alternatively, irradiation stimulates the biological activity and/or bioavailability of medicinal plant components with medicinal properties. For example, sonication and irradiation can be combined in the pretreatment of willow bark to stimulate salicin production.

Nutraceuticals

In some embodiments, fermentation intermediates (eg, products that include high concentrations of sugars and carbohydrates) can be supplemented to create a nutraceutical.

For example, fermentation intermediates can be supplemented with calcium to create a nutraceutical that provides energy and helps improve or maintain bone strength.

Co-products

Lignin residue

As described above, the lignin-containing residues after the main and pretreatment processes have value as high/medium energy fuels and can be used to generate power and steam used in manufacturing processes. However, such lignin residues represent a new type of solid fuel, the demand for which outside the plant is small, and the cost of drying them for transportation only leads to a decrease in their potential value. In some cases, gasification of lignin residues can convert them into a higher value product with lower costs.

Other co-products

Cell material, furfural, and acetic acid have been identified as potential co-products for biomass-to-fuel equipment. Interstitial cellular material can be valuable, but may require extensive purification. Markets for furfural and acetic acid are relevant, although it is unlikely that they are large enough to absorb the output of a fully commercialized lignocellulosic ethanol industry.

Examples

The examples presented below are intended to illustrate and not limit the ideas of this description.

Example 1 - Production of fibrous material from paper with a multilayer coating

A 1,500 lb (680 kg) slipway of clear half-gallon (1.9 L) juice cartons made from unprinted white kraft paperboard with a bulk density of 20 lb/ft? (0.32 g/cm3), received from Ipiegpaiop! Rare Each carton was folded flat and then fed into a Z Pr Riipsi Vatsody shredder at a rate of approximately 15 to 20 pounds per hour (6.8-9.1 kg/hr). The shredder was equipped with two 12-inch (30-cm) rotating blades, two fixed blades, and a 0.30-inch (0.8-cm) discharge screen.

The distance between the rotating and fixed blades was set at 0.10 inches (0.25 cm). The shredding material exiting the device resembled confetti, 0.1 inch (0.25 cm) to 0.5 inch (1.3 cm) wide, 0.25 inch (0.6 cm) to 1 in. (2.5 cm) and a thickness equivalent to that of the parent material (approximately 0.075 in. (0.2 cm)).

The candy-like material was fed into a cutting device with a rotating knife Mipsop, model

ZS30. Model 5C30 is equipped with four rotating blades, four fixed blades and a discharge screen with 1/8 inch (0.32 cm) holes. The gap between the rotating and fixed blades was set at approximately 0.020 inch (0.05 cm). A rotating knife cutter crushed the confetti-like fragments with the blade tips, breaking the fragments and releasing the fibrous material at a rate of approximately one pound per hour (454 grams per hour). The fibrous material had a VET surface area of 0.9748 mg/g w/- 0.0167 mg/g, a porosity of 89.043795 and a bulk density (at 0.53 psi abs. (3.7 kPa)) 0.1260 g/ml. The average fiber length was 1.141 mm and the average fiber width was 0.027 mm, giving an average G/0 of 42:1. A picture of the fibrous material, obtained using a scanning electron microscope at a magnification of 25X, is presented on

FIG. 26.

Example 2 - Production of fibrous material from bleached kraft cardboard

A 1,500 lb (680 kg) slipway of pure bleached kraft paperboard having a bulk density of lb/ft? (0.48 g/smu), received from Ipiegpaiopai! Rare The material was flattened and then fed into a Z Pr Ripsp Vacoi shredder at a rate of approximately 15 to 20 pounds per hour (6.8-9.1 kg/hr). The shredder was equipped with two 12-inch (30-cm) rotating blades, two fixed blades, and a 0.30-inch (0.8-cm) discharge screen. The distance between the rotating and fixed blades was set at 0.10 inches (0.25 cm).

The shredding material exiting the device resembled confetti, 0.1 inch (0.25 cm) to 0.5 inch (1.3 cm) wide, 0.25 inch (0.6 cm) to 1 in. (2.5 cm) and a thickness equivalent to that of the parent material (approximately 0.075 in. (0.2 cm)). The confetti-like material was fed into a cutting device with a rotating knife Mihopp, model 5C30. The discharge screen had 1/8 inch (0.32 cm) openings. The gap between the rotating and fixed blades was set at approximately 0.020 inch (0.05 cm). The rotating knife cutter crushed the confetti-like fragments with the blade tips, breaking the Fragments and releasing the fibrous material at a rate of approximately one pound per hour (454 grams per hour). The fibrous material had a VET surface area of 1.1316 mg/g /- 0.0103 m2/g, a porosity of 88.328595, and a bulk density (at 0.53 lb/sq. in. abs. (3.7 kPa)) of 0 ,1497 g/ml. The average fiber length was 1.063 mm and the average fiber width was 0.0245 mm, giving an average G/O of 43:11. A picture of the fibrous material, obtained using a scanning electron microscope at a magnification of 25X, is presented in FIG. 27.

Example C - Production of twice-shredded fibrous material from bleached kraft paperboard

A 1,500 lb (680 kg) slipway of pure bleached kraft paperboard having a bulk density of 30 lb/ft (0.48 g/cm3) was obtained from Ipiegpaiopai! Rare The material was flattened and then fed into a Z Pr Ripsp Vatsody shredder at a rate of approximately 15 to 20 pounds per hour (6.8-9.1 kg/hr). The shredder was equipped with two 12-inch (30-cm) rotating blades, two fixed blades, and a 0.30-inch (0.8-cm) discharge screen. The distance between the rotating and fixed blades was set at 0.10 inches (0.25 cm).

The material coming out of the shredder resembled confetti (see above). The candy-like material was fed into a cutting device with a rotating knife Mipsop, model 5С30.

The discharge screen had 1/16 inch (0.16 cm) openings. The gap between the rotating and fixed blades was set at approximately 0.020 inch (0.05 cm). A rotating knife cutter crushed the confetti-like fragments with the blade tips, breaking the fragments and releasing the fibrous material at a rate of approximately one pound per hour (454 grams per hour). The material obtained after the first crushing was again fed into the same plant described above and crushed again. The resulting fibrous material had a VET surface area of 1.4408 mg/g w/- 0.0156 me/g, a porosity of 90.899895 and a bulk density (at 0.53 lb/sq. in. abs. (3.7 kPa) ) 0.1298 g/ml. The average fiber length was 0.891 mm and the average fiber width was 0.026 mm, giving an average G/O of 3471. A scanning electron microscope image of the fibrous material at 25X magnification is shown in FIG. 28.

Example 4 - Obtaining three-fold shredded fibrous material from bleached kraft cardboard

A 1,500 lb (680 kg) slipway of pure bleached kraft paperboard having a bulk density of lb/ft? (0.48 g/smu), received from Ipiegpaiopai! Rare The material was flattened and then fed into a Z Pr Ripsp Vacoi shredder at a rate of approximately 15 to 20 pounds per hour (6.8-9.1 kg/hr). The shredder was equipped with two 12-inch (30-cm) rotating blades, two fixed blades, and a 0.30-inch (0.8-cm) discharge screen. The distance between the rotating and fixed blades was set at 0.10 inches (0.25 cm).

The material coming out of the shredder resembled confetti (see above). The candy-like material was fed into a cutting device with a rotating knife Mipsop, model 5С30.

The discharge screen had 1/8 inch (0.32 cm) openings. The gap between the rotating and fixed blades was set at approximately 0.020 inch (0.05 cm). A cutting device with a rotating knife crushed confetti-like fragments with the edges of the blades. The material obtained after the first crushing was again fed to the same plant and the sieve was replaced with a sieve with 1/16 inch (0.16 cm) openings. The material was crushed. The material obtained after the second crushing was again fed to the same plant and the sieve was replaced with a sieve with openings of 1/32 inch (0.08 cm). This material was crushed. The resulting fibrous material had a VET surface area of 1.6897 mg/g /- 0.0155 m"/g, a porosity of 87.716395 and a bulk density (at 0.53 psi abs. (3.7 kPa) ) 0.1448 g/ml. The average length of the fibers was 0.824 mm and the average width of the fibers was 0.0262 mm, giving an average YH/O0O of 32:1. A scanning electron microscope image of the fibrous material at 25X magnification is presented in Fig. FIG. 29.

Example 5 - Production of densified fibrous material from bleached kraft paperboard without the addition of a binder

The fibrous material was prepared according to Example 2. Approximately 1 pound (454 g) of water was sprayed for every 10 pounds (4540 g) of fibrous material. The fibrous material was compacted using a Seiyogpiya Reyei Mi 1100 pellet press operating at 7520. Pellets having a bulk density ranging from about 7 lb/ft (0.11 g/cm") to about 15 lb/ft were obtained. (0.24 g/cm3).

Example 6 - Production of compacted fibrous material from bleached kraft cardboard with a binder

Fibrous material was obtained according to example 2.

The initial solution of ROI MOH"M UU5E, M10 (polyethylene oxide) was prepared in water at a concentration of 2 wt.

What

Approximately 1 pound (454 g) of stock solution was sprayed for every 10 pounds (4540 g) of fibrous material. The fibrous material was compacted using a Saijogpia granulation press

Reiiei Miii 1100 operating at 752C. Granules having bulk densities ranging from about 15 lb/ft (0.24 g/cm3) to about 40 lb/ft (0.64 g/cm3) were obtained.

Example 7 - Reduction of the molecular weight of cellulose in fibrous kraft paper by gamma radiation with minimal oxidation

Fibrous material is obtained according to example 4. Fibrous kraft paper is compacted according to example 5.

Compacted granules are placed in a glass ampoule with a maximum capacity of 250 ml. The glass ampoule is vacuumed in a deep vacuum (107 torr) for 30 minutes, and then filled again with argon gas. The ampoule is sealed in an argon atmosphere. The pellets in the ampoule are irradiated with gamma radiation for about 3 hours at a dose level of about 1 Mrad per hour to produce an irradiated material in which the cellulose has a lower molecular weight than the source material of fibrous kraft paper.

Example 8 - Reduction of the molecular weight of cellulose in fibrous kraft paper by gamma radiation at maximum oxidation

Fibrous material is obtained according to example 4. Fibrous kraft paper is compacted according to example 5.

Compacted granules are placed in a glass ampoule with a maximum capacity of 250 ml. The glass ampoule is filled in an air atmosphere. The granules in the ampoule are irradiated with gamma radiation for about 3 hours at a dose level of about 1 Mrad per hour to produce an irradiated material in which the cellulose has a lower molecular weight than the source material of fibrous kraft paper.

Example 9 - Electron beam processing

The samples were treated with an electron beam using an in-storage accelerator with undamped KpodoihopF TT200 delivering electrons with an energy of 5 MeV at an output power of 80 kW. Table 1 describes the parameters used. Table 2 describes the nominal dose used for IO of the sample (in Mrad) and the corresponding dose delivered to the sample (in kGy).

Table 1

Kpodoihop parameters? TT200 bundle! and w full width at half maximum

Energy dissipation at 10 MeV (EMUNM) 300 keV

Beam power at 10 MeV guaranteed operating range from 1 to 80 (Energy consumption 77711111

State of readiness (vacuum and cooling OM)

With a beam power of 50 kW "210 kW

With a beam power of 80 kW -260 kW

SystemavE 11111111 107.5:1 MG

Tpotvop TN7VI

Scanning ruplor.io 77777111

Nominal scan length (measured at 120 cm 25-35 cm from the window) - 5 gu - - scan

Nominal scan frequency (at 100 Hz i 595 maximum scan length)

Uniformity of scanning (within 9090 59, the nominal length of scanning) o

Table 2

Dosages delivered to samples

Total Dose (Mrad) 4 (number associated with sample IO) Delivered Dose (kgr) nnnnnnnnNNNNNNNNNNNNnnnnnnnnnnnnnnnnnnnnnnnn 100.0 150.3 198.3 330.9 529.0 695.9 993.6 "For example, 9.9 kGy was delivered in 11 seconds at a beam current of 5 mA and a linear velocity of 12.9 ft/min.The cooling time between treatments at a dose of 1 Mrad was approximately 2 minutes.

Example 10 - Methods of determining the molecular weight of cellulosic and lignocellulosic materials using gel permeation chromatography

Cellulosic and lignocellulosic materials for analysis were processed according to example 4. Sample materials presented in the following tables include kraft paper (P), wheat straw (MU5), alfalfa (A), cellulose (C), millet (55), grasses (b), starch (57) and sucrose (5). The number "132" in the sample ID refers to the particle size of the material after passing through a 1/32 inch (0.08 cm) sieve. The number after the hyphen refers to the dose of radiation (Mrad) and "05" refers to ultrasonic treatment. For example, the IU sample "P132-10" refers to kraft paper, which was subjected to crushing to the size of particles of caliber 132 and irradiated with a dose of 10 Mrad.

For samples irradiated with an electron beam (e-beam), the number after the hyphen refers to the amount of energy delivered to the sample. For example, the "P-100e" IOU refers to kraft paper, which received an energy dose of approximately 100 Mrad or approximately 1000 kGy (table 2).

Table C

Peak average molecular weight of irradiated kraft paper

Dosage! average

Sample Source Sample IO (Mrad) Ultrasound2 MM Standard Deviation 1 RIZA | 7777170777717171717717171717171 no 17 32853:10006 SH 1 CUT | 7107/1771 63982468

Kraft paper / RIZALOO | -/lOb/Y7777771717y11111111171111v444zh580 about RIZALVY | 181 17777771771771111111117111116668zh77О about RIZAOV | 70... 1 yes 1777 zoo51o13Ш "Low doses of irradiation apparently increase the molecular weight of some materials.

Dosage level - 1 Mrad/h. 2Treatment for 30 minutes with 20 kHz ultrasound using a 1000 W horn in recirculation conditions where the material is dispersed in water.

Table 4

Peak average molecular weight of e-beam irradiated kraft paper

Average MM standard

Kraft paper

Table 5

The peak average molecular weight of materials irradiated with gamma radiation is Ce | No | zne of Gena (Mrad) deviation

MUVIZA77771711117117177111117011111171771111 no | 14074115175191.ЖЖГ-: 11111111 39145к3425 -: now I o Pl PO po PO EE All PO

MUVIZatoya 71111110 1711111126040х532407/Г

MUVIZatov 7 1711117111117007 17717171 17111111236203453 Г-:ХЗ

АТЩЖ2 77 11171171771111170111171171717717171711717и71111111 17111 16048865151701.YШ-:б 11111111 37525311 18111111 28535490

АТЗаЛО7777111711717111117101171771111117и111111171111150853:1665.Y.-:ZЮО 11111111

АТ3З200И7717111117111117007 17717171 17111111138291х2235 .-:З 1 а11и111111и11111111111гавти5 вСІЗА ///177711171717771717171717017717117171771111117и71111111711111557360ж83693 щЩщ ( 11111111 42594544147/7 18111711 3268:249...7:ОО вС13а107 7777/1771 1717771711177107777717771717171717и71111111 1711111 вовввхе1311ШЮШ вс132-1007 7 17771711171777111170071777717117и1111111171111112234553797....Ш:Н. 1 1 1 1 1

"Peaks merge after processing. "Low doses of radiation apparently increase the molecular weight of some materials.

Dosage level - 1 Mrad/h. hTreatment for 30 minutes with 20 kHz ultrasound using a 1000 W horn in recirculation conditions where the material is dispersed in water.

Table 6

Peak average molecular weight of materials irradiated by e-beam at Yuzrazka | Mapika | Dosage | Mean MM and standard deviation

But 1 a-1e 1 v 0-09 1-8

Gel permeation chromatography (GPC) is used to determine the molecular weight distribution of polymers. During SPC analysis, a polymer sample solution is passed through a column filled with a porous gel that traps small molecules. The sample is separated based on molecular size, with larger molecules eluting faster than smaller molecules. The retention time of each component is most often determined by refractive index (RI), evaporative light scattering (EI 5) or ultraviolet radiation (M) and compared with a calibration curve. The resulting data are then used to calculate the molecular weight distribution for the sample.

Molecular weight distribution is used instead of individual molecular weight to characterize synthetic polymers. Statistical averages are used to describe this distribution. The most common of these average values are "number average molecular weight" (Mu) and "weight average molecular weight" (Mu).

We are analogous to the standard arithmetic mean associated with a group of numbers. When applied to polymers, We refer to the average molecular weight of the molecules in the polymer. We calculate by assuming a single significance level for each molecule, regardless of its individual molecular weight. We calculate the average according to the following formula, where M; represents the number of molecules with a molar mass equal to M...

MIND,

M, --e----

hmm,

AND

Mu is another statistical identifier of the molecular weight distribution that emphasizes the larger molecules more than the smaller molecules in the distribution.

The formula for the statistical calculation of the weighted average molecular weight is presented below. 2

UM, n. N

M, UM, and

The polydispersity index or RI is defined as the Mu/My ratio. The higher the Ri, the wider or more dispersed the distribution. The lowest value that RI can have is 1. It corresponds to a monodisperse sample, that is, a polymer in which all molecules in the distribution have the same molecular weight.

The peak value of the molecular weight (Mp) is another descriptive feature defined as the nature of the molecular weight distribution. It means the molecular weight that is most common in the distribution.

This value also gives an idea of the molecular weight distribution.

Most CPS measurements are performed against various polymer standards. The accuracy of the results depends on how closely the characteristics of the analyzed polymer match the characteristics of the standard used. The expected reproducibility error between different series of individually calibrated determinations is approximately 5-1095 and is typical of the limited precision of ODS determinations. Thus, SPC results are most useful when comparing the molecular weight distributions of different samples in the same series of determinations.

For lignocellulosic samples, preparation was required before SPC analysis. First, a saturated solution (8.4 wt. 95) of lithium chloride (LI) in dimethylacetamide (DMAC) was prepared.

Approximately 100 mg of sample was added to approximately 10 g of freshly prepared saturated solution

PSMUOMAs and the mixture were heated to approximately 150-170 "C with stirring for 1 hour. The resulting solutions were mainly light yellow to dark yellow in color. The temperature of the solutions was reduced to approximately 100 "C and heated for an additional 2 hours. Then the temperature of the solutions was reduced to about 50 "C and the sample solution was heated for about 48-60 hours. It should be noted that the samples irradiated at 100 Mrad were more easily solubilized compared to their untreated counterparts. In addition, the crushed samples (marked with the number 132) had a slightly lower average molecular weight compared to uncut samples.

The resulting sample solutions were diluted 1:11 using OMas as a solvent and filtered through a 0.45-μm RTEE filter. Then the filtered solutions of the samples were analyzed using CPS using the parameters described in Table 7. The peak average molecular weights (Mw) of the samples, when determined by gel permeation chromatography (GPC), are summarized in Tables 3-6. Each sample was prepared in duplicate and each sample preparation was analyzed in two parallels (two injections), with a total of four injections per sample. Polystyrene standards were used to obtain a calibration curve for a molecular weight scale of approximately 580 to 750,000 Daltons

ROTA and RU1V EaziSact.

Table 7

Conditions of ORS analysis

Ready! 10 μm Mikhea-V

Columns (3) Z/Mye: 10М-МВ-148-83; 10M-MV-148-84; 10M- mmVv-174-129

Example 11. Surface analysis by time-of-flight mass spectrometry of secondary ions (TOE-5IM5)

Time-of-flight mass spectrometry of secondary ions (TOE-5IM5) is a surface-sensitive spectroscopy that uses a pulsed ion beam (C5 or microfocused CA) to remove molecules from the most distant surface of the sample. Particles are removed from atomic monolayers on the surface (secondary ions). These particles are then accelerated in a "flight tube" and their mass is determined by measuring the exact time it takes them to reach the detector (ie the time of flight). TOE-5IM5 provides detailed elemental and molecular information about the surface, thin layers, contact surfaces of the sample and performs a full three-dimensional analysis. Its applications are widespread, including semiconductors, polymers, paints, coatings, glass, paper, metals, ceramics, biomaterials, pharmaceuticals, and organic tissue. Since TOE-5IM5 is a research method, all elements in the periodic table are detected, including H. The ToB-5IM5 data are presented in Tables 8-11. The parameters used are presented in Table 12.

Table 8

Normalized average intensity of different positive ions of interest (normalized relative to the total number of ions x 10000) 1100 (RIZA 1 | РИЗаАЛО 7 | РИЗаАЛОО//// go ee me mog value value value 28 15177777 17171176 1459 | 2 sh 197 | 89 | USD 81.07

Table 9

Normalized mean intensity of various negative ions of interest (normalized relative to total number of ions x 10000) 11111RIZA | 7111 CUT | 7 RIZ2alLOOG// ry | me || mi value value value 13 sn | 71960017 91 | 1970 | 26 | 1500, | 6

But 24711 127 | 220171199 | 5 | 7 42 1sMo | 7lb 1 071 | 0743 | 0 | 108 | 09

Table 10

Normalized average intensity of various positive ions of interest (normalized relative to the total number of ions x 10000) 11111000 Re | Re | RO | RZOe | Re | R-IObe

Average- Average- Average- Average- Average" Average- t/2 type unknown value known value value value value value value value 271 A | 549 |194| 677 |86| 752 | 371| 761 |158| 516 159) 622 | 166) 271 SeNnz | FIGHTS |205| 551 |59| 645 |165| 597 |152| 707 94) 600 / 55 39 Sznz | 375 |80| 288 | 8 | 379 | 82| 321 |57| 435 61) 417 | Z2 43 Szn; | 717 | 21| 628 | 52| 653 | 172| 6bo | 89| 861 113) 743 | 73 15) Sen; | 49.9 (146) 43.8 |26| 422 | 79| 414 MOLES 27.7 | 8 | 324 105) 128 XuNe | 38.8 | Z) 392 | 19| 352 | 11.8| 31.9 | 78| 212 61) 242 6.8

Table 11

Normalized mean intensity of various negative ions of interest (normalized relative to total number of ions x 10000) 77711100 RVE | Re | ROe | RO | Re | P-IOOe she che ee me che t/2 | type | not known known known known known known known value known value known value 26 CM 719 189) without |261| 281 |101 | 342 292 57.3 28.9| 112 60

Table 12

Parameters ToB-5IM5 18 kV - ions

ToB-5IM5 uses a folded pulse beam of particles (usually С5 or Са) to knock out chemical particles from the surface of materials. Particles formed closer to the impact area tend to become dissociated ions (positive or negative). Secondary particles formed further from the impact area tend to become molecular compounds, usually fragments of much larger organic macromolecules. The particles are then accelerated on their way towards the detector. Since it is possible to measure the "time of flight" of particles from the time of exposure to the detector on the scale of nanoseconds, it is possible to obtain a mass resolution of up to 0.00X atomic mass units (that is, one thousandth of the mass of a proton). Under normal operating conditions, the results of TOE-5IM5 analysis include: a mass spectrum covering all atomic masses in the range

0-10000 a.o.m., the rasterized beam produces distribution maps of any mass of interest on the submicron scale, and depth profiles are formed by removing surface layers by sputtering under the ion beam. Analysis of negative ions showed that the polymer had increased amounts of SMO, SM and MO groups."

Example 12. X-ray photoelectron spectroscopy (XPRS)/electron spectroscopy for chemical analysis (ESSA)

X-ray photoelectron spectroscopy (XPR) (which is sometimes called "E5СА") measures the chemical composition of the upper five nanometers of the surface; in XP5, photoionization energy and energy dispersion analysis of emitted photoelectrons are used to study the composition and electronic state of the surface region of the sample. X-ray photoelectron spectroscopy is based on a single proton entry/electron exit. Soft X-rays stimulate the ejection of photoelectrons, the kinetic energy of which is measured using an electron electrostatic energy analyzer. Small changes in energy are caused by the valence states of atoms during a chemical shift, from which the ejection of electrons originates; thus, the measurement provides chemical information about the surface of the sample.

Table 13

Atomic concentrations (in to) 1111Г1с111о0 1 А ІІ 5

P132 (Square 1)

P132 (Square 2) нны"?:?'?':;BYEYUEISHIIIIIOOOLOOOOVVVVVVVVVVVVVVVVVVVVVVVVVVVVNY

RI32-10 (Square 1)

RI32-10 (Square 2)

P132-100 (Square 1) 62 | 9367 | 09 | 12

P132-100 (Square 2) 61 IYI 369 | 08 | with aNormalized to 10095 detected elements. XP5 does not detect H or Ne.

Table 14

Chemical state of carbon (at 95 C) of ours, 11111 bzrazka7////// |Ї7777777777711117111111111111 1111111

P132 (Square 1

P132 (Square 2) 725 | 49 | tho | 6 ni;

RI32-10 (Square 1) 734 | 42 | 715 | 9

RI32-10 (Square 2)

P132-100 (Square 1) 17127... | 45 | 715 | 9

P132-100 (Square 2) 17125 | 44 | 23 | 9

Table 15

Atomic concentrations (in Ub)a sg 11111115 1Ї147/sma r-1e (Square 1) 75958 2 yushЩ | 379 | 714 | 09 | - district 1e (Square 2) now district 5e (Square 1) 581 | 397 | 714 | 08 | - r-5e (Square 2) 5860 | 397 | 75 | 08 | -

now r-10e (Square 1) r-10e (Square 2) nyninn"ZHINNNNNnnnnnnshshshsh r-50e (Square 1) 5959 2 yЮЩщ | 379 | 714 | 08 | «0 r-50e (Square 2) 594 | 383 | 714 | 09 | "0 nin?" r-70e (Square 1) 63 | 369 | 72 | 06 | "0 r-70e (Square 2) nin r-100e (Square 1) r-100e (Square 2) 605 2 sh | 372 | 714 | 09 | "0 aNormalized to 10095 detected elements. XP5 does not detect H or Ne. "Less than" symbol "«" indicates that accurate quantification could not be performed due to weak signal intensity.

Table 16

Chemical state of carbon (at 95 C) nn necks 1100 Yuzrazka7////// HER! p-1e (Square 1) p-1e (Square 2) now p-5e (Square 1) p-5e (Square 2) ninnpshudyafyeinninninnnshn p-10e (Square 1) p-10e (Square 2) now p-50e (Square 1) 7.29 2 | 45 | 20 | 6 r-50e (Square 2) ninnn"6ЙОИИИООООООООВВВВВВВВВВВВВВВВВВВВВВВВВВВВВВВН r-70e (Square 1) 732 | 45 | 716 | 6 r-70e (Square 2) 795 | 43 | 16 | 6 now r-100e (Square 1) r-100e (Square 2)

Table 17

Analytical parameters

RNI Omapisht 2000

The source of X-rays is monochromatic AiYKa 1486.6 eV 1400x300 μm

Charge correction C15 284 4 ev

XP5 spectra are obtained by irradiating the material with a beam of aluminum or magnesium X-rays while simultaneously measuring the kinetic energy (KE) and the number of electrons leaving the upper 1-10 nm of the analyzed material (see analytical parameters, Table 17). The KhRZ5 method is highly specific to the surface due to the short range of photoelectrons that are excited in the solid. The energy of the photoelectrons leaving the sample is determined using a concentric hemispherical analyzer (CNA) and this gives a spectrum with a series of photoelectron peaks.

The binding energy of the peaks is characteristic for each element. Peak areas can be used (with appropriate sensitivity factors) to determine the composition of surface materials. The shape of each peak and the binding energy may vary slightly due to the chemical state of the emitting atom.

Thus, CHRB5 can also provide information about chemical bonds. XP5 is not sensitive to hydrogen or helium, but can detect all other elements. ChRO requires ultra-deep vacuum (UDV) conditions and is widely used for surface analysis of polymers, coatings, catalysts, composites, fibers, ceramics, pharmaceutical/medical materials, and materials of biological origin. The data of HRO are presented in tables 13-16.

Example 13. Analysis by Raman spectroscopy

Raman spectra were obtained for the surface of fibers from samples: РИ1И32, Р1Т32-100, Р-Те and Р-100е6.

Measurements were carried out using the spectrometer "HarKat" .-M. A NeMe laser (wavelength 632.8 nm) and a grating of 600 g/mm were used for measurements. Measurements were carried out confocally using the backscattering geometry (1802) under the ОІutry5 ВХ40 microscope.

The samples had a Raman spectrum typical of cellulose.

Example 14. Surface analysis by scanning probe microscopy (SPM) using an atomic force microscope (AEM)

The purpose of this analysis was to obtain atomic force microscope (AEM) images of the samples presented in Tables 18 and 19 to measure surface roughness.

Scanning probe microscopy (SPM) is a branch of microscopy that forms images of surfaces using a physical probe that scans a sample. Surface images are obtained by mechanically moving the probe in a raster scan of the sample, sequentially, and recording the probe-surface interaction as a function of probe position. An atomic force microscope (AEM) or scanning force microscope (SEM) is a type of very high-resolution scanning probe microscope that has demonstrated resolution of nanometer-sized fractions more than 1000 times better than the diffraction limit of light. The probe (or the sample under the stationary probe) is usually displaced using a piezoelectric tube. Such scanners are designed for accurate movement in any of the three perpendicular axes (x,y,2). Adhering to the raster, the sensor data form an image of the probe-surface interaction. Sensor feedback is used to maintain the probe at a constant force or distance from the surface of the object. For atomic force microscopy, the sensor is a position-sensitive photodetector that registers the angle of deviation from the laser beam focused on the top of the cantilever.

Table 18

Results of determination of roughness for samples irradiated by gamma radiation

Table 19

Results of determination of roughness for samples irradiated with an e-beam

AEM images were obtained using Mapobsore PP Oitepviop 5000 (Sidya! Ipbigitepiv,

Zapia Vagraga, Saiyogpia, BOTH). The instrument was calibrated against a correlated MI5ZT standard with an accuracy better than 295. MapoRgore silicone tips were used. Used image processing methods that include automatic alignment, plane fit, or collapse.

One random area on the upper part of a single fiber with an area of 5x5 μm was visualized.

Perspective (3-0) images of these surfaces are included at an enlarged vertical scale as indicated in the graphs (FIGS. 29A-29E). Roughness was analyzed and expressed in: (1) root mean square roughness, KM5; (2) average roughness, Ka; and (3) maximum height (peak-to-dip), Klah. The results are summarized in Tables 18 and 19.

Example 15 - Determination of the crystallinity of the irradiated material by the X-ray diffraction method

X-ray diffraction (XRD) is a method by which a crystalline sample is irradiated with monoenergetic X-rays. The interaction of the lattice structure of the sample with these X-rays is recorded, which gives information about the crystal structure that is exposed to the radiation. The obtained characteristic "print" makes it possible to identify the crystalline compounds present in the sample. Using the analysis of correspondence throughout the pattern (She

Kiwimeii Keipetepi) it is possible to carry out quantitative analyzes of samples containing more than one crystalline compound.

Table 20

KKO data including domain size and 9o crystallinity

Each sample was placed on a holder with a zero background and placed in a diffractometer Riir5 RUU1800 using radiation radiation. Scans were then performed in the range from 5" to 50" with a step size of 0.05" and a counting time of 2 hours in each case.

After obtaining the diffractograms, the phases were identified using the Romjaeg Oiigasiuop Riie published by Ipiegpaiop! Sepige Tog riytasiop Weight. In all samples, the identified crystalline phase was cellulose - Ia, which has a triclinic structure.

A distinctive feature for 20 samples is the peak width, which is related to the size of the crystal domain.

The experimental peak width was used to calculate the domain size and percent crystallinity presented in Table 4.

Percent crystallinity (Xe 90) is defined as the ratio of the area of the crystalline phase to the total area under the X-ray diffraction peaks

Ho - p. x10090 (4, SHI A ) where

Ac is the area for the crystalline phase,

Aa is the area for the amorphous phase,

He is the percentage of crystallinity.

To determine the percent crystallinity of each sample, it was necessary to first determine the amount of the amorphous phase. This is done by estimating the area of each diffractogram that can be attributed to the crystalline phase (which is represented by the sharper peaks) and the non-crystalline phase (which is represented by the broad humps below the pattern and centered at 22" and 387).

A systematic process was used to minimize error in these calculations due to broad crystal peaks as well as high background intensity. First, a linear background was applied, and then it was canceled. Second, each of the two Gaussian peaks centered at 22" and 387 with a width of 10-12? was matched with the humps under the crystalline peaks. Third, the area under the two broad Gaussian peaks and other parts of the pattern were determined. Finally, we calculated percent crystallinity by dividing the area under the crystalline peak by the total intensity (after background subtraction).The domain size and 95 crystallinity of the samples as determined by X-ray diffraction (XRD) are presented in Table 20.

Example 16 - Analysis of irradiated materials by porometry

Mercury analysis of pore size and pore volume (Table 21) is based on passing mercury (a non-wetting liquid) into a porous structure at strictly controlled pressures. Since mercury does not wet most substances and does not penetrate the pores spontaneously due to capillary action, it must be driven into the cavities of the sample by applying external pressure. The pressure required to fill the cavities is inversely proportional to the pore size. Only a small amount of force or pressure is required to fill large cavities, while much higher pressure is required to fill very small pores.

Table 21

Analysis of the size and volume of pores using mercury perometry

I nn iii iii iii viv UDO S nik ovi ovio wk ND se Oriv iii

General General SerSlNy Average Average SkUbeMnA evnu penetrable area pameter o sevenmeter o ludmeter o TISTNT, (obstructive) Porosity

The sample goes to the support of the opera ovrybyoo! юЧИПОО ЕМ poro (volume) osplosha; OMA) lb 0/Density (5) imli imlyai (μm) imkm. (μm) KVDuyM o homl)

RIZI ol it: PISHE Pa u PISHE VEK V EK TSN EE 14785 057Mez

RIUTI ze 3.1 46.46 5 MB 106 00001614 phases what ah

RI Nyuyu 5.3983 poza 00 ik 2165493 II BYAZ ZI

REAK 32506 ovo 025 mk 172410 ich chu 002497 12916 830577

RIZI-YUH OO 3787 dya Oh im ZL Pa 2199 sh1x Za037 la okom AJ. poeib fields 1255 Tov

AIZIA 12514 10320 003 tons I give and 0.1765 Yes Yes

A1355-186 recho MNEzOa 0 455906 mon pe gift ryh Ta 13 Z. 8265 0 aT058 I 1.232229 lo TA tovi zb132-105 A ha 00 lb-nob pany mon 1.3457 5 Ta e132-1 25143 0706 ZAT NYA Olyu po?kh 0Olo ge: 775

IA Nyotskh 4 al MULU KG is a supporting BIV 50109 5132-30-05 more 0b3 t for ONKO Kzhyuv 0.L1o55 TO veze chUB132 29920 5447 7e3675 0Omo ve 02775 Ka» obo

URA ZD zno at plaid zno ol ToZoh 0 yabIinschya

HU ZT ZE 005 Yua 03570 41159 by TOR 53.53

A-1e 19535 sound ZAZ ZHE ZHKY 1130 0359 Togo To KyuZ

A-ve oo BAH Od 59 0.053530 12117 pola BZh? TK K5

A-Ie raku and UA C in oo obo 0 PE I Ne yaks

А-5ve by order 1399 0003577 14671 0075 b-ie 24383 7.52 88.5531 pom 12861 031 TKU: 76616

O-yae Z 5400 AK 00225 103 pi Ba BI b-e zani izheh pam pl 15355 pa Shopping Center; then pezk s-yade zr vm ZvBanNh 0 0o315 ieyui 0294 Ba 00tyaozo

В-16 Background ВХ Я ВЯ 0239315 298 0 ИВ cha Zach

R-yae te 1212 bai 1235353 zob 00 Bio och rn vve ENN A UK PY Z M ZAaoTya 17475 At ODU t4906 Za tiho

RAM 59 TK; 040 7Kt; 15823 hall 00 plze Taz za7016 r-1vaye ZL EN el INA ZAES NAME 10740 zt 0 Oeya TV YABLVEO plo Zam zr Ya2shschU TV Ol 15745 za ti16o

B-ie NDI paz ah PAK ZO pu Gu ZB sh--e 03914 000337 541991 boru zeyue osyakh 16052 ZK sh-0e pato 0349 13360 p 47873 Pov Tlo ZO ev 5-30 Ov zaz tsa Om pla pln 15585 IC sh-a0e P .AND? teh 1ULIBM oyu pata away for ZA i-nk 081 5 zb OO TOZEV 0.47 i.MO5 ahmU vei I ey 17631 aa opa Io P.775i rya? 1435

Ka-1e 00720 V Z 5ler om O1587 field 575 526 visa 334 15595 425 ore about pot r ZA le

BI-10e position Mother-in-law 433625 OO pi ED zheya TO but 5i-30g AT Io ZYA152 Azh po ptak Ba? ziyach

Bi-khaye bab 18335 3755 pom 01A pe 15545 Ba vv eNnshe poide da 14 ЗХ НАШУ OPN Ptah 5 Hall

AshoRoge 9520 can reach a maximum pressure of 414 MPa or 60,000 psi. inch abs It has four low-pressure stations for sample preparation and macropore data collection at pressures from 0.2 psi. inch abs (1.4 kpa) to 50 lb/sq. inch abs (345 kpa). It has two high-pressure chambers that collect data at pressures from 25 psi. inch abs (172 kpa) to 60,000 lb/sq. inch abs (414 MPa). The sample is placed in a cup-shaped device called a penetrometer, which is connected to a glass capillary rod with a metal coating. As mercury penetrates the cavities in and around the sample, it moves down the capillary rod. The loss of mercury from the capillary rod leads to a change in capacitance. The change in electrical capacity during the experiment is converted into the volume of mercury, based on the known volume of the rod of the used penetrometer. A variety of penetrometers are available with different bowl (sample) and capillary sizes to accommodate most sample sizes and configurations. In Table 22, below,

defined key parameters calculated for each sample.

Table 22

Definition of parameters

C - - The total volume of mercury penetrating during the experiment. It can agal penetrating : H : sample porosity and sample compression volume agal pore area and cylindrical pore shape filling pressure, typically 0.5 psi. inch abs (3.45 kPa) pressure, typically 60,000 psi. inch abs (414 MPa)

Example 17 - Analysis of particle size of irradiated materials

The method of determining particle size by light scattering is based on the Mie theory (which also includes the Fraunhofer theory). Mie's theory predicts the relationship between intensity and angle as a function of the size of the spherical scattering particles, provided that the other system variables are known and held constant. These variables are the wavelength of the incident light and the relative refractive index of the sample material. The application of Mie's theory provides detailed information on particle size.

Table 23 summarizes the size of the particles when used as parameters of median diameter, average diameter and modal diameter.

Table 23

Particle size during laser radiation scattering (dry sample dispersion)

MKM MKM

Particle size was determined by laser scattering (dry sample dispersion) using a Maimegp Maziegwieg 2000 using the following conditions: feed rate: ZB, disperser pressure: 4 bar, optical model: (2.610, 1.000), 1.000,

The appropriate amount of the sample was fed to the vibrating tray. The feed rate and air pressure were adjusted to ensure that the particles were properly dispersed. The key component is choosing an air pressure that breaks up the agglomerates but does not compromise the integrity of the sample.

The amount of sample required varies with particle size. As a general rule, samples with fine particles require less material than samples with large particles.

Example 18 - Analysis of the surface area of irradiated materials

The surface area of each sample was analyzed using the Misgotegis5 ABAR 2420 system

Asseiegaieuy Zipase Agea apa Rogovziteigu Zuziet. The samples are prepared by primary degassing for 16 hours at 40°C. Next, the headspace (both warm and cold) with helium is calculated, and then the sample tube is again evacuated to remove the helium. Data collection begins after this second evacuation, and it is to set a set pressure that controls how much gas is dosed into the sample. At each set pressure, the amount of gas adsorbed and the true pressure are determined and recorded. The pressure inside the sample tube is measured using a pressure sensor. Additional gas doses continue until the set pressure is reached and ensuring equilibration The amount of gas adsorbed is determined by summing over multiple doses to the sample The pressure and amount define the gas adsorption isotherm and are used to calculate a number of parameters including the surface area of the BET (Table 24).

Table 24

Generalized representation of surface area by gas adsorption e-0505..77777771711111171Г111111111111111711111ои вей С7Ї7711111111111111111111111111111111111Г111111111111111111114961

HEPERYA IS: Z PON KON KON TKU: PI

The BET model for isotherms is a widely used theory for calculating the specific surface area. The analysis involves determining the monolayer capacity of the sample surface by calculating the amount needed to cover the entire surface with one close-packed layer of krypton. To determine the total surface area, the capacity of the monolayer is multiplied by the cross-sectional area of the sample gas molecule. The specific surface area is the surface area of a sample aliquot divided by the mass of the sample.

Example 19 - Determination of fiber length in irradiated materials

Fiber length distribution testing was performed in three parallels on the presented samples using the Tesprar MogEgi GVO1 system. The average length and width of the fibers are presented in Table 25.

Table 25

Generalized presentation of data on the length and width of lignocellulosic fibers

Arithmetic Mean Statistically

And sample average length, adjusted average | Width (mm) weighted length weighted (micrometers) (μm) length (mm) length (mm)

Example 20 - Ultrasonic treatment of irradiated and non-irradiated millet

Millet was crushed according to example 4. Millet was treated only with ultrasound or irradiation with gamma rays at a dose of 10 or 100 Mrad, followed by ultrasound treatment. The obtained materials correspond to 5132-VK (unirradiated), 5132-10-BE (10 Mrad and ultrasonic treatment) and 5132-100-

VC (100 Mrad and sonication) as shown in Table 1. Sonication was performed for each sample for 30 minutes using 20 kHz ultrasound from a 1000 W horn under recirculation conditions. Each sample was dispersed in water at a concentration of approximately 0.10 g/ml.

In FIG. 30 and 31 present a device used for ultrasound treatment. The device 500 includes a transducer 502 connected to a booster 504 connected to a horn 506 made of titanium or a titanium alloy. The horn, which has an insulation 510 made of MITOMF around its perimeter on its processing side, forms a liquid-tight seal with the processing cell 508. The processing side of the horn is immersed in a liquid, such as water, in which the sample to be processed is dispersed. ultrasound treatment. The pressure in the cell is monitored using the pressure sensor 512. During operation, the sample is pushed by the pump 517 from the container 516 through the processing cell and is treated with ultrasound. After sonication, the sample is collected in container 520. The process may be reversible in that the contents of container 520 may be sent through the processing cell and collected in container 516. This process may be repeated several times until the desired level of sample processing is achieved.

Example 21 - Scanning electron microscope images of non-irradiated millet compared to irradiated and irradiated and sonicated millet

Millet samples for scanning electron microscope images were placed on carbon tape and sputter-coated with gold (70 seconds). Images were obtained using a scanning electron microscope with field emission of UEOI. 6500.

In FIG. 32 is a scanning electron microscope image, taken at 1000X magnification, of fibrous material obtained by crushing millet on a rotary knife cutter and then passing the crushed material through a 1/32 inch (0.08 cm) sieve.

In FIG. 33 and 34 show images obtained using a scanning electron microscope at a magnification of 1000X of the fibrous material of FIG. 32 after exposure to gamma rays with a dose of 10 i

100 Mrad, respectively.

In FIG. 35 presents pictures obtained with the help of a scanning electron microscope at a magnification of 1000X, the fibrous material of FIG. 32 after 10 Mrad irradiation and ultrasound treatment.

In FIG. 36 presents images obtained using a scanning electron microscope at a magnification of 1000X, the fibrous material of FIG. 32 after 100 Mrad irradiation and ultrasound treatment.

Example 22 - Fourier transform infrared spectra (ET-IR) of irradiated and non-irradiated kraft paper

The ET-IR analysis was carried out on Misoieitras 400. The results indicate that samples P132, P132-10, RIZ32-100, P-1e, P-5e, P-10e, P-30e, P-7O0e and P-100e correspond to cellulose-based material.

In FIG. 37 shows the infrared spectrum of the kraft cardboard shredded according to example 4, and FIG. 38 presents the infrared spectrum of the kraft paper of FIG. 37 after exposure to gamma radiation at a dose of 100 Mrad. The irradiated sample shows an additional peak in region A (centered at approximately 1730 cm"), which is not detected in the unirradiated material. It should be noted that an increase in the absorption level of carbonyl groups at "1650 cm" was detected in samples from P1T32 to P132-10 and up to P132-100. Similar results were observed for samples P-1e, P-5e, P-10e, P-30e, P-7Oe and P-1006.

Example 23 - Proton and carbon-13 nuclear magnetic resonance spectra (H-NMR and "ZS-NMR) of irradiated and non-irradiated kraft paper

Sample preparation

Samples P1I32, P1I32-10, RTI32-100, P-1e, P-5e, P-10e, P-ZOe, P-70e and P-100e were prepared for analysis by dissolving in DMSO-IV with 295 tetrabutylammonium fluoride trihydrate. Samples exposed to lower exposure levels were essentially less soluble than samples exposed to higher exposure levels. Unirradiated samples formed a gel in this mixture of solvents, but heating to 60 "С allowed peaks in the NMR spectra. Samples exposed to higher levels of irradiation were soluble at a concentration of 1095 wt./wt.

Analysis

The H-NMR spectra of the samples at a concentration of 15 mg/ml showed a distinct, very broad resonance peak with a center at the level of 16 ppm (FIG. ZBA-38)). This peak is characteristic of the exchange proton -OH for enols, and it was confirmed by the "420 oscillation". Model compounds (acetylacetone, glucuronic acid and ketogulonic acid) were analyzed and confirmed that this peak was indeed an exchangeable enol proton. This enol peak presented was highly sensitive to the effects of concentration, and the authors of this invention were not able to conclude whether this resonance was due to the enol or perhaps the carboxylic acid.

The proton resonances of the carboxylic acids of the model compounds were similar to the proton resonances observed in the case of treated cellulose samples. These model compounds had an upwardly shifted region of 5-6 m.h. The preparation of P-100e in higher concentrations (1095 wt./wt.) led to a sharp decrease in the shift of the region to the region where the resonances of the carboxylic acids of the model compounds (6 ppm) were detected (FIG. 38M). These results lead to the conclusion that this resonance is unreliable to characterize this functional group, however, the data indicate that the number of exchangeable hydrogen atoms increases with increasing sample exposure. Also no vinyl protons were detected.

C-NMR spectra of the samples confirm the presence of the carbonyl group of the carboxylic acid or a carboxylic acid derivative. This new peak (at 168 ppm) is not present in the untreated samples (FIG. 38K). The long-delay C-NMR spectrum provided the possibility of quantitative determination of the signal for P-100e (FIGS. 381I-38M).Comparison of the integration of the carbonyl group resonance with the resonances at about 100 (C1 signals) indicates that the carbon ratio of carbonyl groups to C1 is 13.8, or about 1 carbonyl group for every 14 glucose units Chemical shift at 100 ppm correlates well with glucuronic acid.

Titration

Samples P-100e and P1132-100 (1 g) were suspended in deionized water (25 ml). The alizarin yellow indicator was added to each sample while stirring. P-100e was more difficult to wet.

Both samples were titrated with 0.2 M Mahon solution. The result was barely noticeable and was confirmed using pH paper. The initial pH value of the samples was 4 for both samples. For P132-100, 0.4 milliequivalent of hydroxide was required, which gives a molecular weight for the carboxylic acid of 2500 a.m. If 180 a.o.m. is used for the monomer, then this indicates that there is one carboxylic acid group for 13.9 monomer units. Similarly, P-100e required 3.2 milliequivalents of hydroxide, which, according to calculations, corresponds to one carboxylic acid group for every 17.4 monomer units.

Conclusions

The C-6 carbon of cellulose appears to be oxidized to a carboxylic acid (a derivative of glucuronic acid), and this oxidation is surprisingly specific. This oxidation is consistent with an IR band that increases upon irradiation at 1740 cm", corresponding to an aliphatic carboxylic acid. The titration results are consistent with quantitative "3CO-NMR. The increased solubility of the sample at higher levels of irradiation correlates well with the increasing number of carboxylic acid protons.

The proposed mechanism of degradation of "C-6b-oxidized cellulose" is presented below in scheme 1.

Scheme of n a g s-ezkEnelenia nelvalose

Wife.

BUT - -ma a; o t n m OH and sht I

M ma i VI i le: NI n yzhnanya I

Id) soch E che i no od EZNYI "cho shk M ky v na. es Yake no n n zh - M no che! pe Phi A; "KA

Example 24 - Combination of pretreatment with an electron beam and ultrasound

Millet is used as a raw material and is crushed with a cutting device with a rotating Mipsop knife into a fibrous material. The fibrous material is then spread evenly over an open tray made of tin with an area of more than 500 in: (12.7 m). The fibrous material is distributed to a thickness of approximately 1-2 inches (2.5-5 cm) in an open tray. The fibrous material can be distributed in plastic bags at lower radiation doses (less than 10 Mrad) and left exposed on a metal tray at higher radiation doses.

Then individual samples of fibrous material are subjected to successive doses of electron beam irradiation until the total dose is 1, 2, 3, 5, 10, 50 and 100 Mrad. Some samples are maintained under the same conditions as other samples but not irradiated to serve as controls.

After cooling, the irradiated fibrous material is sent for further processing using an ultrasonic treatment device.

The ultrasonic treatment device includes a transducer connected to a booster connected to a horn made of titanium or a titanium alloy. The horn, which has insulation made of MITOMF) around its perimeter on its processing side, forms a liquid-tight seal with the processing cell. The processing side of the horn is immersed in a liquid, such as water, in which the sample to be sonicated is dispersed. The pressure in the cell is monitored using a pressure sensor. In operation, each sample is pumped through a processing cell and sonicated.

To prepare the irradiated fibrous material for sonication, the irradiated fibrous material is withdrawn from any container (eg, plastic bags) and dispersed in water at a concentration of approximately 0.10 g/ml. Ultrasound treatment is carried out on each sample for 30 minutes, using ultrasound with a frequency of 20 kHz from a 1000 W horn in recirculation conditions. After ultrasonic treatment, the irradiated fibrous material is collected in a container.

This process can be repeated many times until the desired level of processing is achieved, based on the monitoring of structural changes in the millet. Next, some irradiated samples are kept under the same conditions as the other samples, but not sonicated, to serve as controls. In addition, some samples that were not irradiated or not treated with ultrasound also serve as controls. Thus, some controls are not treated, some are only irradiated, and some are only treated with ultrasound.

Example 25 - Testing of pre-treated biomass using microorganisms

Specific lignocellulosic materials, pretreated as described herein, are analyzed for toxicity to common strains of yeast and bacteria used in the biofuel industry for the fermentation step of producing ethanol. In addition, sugar content and compatibility with cellulase enzymes are investigated to determine the efficiency of the processing process.

Testing of pre-treated materials is carried out in two phases as follows.

Phase 1. Toxicity and sugar content

The toxicity of pre-treated grasses and paper raw materials is determined in the yeast Zasspagotusez segemisiae (wine yeast) and Rispia iiirii5 (ATSS 66278), as well as in the bacteria 7utotopap tobi (ATSS 31821) and Siozygidiit (eptosePysht (ATSS 31924). Growth studies are carried out for each of the organisms to determine the optimal time of incubation and sampling.

Each raw material is then incubated, in two parallels, with 5. sehemiziaye, R. 5iiryniye, 7. tobiiv5 and S.

Ipeptoseisht in a standard microbiological environment for each organism. For two yeast strains, 5. segemiziae and R. viiryih, UM broth is used. For 7. torbii5 use medium CM and for S. IPeptosePyshSht use medium CM4. For comparison, a positive control is used, with the addition of pure sugar, but without raw materials. In the incubation process during the 12-hour period, a total of five samples are obtained at time points 0, 3, 6, 9 and 12 hours and analyzed for viability (counting in cups for 7. tobia5 and direct counting for 5. segemiviae) and ethanol concentration.

The sugar content in raw materials is determined using high-performance liquid chromatography (HPLC), using a 5podeh"M zudag 5РО810 column, or a Viogai Atipeh? NRH-87R column.

Each raw material (approximately 5 g) is mixed with reverse osmosis (RO) water for 1 hour. The liquid part of the mixture is removed and analyzed for the content of glucose, galactose, xylose, mannose, arabinose and cellobiose. The analysis is carried out according to the protocol of YuOeieptipaiiop oi Bigisichga! Sagropuagaie5 apa Chidpip ip Viota5x5 from Maiopai! Vioepegdu Sepieg.

Phase 2. Compatibility of cellulase

The raw material is tested, in two parallels, using the commercially available AssayeGaze "m 1000, which contains an enzyme complex that reduces lignocellulosic biomass to fermentable sugars, at the recommended temperature and concentration in an Erlenmeyer flask. The flasks are incubated with moderate shaking at approximately 200 rpm . for 12 hours. During this time period, samples are obtained every three hours at time points 0, 3, 6, 9 and 12 hours to determine the concentration of reducing sugars (Nore apa Oeap, Vioitesi ., 1974, 144:403) in liquid part of the flasks

Example 26 - Analysis of sugar concentration using HPLC 13 samples were analyzed for sugar concentration (HPLC) and toxicity relative to C microorganisms (Rispia viii5, zhasspagotusez segemiviae and 2utopakh thorium). Table 26 presents the equipment used for these experiments. Tables 27 and 28 list the sugars (including manufacturer and lot number) used to prepare the HPLC standard and the protocol used to prepare the HPLC standard, respectively.

Table 26

The equipment used in the experiments

Table 27

Sugars used in HPLC analysis

Viospetics

Table 28

Preparation of HPLC standards

Desired concentration Volume of solution K Volume of water Total volume (ml) (mg/ml) P U sugar Maporige (ml) mg/ml mg/ml mg/ml mg/ml mg/ml

Standard for 18.75 ml of 31 25 BU verification 1.5 mg/ml | with a concentration of 4 mg/ml "

Analysis

Each sample (1 gram) was mixed with reverse osmosis water at 200 rpm. and 50 C during the night. The pH value of the sample was adjusted to 5-6 and it was filtered through a 0.2-μm syringe filter. Before analysis, the samples were stored at -20"C to maintain the integrity of the samples.

Observations made during sample preparation are presented in Table 29.

Table 29

Observations during the preparation of samples for HPLC

Used

Loose, difficult to mix

The pH value is low,

RI132-100 1 20 3.19 is difficult to bring to 5.0 using 1On. Masn

RIZ32-08 7705 | 5 юЮщЩщч5Б |64/! Fri and Thu Mon Thu

AI132 11115155 |.

A132-10 17111161 11491111

А132-100 11161531 p I PO PONYA POLO va132 11116155 Her va132-10 11615111 8a132-100 ninninshiisnyshekhly shi va132-10-05 705 | sh5B |5sh2 | (К ва132-100-05 7095 | 5 YushЩщ5Б /|497/ po I PO PON PO

Hu5132 1116 15631111

Mu8132-10 11665111

Му8132-100 Нины ши ССС По "рН of these samples was proved using 1n. Mahon.

The standards were freshly prepared from a 4 mg/ml stock solution of 6 combined sugars: glucose, xylose, cellobiose, arabinose, mannose, and galactose. The mother liquor was prepared by dissolving 0.400 grams of each sugar in 75 ml of Maporyge water (filtered through a 0.3-micron filter). After dissolution, the mother liquor was diluted to 100 ml using a volumetric flask and stored at -

"C. Working solutions of standards at a concentration of 0.1, 0.5, 1, 2, and 4 mg/ml were obtained by serial dilution of the mother solution with Maporyge water. In addition, a standard for verification at a concentration of 1.5 mg was also prepared from the mother solution / ml.

Concentrations of sugars were analyzed according to the protocol of YuOeiegtipaiop oi bBigisiiga! Sagtopuagaiez ip

Viotaz5 (MKEI Viotav55 Rgodgat, 2006), and this protocol is fully incorporated herein by reference. ZNOBEKH BYSAK 5ROV10 SOY MM with an evaporative light scattering detector was used. A check standard (standard at a concentration of 1.5 mg/ml) was analyzed after every 8 injections to ensure that the integrity of the column and detector was maintained throughout the experiment.

The coefficient of variation of the standard curve (value K?) was at least 0.989, and the concentration of the test standards was within 10 95 of the true concentration. HPLC conditions were as follows.

Table 30

Parameters HPLC filter and degassed

Column temperature evaporator temperature 11020, temperature

Atomizer detector temperature 902C "In initial tests, it was shown that better separation was observed when using Maporyge water in the mobile phase instead of a 15/85 acetonitrile:water mixture (the manufacturer does not recommend using more than 20 95 acetonitrile in this column).

The results

The results of HPLC analysis are presented in Tables 31, 32 and 33.

Table 31

Sugar concentration expressed as mg/ml and mg/g of extract

Xylose Arabinose Glucose Galactose Mannose (see Cellobiose

I sample MmM-150 MM--150 MmM--180 (see glucocog I MM - r С5НиоО5 С5НиоО5 SeNiGov glucose) mo/mlMo te Si2NggOchi

Monosaccharide | Monosaccharide | Monosaccharide | mg/ml:mg/g I Disaccharide 11111 dmg/mlo|msg mg/ml o|mg/g mg/ml o|mgg mg/ml mg/g |mg/ml mg/g |mg/ml mg/g

RGS

R-iz2 | because |000| 000 | 000 000 | 000 |о000ц|000| 00010001 000 рР-132-10| 0.00 0000000 | 000 | 0.34 | 860 | 000sh|000| 000 10.0010093 tyu (095706 obo ooo lake howling

PHELIMNY DUST FIELD CAPTURE COLUMN HORSE HORSE HORSE HORSE HORSE HORSE NOAH a-132 12.66| 0.34 |5.04| 092 113.76 000 7132-10 T6.04 tab! 0001. by | ою ою овл зве сити ооо оо ов |v on a-132- tive 0990573 089 yav 035 vaz 0001000 000 ooo oo a-132- 100-085 0.37 | b22 | 0.35 | 5.90 | 0331543) 040 | 6.701 0.39

A 11111

A-132 20.39 0.00 | 0.00 | 1.08 | 16.22 16.02| 000 Yu-KB

A-132-10 17.87| 000 | 000 | 000 | 000 1000 Sh|0.00 0.37 |5.52 1000 t Pebten oo oyu o le (in Geo o o (of

KEN SAWS PON POLON COLUMN HORSE KON KON KON HORSE KON HORSE NOAH muz-132 049 | 741 | 041 | 65,039 Sh|590,000 0001000 |000,000 of the above mentioned Cedo ov ve ob

Table 32

The concentration of sugar, expressed in 95 of paper and o

Sugar concentration (5 dry RIZ2 P132-10 P132-100 RI32-08 samples) chelobiose //--:777777177171000 |... 081 | 072 2 Sh | 000 glucose 77777711 000 | ...yuD086 | 067 | 056 xylose. ///7777777777771771711000 | ..yuYyuD000 2 | YuyuYUCROO | 058 galactose ///////7777777771771711000 |... ю000 2 | Yu.S 000 oarabinosai//-/--:777777171111000 |... 000 | .--у000 2 | B fuTsbo manosai 77777777 17111000 | ...yuD000 2 2 yu | yush(000 | 000

Table 33

Sugar concentration expressed as 95% of the entire sample

Sugar concentration (90 A132- | A132- za132- | Ba132- | 5Сс132- | BS132- M5132- dry 7132) 30 | h00 96192, 30 | 00 10-05 |100-05 791921 70 samples) cellobiose 0.00 0.00 | 1.22 | 0.00 | 0.00 | 0.67 | 000 | 0.65 | 000 | 0.00 glucose 1.62) 0.00 | 0.52 | i27 | 1.60 | 0.61 / 0.54 | 0.59 | 0.59 | 10 oxylose (2.04 1.79 | 1.61 | 0.59 | 0.75 | 0.00 0.57 | 000 | 0.74 | 0.85 galactose 0.58 0.00 | 0.00 | 0.50 | 0.52 | 0.00 / 000 | 0.54 | 000 | 051 arabinose 0.00 0.00 | 0.00 | 0.57 | 0.62 | 0.55 | 0.65 | 0 .62 | 0.62 | 0.60 (mannose 1.60) 0.55 | 0.00 | 1.38 | 1.47 | 0.83 | 000 | 0.67 | 000 | 0.76

Example 27 - Toxicity study

Twelve samples were analyzed for toxicity against a panel of three ethanol-producing cultures. In this study, glucose was added to the samples to determine the difference between starvation of the cultures and toxicity of the samples. The thirteenth sample was tested for toxicity relative to

Rispia "iiiriy5. A summary of the protocol used is given in Table 32. A description of the chemical reagents and equipment used in toxicity testing is presented in Tables 34-36.

Table 34

Toxicity testing conditions

Zasspagotusev's variable butotopav is a genus of MAVI virians. tobiiv is segregating U-7124

ATSS 31821 ATSS 24858

Inoculation volume (ml)

Incubation temperature

Rotational speed

The volume of an Erlenmeyer flask

The volume of the medium

Total incubation time (hours)

Ethanol analysis (hours) 24, 30, 36 24, 30, 36 24, 36, 48 24, 36 24, 36 24, 48

Table 35

Reagents used for toxicity testing

Nitrogen base and

Yeast extract (used for Wesyup biskippop 288620 4026828

Z. segemiziae

Yeast extract (used for R. Vesyup biskippop 212750 7165593 viiriiiv and 7. tobiiv

Table 36

Components of U5I used in the study in a rotating flask

Testing was performed using three microorganisms as described below. zasspagotusez segemiviae ATOS 24858 (Ategisap Toure Siyige SoiPesiop)

The culture of 5. segemiziae on a slanted medium was obtained from a rehydrated lyophilized culture obtained from ATSS. Part of the culture material on the cut medium was streaked onto UM agar broth at a concentration of 20 g/l (pH 5.0) and incubated at 30 "C for 2 days. In a 250 ml Erlenmeyer flask containing 50 ml of medium ( 20 g/l of glucose, 3 g/l of yeast extract and 5.0 g/l of peptone, pH 5.0), one colony was inoculated from a UM dish and incubated for 24 hours at 25 °C and 200 rpm. After growth for 23 hours, samples were taken and analyzed for optical density (600 nm in UV spectrophotometer) and purity (Gram stain). Based on these results, one flask (called the seed flask) was selected with an OD of 14.8 and pure Gram stain was selected to inoculate all flasks tested.

Test containers were 500 ml Erlenmeyer flasks containing 100 ml of the sterile medium described above. All flasks were autoclaved at 121°C and 15 psi (0.1 MPa) prior to addition of test materials. Test materials were not sterilized because autoclaving alters the contents of the samples. Test samples were added at the time of inoculation (rather than before) to reduce 1 ml (1 95 vol/06) of inoculum was added to each flask to supplement the samples tested.The flasks were incubated as described above for 36 hours.

Risnia viiriiv MAV x-7124 (АН5 Sipyshte SoPesiyop)

The culture of R. zir on a slanted medium was obtained from a rehydrated lyophilized culture obtained from AK5 Siyige SoPesiop. Part of the culture material on the cut medium was streaked onto UM agar broth at a concentration of 20 g/l (pH 5.0) and incubated at 30 "C for 2 days. In an Erlenmeyer flask with a volume of 250 ml containing 100 ml of medium ( 40 g/L glucose, 1.7 g/L nitrogenous yeast base, 2.27 g/L urea, 6.56 g/L peptone, 40 g/L xylose, pH 5.0), inoculated a small amount of material from a cup and incubated for 24 hours at 25 °C and 125 rpm. After growth for 23 hours, samples were taken and analyzed for optical density (600 nm in a UV spectrophotometer) and purity (Gram stain). Based on these results, one flask (called the seed flask) with an OO of 5.23 was selected and pure Gram stain was selected to inoculate all flasks tested.

Test containers were 250 ml Erlenmeyer flasks containing 100 ml of the sterile medium described above. All flasks were autoclaved at 121°C and 15 psi (0.1 MPa) and filter-sterilized (0.22-μm filter) medium was added to the flasks prior to addition of test materials. Test materials were not sterilized because autoclaving alters the contents of the samples. , and filter sterilization is not suitable for sterilization of solids. Test samples were added during (rather than before) inoculation to reduce the possibility of contamination. To supplement the test samples, 1 mL (1,905 v/06.) of material was added to each flask of the seed flask. The flasks were incubated as described above for 48 hours. 7utotopazus torbia ATOSS 31821 (Ategtisap Ture Siyige)

The culture of 24. thorium on a slanted medium was obtained from a rehydrated lyophilized culture obtained from ATTS. Part of the culture material on the cut medium was streaked onto OME cups (glucose 20 g/l, yeast extract 10 g/l, agar 20 g/l, pH 5.4) and incubated at 30 "С and 595 СО" for 2 days. In a 20-ml tube with a screw cap containing 15 ml of medium (25 g/l glucose, 10 g/l yeast extract, 1 g/l MaZO» 7H2O, 1 g/l (MNa2650x, 2. g/l

KnegRO", pH 5.4), one colony was inoculated and incubated for 24 hours at 30 "C without rotation.

After growth for 23 hours, samples were taken and analyzed for optical density (600 nm in

UV spectrophotometers) and purity (Gram's stain). Based on these results, one tube (00 1.96) was selected to inoculate another seed flask. Another 125 ml seed flask containing 70 ml of the medium described above was inoculated with 700 μl (1 95 v/v) and incubated for 24 h at 30 °C with shaking. After growth for 23 h, samples were taken and analyzed their relative optical density (600 nm in a UV spectrophotometer) and purity (Gram stain).Based on these results, one flask (called the seed flask) with an OD of 3.72 was selected to inoculate all flasks tested.

Test vessels were 250 ml Erlenmeyer flasks containing 100 ml of the sterile medium described above, except for the addition of 5 g/l yeast extract. All flasks were autoclaved at 121 C and 15 lb/sq. inch (0.1 MPa) and filter-sterilized (0.22-μm filter) medium was added to the flasks before adding the test materials. The test materials were not sterilized because autoclaving alters the contents of the samples and filter sterilization is not suitable for sterilizing solids. Test samples were added during inoculation to reduce the possibility of contamination. To supplement the tested samples, 1 mL (1 95 vol/vol) of seed flask material was added to each flask. The flasks were incubated as described above for 36 hours.

Analysis

Two samples were analyzed for cell concentration (using streaking for 2. tobii5 and direct counting (hemocytometer and microscope for 5. segemiviae and P. 5iii5).

Appropriately diluted samples of 7. tobiije were streaked onto plates with yeast extract with dextrose (glucose 20 g/l, yeast extract 10 g/l, agar 20 g/l, pH 5.4), incubated at 30 "С and 595 СО » for 2 days and the number of colonies was counted. Appropriately diluted samples of 5. segemisiae and P. 5Priiis were mixed with 0.05 95 trypan blue and placed in a hemocytometer

Meshybrapeg. Cells were counted at 40X magnification.

Three samples were analyzed for ethanol concentration using the U5I Viospet analyzer based on alcohol dehydrogenase analysis (U5I, Ipiegosiepse). The samples were centrifuged at 14,000 rpm. for 20 minutes, and the supernatant was stored at -20 "C to preserve integrity. Before analysis, the samples were diluted to an ethanol concentration in the range of 0-3.2 g/L. A standard in the form of ethanol at a concentration of 3.2 g/L was analyzed approximately every 30 of the samples to ensure that membrane integrity was maintained throughout the assay.The optical density (600 nm) of the samples is not reported because the solid test samples interfered with absorbance measurement due to turbidity of the samples and was inaccurate.

Results of ethanol analysis

To compare each sample with the control for each microorganism, the output was used (Tables 37-39). However, the output cannot be used for comparison between strains. When comparing the strains, it was necessary to analyze the total concentration of ethanol. When analyzing the 95 data, a yield less than 80 95 may indicate toxicity when accompanied by low cell counts.

The equation used to determine the 95 output is:

Fo output - (ethanol in the sample/ethanol in the control)x100.

Table 37

Ethanol concentration and 95% yield when using zasspagotusezh sehemiziye

Concentration. Concentration. Concentration. 00000 anode out! Sstanoluf Gevivl| stanol(ilu Tykh vi32 | 753 | 186 | bo 1 146 | 399 2 shЩ | 25 mu513a-10 | 77749 | 172 | -(660 | 146 | 455 | 246

Table 38

Ethanol concentration and 9o output when using Rispia 5iiiriiii5

Sample and Concentration. Concentration. Concentration.

RIZALOOI | 52 247 | -(8b | 472 | 133 | 288

A132-10 | 152 1710 | 771857. 1033 | 2 - 260 2юЮюЮХ(| 565

A132-100 | 10.9 514 | 16.97. 923 | sh 222 | 483 visa | 77771780 1375 | 2 sh-(129 | 713 | 2 sh133 | 288 6132-10 | scrap 0 476 | 160 884 | 2 sh 223 2 sh | 485 132-100 | 86 | 406 | 152 837 | 216 | 470 mU5IZ2 | 98 yuyu 460 | 149,820 | 2 sh- 179 7 sh | z89

Sample A" | 132 156 | 190 166 | 206 | 160

The samples in bold were the highest ethanol producers, over 20 g/L, and had concentrations similar to those in wood hydrolysates (Zgeepaiy

N.K. apa dentiv T.MU., Viotezoshgse Tesppoiodu 72 (2000) 253-260). "Analyzed in a later experiment with a rotating flask.

Table 39

Ethanol concentration and 90% yield when using utotopap tobias5

Sample and Concentration. Concentration. Concentration.

RIZ2 /17777717171775 | 85 | ЩЩЩЩ 68 2 ЩЩЩ(| 84 | ЩЩ 75 | 9

CUTTER 177777/775 | 85 | sh 48 sh | 59 | 6 yush 68 | 84

AT32 17777796 77091... 83 | 103 | 2 sh-9 | 2

UVIZa 7 177777111779 | 89 | 2 yushushush 71 | 88 | 2 shshshch 77 | 94

MUvIza-10o1777771782 7 | 93 | 2 sh 68 7 shf(| 85 | 6 sh 73 sh | 90

MU5I3Za-100 77 87 | 98 | (69 2 yush | 86 | 83 | 102 visa 17777787 | 99 | 2 yush 71 | 88 | 5 yush "81 | 995 sizalo | (78 | 88 | 70 | 88 | 73 | 90 sizalob | 86 98 | 78 | 98 | 83 | 102

Control | 88 | 100 | (80 5 yu KmKEBu)| l00 | 8 7 | 00

Results of cell concentration analysis

Fo cells are used to compare each sample with the control for each organism (Tables 40-42). However, 95 cells cannot be used for comparison between strains. The total cell concentration had to be used when comparing strains. In data analysis 95 a yield of less than 70,956 may indicate toxicity when accompanied by a low concentration of ethanol. The equation used to determine the 95 output is:

Fo of cells - (number of cells in the sample/number of cells in the control)x100.

Table 40

The results of the analysis of the concentration of cells Zasspagotusez segemiviae

Sample i Concentration of cells about . Concentration of cells about . (x108/ml) Uo Cell (x108/ml) Uo Cell

RESPONSE 2

P132-10

RI32-100 71895 | 3 | 199 | 66 2 "Fe"e

A132

A132-10

A132-100 visa p132-10 p132-100

MU5132

M5132-10

MU8132-100

Table 41

The results of the analysis of the concentration of Rispia 5iiiiiiiii5 cells

Sample i Concentration of cells about . Concentration of cells about . (x108/ml) uyu Cell (x108/ml) uyu Cell

RESPONSE 2

P132-10

RI32-100

A132

A132-10

A132-100 siz2 s132-10 71171228... |... 17771117198 17111142 2 s132-100

MU5132

M5132-10

MU8132-100

Table 42

The results of the analysis of the concentration of cells of 2utotopov tobias5

Concentration of cells about . Concentration of cells about . 5 Cells C«tov/ml) 5 Cells

RIZ2 7708. | ..YuyuYuYuYD86 2 sh | 297 | 66

RI32-10 21.80 7777264. |... 437 198 2 Sh

RI32-100

A132

A132-10

A132-100 77778960 | .-.YuyuYuyuYuyu6 7 177711420 17777794 2 visa 7748. .| ..ЮюЮюЮЙ90 5 5ющЩ | 384 | 86 s132-10 14.75 s132-100 1111600 .YuuYuuIille 112551... 57 2 sh ("

MU5132

MU8132-10 13.20

MU8132-100

Example 28 - Fermentation in a rotating flask of cellulose samples using R. 5iirie

Summary o Thirteen samples were tested for ethanol production in R. ziirizis culture without added sugar.

They were tested in the presence and absence of cellulase (enzyme complex AssePegazetF 1000,

Sepepsog). The equipment and reagents used for the experiment are listed below in Tables 43-45.

Table 43

Equipment and frequency of maintenance maintenance. . V. Vgatsp Vioiesii,

Opisat spectrophotometer, OMZ00

Analyzer U5I Viospet Ipiegzsivpse, U5I

Table 44

Components of U5I used in the study in a rotating flask

Membrane for ethanol U5I 2786 071100153

Standard for ethanol U5BI (3.2 g/l) 2790 012711040

Buffer for ethanol U5I 2787 07мМ1000053, 07100215

Table 45

Reagents used for fermentation in a rotating flask ospoЯАВ Spetivig 9472706 АО-7284-43

Vesiop Oiskipvzop 291940 7128171

Vesiop Oiskipzop 211677 4303198

Broth UM Vesiop Biskipbop 271120 6278265

Enzyme complex

Asseiegazef AsseiIegaze 1000 1600794133 ! ! 1304473 s-5400 107НО245

The culture of R. viryii5 on a slanted medium of MAKKI m-7124 was obtained from a rehydrated lyophilized culture obtained from AK5 Siyige SoPesibp. Part of the culture material on the cut medium was streaked onto yeast and mold broth (UM) and agar at a concentration of 20 g/l (pH 5.0) and incubated at 30 "C for 2 days. In an Erlenmeyer flask with a volume of 250 ml, containing 100 ml of medium (40 g/l glucose, 1.7 g/l yeast nitrogen base, 2.27 g/l urea, 6.56 g/l peptone, 40 g/l xylose, pH 5.0), one colony was inoculated and incubated for 24 hours at 25 C and 100 rpm After growth for 23 hours, samples were taken and analyzed for optical density (600 nm in UV spectrophotometer) and purity (Gram stain). of the results, one flask (called the seed flask) with an OO of 6.79 was selected and pure Gram stain was selected for inoculation of all tested flasks.

The tested containers were 250 ml Erlenmeyer flasks containing 100 ml of medium (1.7 g/l nitrogenous yeast base, 2.27 g/l urea and 6.56 g/l peptone). No sugar (glucose or xylose) was added to the growth medium of the flasks. All flasks were autoclaved at 121 °C at 15 psi (0.1 MPa) and filter-sterilized (0.22-μm filter) medium was added to the flasks prior to addition of the test materials. The test materials were not sterilized because autoclaving alters the contents of the samples. and filter sterilization is not suitable for sterilization of solids. The test samples (listed in Table 46) were added at the time of inoculation (rather than before) to reduce the possibility of contamination. To supplement the test samples, 1 ml (1 95 vol.) was added to each flask. /06.) of seed flask material. The flasks containing sample P132-100 required the addition of 0.4 ml of 1 M Mahon to bring the pH to 5.0. The flasks were incubated at 30 "C and 150 rpm. within 96 hours.

One set of duplicate flasks per starting material contained the AssePegaze?t enzyme complex (1.25 ml per flask, the highest recommended dose is 0.25 ml per gram of biomass,

Sepepsog) to attempt simultaneous saccharification and fermentation (551E). Another set of duplicate flasks did not contain the enzyme complex AssayIegazeFf). A total of 52 flasks were analyzed.

Six control flasks were also analyzed. The positive control flasks contained powdered cellulose 5oiYikanios 200 IU (batch 2 OSHA158072, Ipiegpaiopai! Rirbeg Sogrogaiop) at a concentration of 2.5 grams per 100-ml flask (25 grams per liter) with and without the addition of AssayiIegazef enzyme complex. In addition, a control containing only sugar (glucose and xylose) was used.

Table 46

Amount of each starting material added to each flask

Analysis

The samples were analyzed for ethanol concentration (Tables 47, 48 and 49) using the M5I Viospet analyzer based on alcohol dehydrogenase analysis (U5I, Ipiegesiepse). The samples were centrifuged at 14,000 rpm. for 20 minutes and the supernatant was stored at -20 "C. Prior to analysis, the samples were diluted to an ethanol concentration in the range of 0-3.2 g/L. A standard in the form of ethanol at a concentration of 2.0 g/L was analyzed approximately after every 30 samples for assurance in that the integrity of the membrane was preserved during the analysis.

The results

Table 47

Results for control flasks

11111111 | ha4hours | Zbhodin | 48 hours Hours

Containing glucose, without cellulose,

Eva NSS SI SETY NS (ZoiKagios), without sugar, without enzyme

Containing crystalline cellulose (ZoiYKagios) in a concentration of 25 g/l, without sugar, with the addition of 6.56 7.88 9.80 8.65

AsseiIegazeF

Table 48

Results for rotary flasks without enzyme complex AssayiIiegazetF 1000

RIZA 77777771 17711171009 | .-.yu.000 2 yuyu yurKyuBr7/Yur 000 | ole

RIZALOOI 77777771 17711009. |. ..ю001 юЮющ| 000 | 002 visa 77777771 17111040 | 7711009 1777 024 | 042 visaYAO 77777771 17111069 | 042 1 022 | 024

Table 49

Results for rotary flasks with AssayEgaseF 1000 enzyme complex

MUvIZ32 77777111 11111789 7111147 177171111о7 | 1099

Example 29 - Analysis with cellulase

Brief summary

Thirteen samples were tested for sensitivity to cellulase using industrial cellulase (AssePegazeft 1000, Sepepsog) under optimal conditions of temperature and pH.

Protocol

The protocol is a modification of the MEE "Iarogaigu Apaiuiisa! Rgosedite GAR-009 Epgutaiiis sasspagiisantsop oi GidposePshovis Viotav5". A sample of the material was added to 10 ml of 01 M sodium citrate buffer (pH 4.8) and 40 mg/ml tetracycline (to prevent bacterial growth) in a 50-ml test tube in duplicate. The amount of sample added to each tube is listed in Table 50.

Some samples were difficult to mix (P132, P132-10, PI32-100), so they were added at a lower concentration. Also included was a positive control in the form of 0.2 grams of powdered cellulose from ZoYKanios 200 IU (batch Z OA158072, Ipiegpaiopa! Ribeg Sogrogaiop) and a negative control (no sample). A sufficient amount of reverse osmosis (RO) water was added to the test tubes, bringing the volume to only 20 ml. Both sodium citrate buffer and water were heated to 50 °C before use.

The enzyme AssayEgaheF 1000 was added to each test tube at a dosage of 0.25 ml per gram of biomass (the highest dosage recommended by Sepepsog). The test tubes were incubated at an angle of 45" at 150 rpm and 50 "C (recommended by Sepepsoi) for 72 hours. Samples were taken after 0, 3, 6, 9, 12, 18, 24, 48 and 72 hours (tables 52 and 53), they were centrifuged at 14,000 rpm. for 20 minutes and the supernatant was frozen at -20 "C. The concentration of glucose in the samples was analyzed using the U5Ii analyzer

Viospet (Ipiegsiepse) using the conditions described in Table 51. A standard glucose solution, 2.5 g/L, was prepared by dissolving 2,500 grams of glucose (Zidta Ca 57528-5K0, lot Ж: 107Н0245) in distilled water. After dissolution, the total volume was brought up to 1 l with distilled water in a volumetric flask. The standard was freshly prepared and stored at 4 °C.

Table 50

Number of each sample added (Negative control/-/:/777777711 11111110

Table 51

Components of U5I used in the study in a rotating flask

The results

Table 52

Results of analysis with cellulase

Glucose concentration (mg/ml) during incubation (hours)

Ro about |z | 6 | 9 | 12 | 18 | 24 | 48 | 72 p132-100

MU5132

M5132-10

Mu5132-100 0.70 | 2.76 | 3.63 4.59 | 4.78 | 5.29 | 5.96 | 6.99 | 743 samples)

Positive control 0.17 | 2.38 | 3.65 | 4.71 | B25 | 5598) 719 | 926 | 986 zZoikKaNios

Erafik 1. Glucose concentration (the best I prolunentni 18.00 pe et te ee ee ee tekteteent sh zde | t shi sh ra 12.Yu pi nn | -- ТО

There is. I shi Shk son | | s MIDYAZALOV le y.O0 en nn nin nn i E x Bonnia with a VISA

Kok NK KUSH: WE E schi and. Cheers still Positive coktros 4 ty 10 yaVoa Bio) o ho 40 vo vo

Hours!

The amount of cellulose split in the test tube was calculated as follows: g/ml glucose 20 ml (sample volume) x 0.9 (to correct for the water molecule added during cellulose hydrolysis).

The percentage of the total sample released as glucose (in Table 53 below) was calculated as follows: cellulose/g sample added (for details, see Table 5) x 100

Table 53

Results of analysis with cellulose

Percentage of total sample released as glucose (905) over time

Number of incubation sample (hours) oi with HER 6 | 9 | 12 | 18 | 24 | 48 | 72

RAZ2 14.98 | 25.16 | 31.36 | 34.85 | 39.38 | 43.81 | 54.29 | 56,27

R1I32-10 12.02 | 18.25 | 22.97 | 25.06 | 27.00 | 32.29 | 40.43 | 41.87

RI132-100 13.79 | 20.98 | 26,28 | 29.02 | 34.06 | 38.45 | 45.65 | 49.61 dIZ2

AIZ32-10 0.94| 425 | 542 | 629 | 682) 756 | 88 | 9.60 | 9.89

A132-100 11.57 | 12.58 s132 s132-10 074| 34 | 3.91 | 466 | 482) 4.99 | 5b2 | 6.60 | 720 p132-100 11.50 | 13.01

E

M5132-10

Mu/5132-100 10.99 |11.42| 12.67 | 1426 | 16.73 | 17.78

Positive control 129 | 21.15 32.72 42.30 | 47.07) 53.73 64.53 | 83.16 | 88.56 zZoikKaNios

Example 30 - Fermentation in a rotary flask using Rispia «iiryiv

Brief summary

A rotary flask fermentation using Rispia viiirie was performed using the four cellulosic materials with the highest 95 yield based on Table 36.

Protocol

The experiments were carried out according to the parameters given in tables 54-56.

Table 54

Equipment and frequency of maintenance maintenance

Table 55

Components of U5I used in the study in a rotating flask

Table 56

Reagents used for yeast rotary flask fermentation

Obtaining seed material

For all spinner flask experiments presented below, seed flasks were prepared using the following method.

Working bank of cells of R. 5iiryie MKK. y-7124 was obtained from a rehydrated lyophilized culture obtained from AK5 Siyige SoPesiop. Cryoflacons containing the culture of R. 5irnie in 15 95 vol./06b. glycerol, stored at -75 "С. Part of the thawed material of the working bank of cells was streaked onto broth for yeast and mold (UM) and 20 g/l agar (pH 5.0) and incubated at 30 "С for 2 days. Cups were kept for 2 days at 4 "C before use. In a flask

250 mL Erlenmeyer flask containing 100 mL medium (40 g/L glucose, 1.7 g/L yeast nitrogen base, 2.27 g/L urea, 6.56 g/L peptone, 40 g/L xylose , pH 5.0), one colony was inoculated and incubated for 24 hours at 25 °C and 100 rpm. After growth for 23 hours, samples were taken and analyzed for optical density (600 nm in a UV spectrophotometer) and purity (Gram's stain).

Based on these results, one flask (called the seed flask) with an optical density of 4 to 8 and with pure Gram stain was used to inoculate all tested flasks.

Three experiments were conducted using samples AT132-10, A132-100, 1132-10 and 5132-100. In experiment 1, these four samples were tested with respect to the concentration of ethanol at different concentrations of xylose and at constant concentrations of glucose. In experiment 22, these four samples were tested against the concentration of ethanol at twice the concentration of the starting material used in the experiments presented in Table 36. Finally, in experiment 23, these four samples were tested against the concentration of ethanol at different concentrations of both xylose and glucose, simultaneously.

Experiment I - Variation of xylose concentration

Four cellulosic samples (A132-10, A132-100, (1132-10 and 132-100) were tested at different concentrations of xylose, as presented in Table 57, below.

Table 57

The composition of the medium in the flasks of the experiment 41 1 bovxylov.7/// | 77777777 000777777777111111Ї1711111111111111400С1

The tested vessels (a total of 40, 250-ml Erlenmeyer flasks) contained 100 ml of medium.

Five different types of media were prepared with the amount of xylose and glucose given in Table 57.

In addition, the medium contained 1.7 g/L of nitrogenous yeast bases (Vesiop bisKipwop 2291940), 2.27 g/L of urea (ZSPoOYIAK SPpetivighu 59472706) and 6.56 g/L of peptone (Vesiop Oiskipwop 25211677). All flasks were autoclaved at 121 °C and 15 psi (0.1 MPa) and filter-sterilized (0.22-μm filter) medium was added to the flasks before adding the test materials. The flasks were kept at room temperature for 4 days and inspected regarding contamination (turbidity) prior to use. The test materials were not sterilized because autoclaving alters the contents of the samples and filter sterilization is not suitable for sterilization of solids. 100 mL) was added during (rather than before) inoculation to reduce the possibility of contamination. To supplement the tested samples, 1 mL (195 vol./06.) of seed flask material was added to each flask. The flasks were incubated at 30 "C and 150 rpm within 72 hours.

Unfortunately, one flask (sample A132-100 with 100 95 xylose) was broken during testing. Thus, all results after 24 hours of incubation are described for one flask. After incubation for 72 hours, 100 95 of the original amount of cellulosic material (5.0 g) was added to flasks with 100 95 xylose (7 flasks in total, one flask containing sample A132-100 was broken) and incubated as described above, for an additional 48 hours.

Table 58

Addition of starting material to flasks with 100 95 xylose at an incubation time of 72 hours

Analysis

Samples were taken from 40 tested flasks at incubation times of 0, 6, 12, 24, 36, 48 and 72 hours. In addition, samples were taken 24 and 48 hours after the addition of the second amount of starting material to the flasks with 100 95 xylose (see Table 58).

A total of 292 samples were analyzed for ethanol concentration using the U5i analyzer

Viospet based on alcohol dehydrogenase analysis (U5I, Ipiegesiepse). The samples were centrifuged at 14,000 rpm. for 20 minutes and the supernatant was stored at -20 "C. It should be noted that at time 0 the samples had to be filtered through a 0.45-μm syringe filter. Before analysis, the samples were diluted to an ethanol concentration in the range of 0-3.2 g/l. A 2.0 g/L ethanol standard was analyzed after approximately every 30 samples to ensure that membrane integrity was maintained.

A total of 47 samples were analyzed for the number of cells. Samples were taken after incubation for 72 hours and 48 hours after addition of additional cellulosic material. Appropriately diluted samples were mixed with 0.05 95 trypan blue and loaded into a Meibaceg hemocytometer.

Cells were counted at 40X magnification.

Experiment 22 - Analysis of 2X concentration of the starting material

Test vessels (a total of 8, 250-ml Erlenmeyer flasks) contained 100 ml of medium.

The medium contained 40 g/l of glucose, 40 g/l of xylose, 1.7 g/l of nitrogen bases of yeast (Vesiop biscipbop

291940), 2.27 g/l of urea (ZSPOYAK SPpetizig 29472706) and 6.56 g/l of peptone (Vesiop bisKipbop y211677). Flasks were prepared as in experiment 21. The tested samples (A132-10, A132-100, 1132-10 and

Cs132-100 at a concentration of 10 g per 100 ml) was added during inoculation (and not before it) to reduce the likelihood of contamination. To supplement the tested samples, 1 ml (1 95 vol./06.) of seed flask material was added to each flask. The flasks were incubated at 30 °C and 150 rpm for 72 hours.

Analysis

Samples were taken from 8 tested flasks at incubation times of 0, 6, 12, 24, 36, 48 and 72 hours. Ethanol analysis of 56 samples was performed according to experiment 1 and its results are presented in table 59.

Cell counts were performed on a sample obtained after 72 hours according to experiment I, and its results are presented in Table 60.

Table 59

Ethanol concentration in flasks with doubled amount of starting material 0111111111111111711111198 11026 | 012 | 0 0061111111111111171111111175 | 1021 | 020 | oo 12111111 2y61 1711073 | 069 | 031

Table 60

Cell concentration at the time of incubation of 72 hours in flasks with a doubled amount of starting material

Experiment 23 - Variation of xylose and glucose concentrations

Four cellulose samples (A132-10, A132-100, 0132-10 and (c:132-100) were tested at different concentrations of xylose and glucose, as presented in the table below (table 61).

Table 61

The composition of the medium for the flasks of the experiment YAZ 1 b9tsuvru.//// | 77777777777171007777111111Ї11111111111111101

Test vessels (a total of 32, 250-ml Erlenmeyer flasks) contained 100 ml of medium.

Four different types of medium were prepared with the amount of xylose and glucose given in Table 61.

In addition, the medium contained 1.7 g/l of nitrogenous yeast bases (Vesiop bisKipzop Zh291940), 2.27 g/l of urea (ZSPOYAK SPetizigu 29472706) and 6.56 g/l of peptone (Vesiop OisKipvop 211677). Flasks were prepared according to experiment Y1. The tested samples (A132-10, AT132-100, 1132-10 and 132-100) were added during inoculation (and not before it) to reduce the possibility of contamination. To supplement the tested samples, 1 ml (1 95 vol./06.) of seed flask material was added to each flask. The flasks were incubated at 30 °C and 150 rpm for 72 hours.

Analysis

Samples were taken from 32 tested flasks at incubation times of 0, 6, 12, 24, 36, 48 and 72 hours. (see tables 62-65). A total of 224 samples were analyzed for ethanol concentration using the M5I Viospet analyzer based on alcohol dehydrogenase analysis (U5I, Ipiegesiepse). The samples were centrifuged at 14,000 rpm. for 20 minutes and the supernatant was stored at -20 "C. It should be noted that some samples were required to be centrifuged and then filtered through a 0.45-μm syringe filter. Before analysis, the samples were diluted to an ethanol concentration in the range of 0-3.2 g / l.

A standard in the form of ethanol at a concentration of 2.0 g/L was analyzed after approximately every 30 samples to ensure that the integrity of the U5I membrane was maintained.

Table 62

Results relative to ethanol for sample A13210: taking n and 10 55 sho | 50 100 чо : tab 000255 Because the sample koilozio KONLOZM O ksnlozn o BSYLOZNO kenlozno ospukriv? and about Nukri? oh pukrin? and nests?

In you ovo oo 053 85708605,

SHO В В В ВВ ВВ

112 1.72: 186 ANNI LI Shk EN M I ME MN U M SHE i 24 15.55: 15.90 17.05 7.05 1695. 102 488 9U7 1 1335

SHOV | L7.LO 11740 2025 2135 2095129 427 | 89901755 her 18.40 1 1705 lei: 8300 1 BV 147 03035 1 833 1 180 2-72... 1515 15.55) 1925 2185 Rob Mao li and codeine What ' What and . : 2315 What | What | What | What

This is dksen su MON KIM KANA MANNYA KAK MAK AKA KAK KANA idolization ! ! | | : | and Analysis from experiment 73.

Table 63

Results relative to ethanel for sample A132-00 about vzla | 05 | 85 | 255 BO 4005 05 and 25 50 o Zrahkha | Zheilozy | Keplozo xylozno konlozo o Keplozno pukriv? about sugar? oh oh nukri? 0 buds? 9 009 017 0 08 20.14 0.09 03 6 bl 0 015 0 015 1 04 b 1011003 42068100 85 ovo 56055

SyYa00019.90 15.70 16.50 16.05 1460708 03337995 16.00 | 1790 185.00 945 17509 92 2.85 87 | 150

WED 16.70 19.30 2515 2700 147 1 754 1 1550 s 21465 | 1535 18.65 10 yav 060511 "After 34 | | ' hours What . | Щ Щ ' 24 vahs What What | Щ | and addition ! and : ! rolls -e | yan 2avom - s nElya | | and ! addition | | ! y t Analysis from experiment 73.

All results are based on the analysis of one column.

Tailynya 64

Results relative to ethanol for sample 5132-10

My "? moms and ach 01000111... ZhontsenIrashia vIVNOLU I) 00000 io sample xylose xylose xylose Xylosen| venlozi about opukrin? | sugar? about Nukri? | sugar? 9. 1 009 | 008 | 008 | 008 | 008 | 005 | 005 1 005 | 006. 6

LOTTO 1096100 087 | Ла ов лою ши Ж EE LY SNY vy В В Л НЕД А КЛ ВВ ДН с вв АН 36.15.10) 17.45 | 16.80. 18.75 | 2215 |) 009 302 869 | 1655. 48 15.95 18.90 | and25 2110 | 80 0.07 05 her vlo 185.50

SOY 001350 15.80 | 1855 2155 | db | because OBL and After 24 as -- - I 28.35 I-- - | - - they are worthy!

Her after ! (adding kA AN PON PON KOH KOKO KKKN A pi and After 48 - - - - wai - - ! - - and tdognny ! same Analysis from hxperiment 73.

Table 65

Results regarding ethanol for sample 13210 wells 10-:5:А5 544 -А-4- ж 52 5 25 2 52 56 6 06 5 Ethanol concentration E puvr nukriv? sugar? 6 ad 1 vo; Z 1 obov 1 EE bo b.bE

SHVL LA 1 LA VV 80600186

ShVL MVV vv 48 840 her 1700 chad 1 2255: 2445 KE SV tv 158 on. PIKA NE NIK SCHA MDE ME AS SY KODAK MONE NAU NI ME

In 24 7! It's time after - and r pt | - E za za t- E - sh pt daolavaniya! : and N N holopElya | w E - - | w 1 24vo w i - - -- lololavaniya VEY : t Analysis from experiment 3.

Sampling was carried out after incubation for 72 hours for cell counting (see tables 66-67). Appropriately diluted samples were mixed with 0.05 95 trypan blue and loaded into a Metzbaceg hemocytometer. Cells were counted at 40X magnification.

The results

To inoculate all tested flasks, experiment! and Zh2 used one seed flask.

The measured optical density (600 nm) in the seed flask was 5.14 and the cell concentration was 4.65x108 cells/ml (Tables 65-66). Thus, the initial concentration of cells in the tested flasks was approximately 4.65x105 cells/ml.

For the inoculation of the flasks of experiment 23, a second seed flask was used. The optical density (600 nm) in the seed flask was 5.78 and the cell concentration was 3.75x108 cells/ml. Thus, the initial concentration of cells in the tested flasks was approximately 3.75x105 cells/ml.

Table 66 knlOozZy

The number of cells after incubation for 72 hours is therefore | B 50 400 035 0 o 255 50

OBSNLOZIO oat gates 0 BSNLOZNO Ksplozio: KSKLOZI sugar. nUKrU sugar : nekru

ATO 0379635) 52 499 405 056 025 055156 where are we? oh 382 ol el ЕС 2132100 653. 4085 484 447 5295 ОО 033 088 0222 "Samples were significantly contaminated after growth for 72 hours. This is expected because Rispia did not grow well without added sugar and contaminating organisms (from non-sterile samples) were able to outgrow Rispia .

Table 67

Cell numbers after incubation for 48 hours after addition (100 95 xylose and glucose)

Cell concentration (x108/ml)

A132-10 10.17 dIZ32-100 c132-10 c132-100

Example 31 - Toxicity testing of lignocellulosic samples relative to P. 5iiiriiii5 and 5. segemisiae

Brief summary

Thirty-seven samples were analyzed for toxicity against two ethanol-producing cultures, Sasspagotusez segemiziae and Rispia 5iiryi5. In this study, glucose was added to the samples to determine the difference between starvation of the cultures and toxicity of the samples.

Table 68

Conditions for toxicity testing

Variable Zasspagotusee sereuvive ATOS Risnia viiriyiv MAVI -7124

Inoculation volume (ml) 0.5-1 (target: 6-7x105 cells/ml) 1 (target: 3-4x105 cells/ml)

Incubation temperature (12C

Rotational device speed (rpm) 200 125 5000-ml Erlenmeyer flask 250-ml Erlenmeyer flask

The volume of the medium

Total incubation time 79 79 (hours)

Ethanol analysis (hours) 0, 6, 12, 24, 36, 48, 72 0, 6, 12, 24, 36, 48, 72

Counting cells (hours) 24.72 24.72

Protocol

A summary of the protocol used is given in Table 68. A description of reagents used in toxicity testing is given in Table 69. For each week of testing, two control flasks (without sample addition) were obtained for each microorganism. A total of 82 flasks were analyzed.

In the course of experiments during the first 24 hours of incubation in flasks with R. 5iiryh containing samples

C, b-ie, C-5e, and C-10e, neither ethanol nor cells appeared. To confirm the results, the test was repeated. The second test confirmed some inhibition of the growth of R. 5iiirich when adding the samples to the flasks

C, STE, SBE Her STANDS.

Table 69

Reagents and materials used for toxicity testing

Table 70

Components of U5I used for toxicity studies

Samples to be Tested

The seven samples tested (all marked "C") were ground using a coffee grinder suitable for small samples. The samples were crushed to a comparable particle size (between samples) by visual inspection. Sample number C-10e was easily crushed into small particles.

All samples were added to the flasks at a concentration of 50 grams per liter, with the exception of six "P" samples (25 grams per liter). These samples ranged in color from white to off-white and visually appeared loose, and at a concentration of 50 grams per liter the flasks did not mix properly (not enough free liquid). Samples "5" dissolved easily and could subsequently be added to the flasks at higher concentrations. Samples "A" and "c" could be added in the amount of 100 grams per liter.

Testing was performed using two microorganisms as described below. zasspagotusez segemiviae ATOS 24858 (Ategisap Toure Siyige SoPesiop)

The working cell bank of 5. segemisiae ATSS 24858 was obtained from a rehydrated lyophilized culture obtained from Ategisap Ture Siyige SoPesip. Cryoflacons containing culture 5. segemisiae in 1595 vol./vol. glycerol, stored at a temperature of -75 "С. Part of the thawed material of the working bank of cells was streaked onto broth for yeast and mold (UM) "t 20 g/l agar (pH 5.0) and incubated at 30 "С for 2 days. A 250-ml Erlenmeyer flask containing 1 ml of medium (20 g/l glucose, 3 g/l yeast extract and 5.0 g/l peptone, pH 5.0) was inoculated with one colony from a plate with UM and its incubated for 24 hours at 25 "C and 200 rpm. After growth for 23 hours, samples were taken and analyzed for optical density (600 nm in a UV spectrophotometer) and purity (Gram stain). Based on these results, one flask ( called a seed flask) with 00 9-15 and pure Gram stain was used to inoculate growth flasks.

After growth for 23 hours, the culture flask had a low OO (5.14) and cell count (1.35x108 cells/ml). It should be noted that the colony taken from the seed plate was smaller than usual. Thus, 0. ml of inoculum was added to each tested container (as opposed to the planned 0.1 ml).

Test containers were 500-ml Erlenmeyer flasks containing 100 ml of the sterile medium described above. All flasks were autoclaved at 121°C and 15 psi (0.1 MPa) prior to addition of test materials. Test materials were not sterilized because autoclaving alters the contents of the samples. Test samples were added at the time of inoculation (rather than before) to reduce 0.5-1.0 ml (0.5-1 95 vol./06) of inoculum material was added to each flask to supplement the tested samples.The flasks were incubated as described above for 72 hours.

Risnia viiriiiis (AN5 Siyitge SoPesiiop)

Working bank of cells of R. 5iiryie MKK. y-7124 was obtained from a rehydrated lyophilized culture obtained from AK5 Siyige SoPesiop. Cryoflacons containing the culture of R. v5iiryie in 15 95 vol./06b. glycerol, stored at -75 "C. Part of the thawed materials of the working bank of cells was streaked onto broth for yeast and mold (UM) and 20 g/l agar (pH 5.0) and incubated at 30 "C for 2 days. Cups were kept for 5 days at 4 "C before use. In a flask

250 mL Erlenmeyer flask containing 100 mL medium (40 g/L glucose, 1.7 g/L yeast nitrogen base, 2.27 g/L urea, 6.56 g/L peptone, 40 g/L xylose , pH 5.0), one colony was inoculated and incubated for 24 hours at 25 °C and 125 rpm. After growth for 23 hours, samples were taken and analyzed for optical density (600 nm in a UV spectrophotometer) and purity (Gram's stain).

Based on these results, one flask (called the seed flask) with an optical density of 5-9 and pure Gram stain was used to inoculate all flasks tested.

Test containers were 250-ml Erlenmeyer flasks containing 100 ml of the sterile medium described above. All flasks were autoclaved at 121°C and 15 psi (0.1 MPa) and filter-sterilized (0.22-μm filter) medium was added to the flasks prior to addition of test materials. Test materials were not sterilized because autoclaving alters the contents of the samples. , and filter sterilization is not suitable for sterilization of solids. Test samples were added at the time of inoculation (rather than prior to inoculation) to reduce the possibility of contamination. To supplement the test samples, 1 ml (1 95 vol/06.) of material was added to each flask The flasks were incubated as described above for 72 hours.

Analysis

Samples were taken from seed flasks immediately before inoculation and from each flask after 24 and 72 hours and analyzed for cell concentration using direct counting. Appropriately diluted samples of 5. segemiviae and R. "iiryi5 were mixed with 0.05 95 trypan blue and loaded into the Meshybaceg hemocytometer. Cells were counted at 40X magnification.

Samples were taken from each flask at time points of 0, 6, 12, 24, 36, 48 and 72 hours and analyzed for ethanol concentration using the U5I Viospet analyzer based on the alcohol dehydrogenase assay (U5I, Ipiegesiepse). The samples were centrifuged at 14,000 rpm. for 20 minutes and the supernatant was stored at -20 C. Before analysis, the samples were diluted to an ethanol concentration of 0-3.2 g/l. A 2.0 g/L ethanol standard was run approximately every 30 samples to ensure membrane integrity was maintained throughout the run.

Calculation

The following calculations were used to compare the number of cells and ethanol concentration with control flasks:

Fo output - (ethanol concentration in the tested flask/ethanol in the control)x100 95 cells - (number of cells in the tested flask/number of cells in the control flask)x100.

The results

The inoculum of 5. segemisiae had an optical density (600 nm) of 5.14 and a cell concentration of 1.35x108 cells/ml. Half of the seed flask material was added to each of the test flasks. Thus, the initial concentration of cells in each flask was 6.75x105/ml. During the second week of testing, the seed flask with 5. segemisiae had an optical density (600 nm) of 4.87 and a cell concentration of 3.15x107 cells/ml. One ml of seed flask material was added to each of the test flasks. Thus, the initial concentration of cells in each flask was 6.30x105/ml. The pH value in the flasks with 5. segemiziae at the time of sampling at 0 hours is presented in Table 71. The pH value of the contents of the flasks was within the optimal pH values for the growth of 5. segemiziae (pH 4-6). No pH adjustment was necessary.

Pt rennia BE KOLBYah 5 segemiviae B MOMENT Vayattya treason

The number is 331 The number appears rn Kk

BRA 117711 5oda | 777777771sya 7 | 5Abe7/ Kk 1111111 RE 77/17 yaya | 77777771 SLEE 1771711 554 K 1111111 RUE | 777 5Ode | Sat | 55095 Kk 77171718 77 4986 | 77777711 CE: | 538395 Kk 11111 ROB. | 777 46b7ya | 777 sob | 529 K

R 11111 ROB. 1717171 yayaz | 7777 s5OeE 1717171 48095 Kk

EE11111va1111111111771115yadba | 7 stoova /////| 46bb5/-/k 111111 1ЕБЕ | 7775475 | 77777778 | 75 111111 o5Be | 777 54ba/// | 7 5LE | 50b95 Kk

R 77771111 el0Э 77171717 5395 77771711 BTOE: 17777 496977 Кк 77771717 e50Б5 7775075 | B LOEE | 4945 Kk

B A1111111117111152a | 7777111 BTOB. | 5688 Kk 71111111 AEN | 7775695 | at 5T5OB. | 4488 -/ Kk 1111111 AEN | 7775629 | BOB IS | 4038 Kk

RE AL 1777 561a | 0 controlAv/// | 5029 -/К 11111 SUBSCRIBE | 777 5jaa | 0 controlbed//// | 5048 K

BO in

PPO" 8" refers to sucrose 1!

РРО" Applies to CHI corn!

RISOK KhM junior

The concentration and yield of ethanol in flasks with 5. segemisiae are presented in Tables 72 and 73. The highest concentrations of ethanol were produced by series 5.

Table 72

The concentration of ethanol in flasks with 5. segemiviae

Ethanol concentration (g/l) at the following time points (hours)

Momerzrazha Go GT 6 | 12 | 24 | with 6 | 48 | 72 7777 R 7 | 002 | 004 | 038 | 587 | 786 | 541 | 7.04

RIOE 006 | 005 | 065 | 663 | 7.66 | 557 | 140

RBOE

RIOOE

10.20 | 824 | 666 | 284 10.20 10.00

STO a5OE 7 BUT | 034 | 044 | 079 | 966 | 89 | 718 | 264 7 ABE | 055 | 045 | 099 | 944 | 896 | 756 | 380

ATOE

ABOE

10.10 15.90 / 81E | 005 | 006 | 031 | 724 | 952 | 1270 14.90 11.90 19.00

ZI0E 11.50 19.30

ZZOE 003 | 005 | 009 | 594 | 7.97 | 1120 | 2040

"ZBOE" 13.00 18.30 5IOOE 13.80 12.70

7117 C1E | 000 | 006 | 037 | 893 | 786 | 599 | 7.37 717 s50E | 003 | 006 | 044 | 931 | 779 | 578 | 7.79 words | 003 | 006 | 058 | 906 | 685 | 595 | 7.09 77/78 6 south k| (| 002 | 005 | 099 | 854 | 6bo | 509 | 042 8 ISOE | 003 | 004 | 071 | 847 | 696 | 489 | 021 "were analyzed for 2 weeks.

Table 73

The output in flasks with 5. segemizes

Exit (95) in the following moments of time (hours)

R 1771717111108.9,.7.7.ЮЮЮЯ |. у1076 | 684 | 160 2 sh "( 1 RIE /й177777717946 | 1099 2 уХХЦЦ- | 650 2 2 щХХщ | 89 1 RE 1777711 1640 | 873 | 430 | 06 108.1 106377 | ..ЮюЮюЮююД966 2427 | ююХ9 2 4ую 1

"were analyzed for 2 weeks.

The concentration of cells and 95 cells in flasks with 5. segemiviae are presented in table 74. Large numbers of cells were observed in all flasks; however, not all cells apparently produced ethanol.

Table 74

Number of cells 5. segemiviae and 95 cells (cells x 108/ml) control) x 100

R G7717171171062 | .юЮю0964.. | .ЮюЮюЙ977 юю | .YuyuYufyu1390 2 shSh

OR | 066 | 762 | 7710355 2 Shshshsh | 2346 .55E | 023 | (b | 365 2 YushShsh | 1434 8vBoye | 044 | .-.yuYy090 | 865 2 sshShsh | 1965 85 T3OE | 059 | 060 | 98 | 869 about controlA" | 066 | 042 | 131.2

And controls" | 035 | 050 | 6950 (y | 1О8

The seed flask with R. 5yrije had an optical density (600 nm) of 5.01 and a cell concentration of 3.30x108 cells/ml. One ml of seed flask material was added to each of the test flasks. Thus, the initial concentration of cells in each flask was 3.30x105/ml. During the second week of testing, the culture flask with R. 5iiryi5 had an optical density (600 nm) of 5.45 and a cell concentration of 3.83x108 cells/ml.

One ml of seed flask material was added to each of the test flasks. Thus, the initial concentration of cells in each flask was 3.83x106/ml. The pH value in the flasks with P. 5iiiriy5 at the time of sampling at 0 hours is presented in Table 75. The pH value of the contents of the flasks was within the optimal pH values for the growth of P. 5iiirikis (pH 4-7). No pH adjustment was necessary.

PT table in pH values. in pits from the River at the time of taking samples at 0h:

B essay Y77717171717111se1111111117111111115Ovbe11 11 RE | 7777777 4875 | 7777771 SLEE 17777711 530977 11117 RE | 77777 80577771 77777111 с5ЕН 17777711 52098

ROB | 77777 yav | 777 sov. 17777771 5Obe77 11 RBOYE | 7777 yadbe | sob 1777777 4898 tin | 77777 yards | 7 s5OE 1777777 4lOv u

Gr 611117 771111115d5. | 7 SslobvN //777777745987777

Br elEeeE | 7777777 5th | 777775 17777711 493877 11111 o5EB6 | 777775 | OO 5PLEE 17777777 49087777 o 6lovYa | 77777753» | 52 17777 4818 o e5oBya | 7777 yav 77771 BLOVE 1777777 4838777 2 AB 6bBYAE 771115 1777777 40187 1111 BUT | 777777 566 | BT 177 4me 11111 ABE | 777777 56027 | 7777 BOS 1777777 4079 777 11111 ABYA | 77777558 | konolaya 1 49387 111 ABOVV | 77 569 | condolbe/// | 40187 in 18111175 11111 5lEE | 777777 4845777 r 11111555 | 4865677 r 11185. | 77 485 r 11 85 MY | y/y 45157 r 111550 | year 40757

There are 7 5 loev | i 0v5 77 v

The concentration and output of ethanol in flasks with P. 5iiirii5 are presented in tables 76 and 77. The highest concentrations of ethanol had series C and A. Flasks with C-Zb0e, C-50e and C-100e also contained high concentrations of ethanol. The concentration of cells and 956 cells in the flasks with P. viiriije are presented in Table 78. In the flasks with the designation 5, low concentrations of cells were observed. Low numbers of cells were also observed in the flasks containing samples C, C1E, C5E and C10OE at the time of sampling 24 hours.

Table 76

Ethanol concentration in flasks with R. viiriii5

Ethanol concentration (g/l) at the following time points (hours)

Momerzrazha Go 1 6 Y 12 | 24 | with | 48 | 7,777 R. 777 17170001 | 005 | 026 | 498 | 8.57 | 1410 | 1700 12.40 | 17.30 12.40 | 1560 | 18.60

RIOE 13.00. | 17.00

RBOE 10.50. | 16.90

RIOOE 10.50 | 715.60 | 18.80 10.80 | 17.70 | 1940 | 25.40 1220 | 19.60 | 23.80 | 28.60 12.50 | 90 | 2450 | 2750

STO 10.20 | h9l0 | 22.90 | 2820

Oboe 18.40 | 2220 | 28.00 16.10 | 1940 | 7:30 p.m 18.60 16.10 | 2240 | 27.60 | 27.70 10.60 | 2210 | 27 lo | gyo

7 C30E | 001 | 003 | 060 | 71230 | 2120 | 2600 | 2720 7.87 6yuYUB | 003 | 003 | 0.37 | 6b6o | 71070 | 13.50 | 10.90 8 PE 1001 | 000 | 048 | 524 | 9.37 | 1250 | 1570 controlA" | 002 | 005 | 0.69 / 486 | 869 | 1110 | 1640 "were analyzed for 2 weeks.

Table 77

Output in flasks from R. v5iiriiv

Exit (95) in the following moments of time (hours)

R Y771111712003 | 71147171 151.0 | 714782 2 / ROOE | 906 2 юЮ-| 1406 | 167.0 | 163.5 2 Ki

7 с5Е'77717171717109777 | ..ЮюЮюЮ7Й7Д38 | 1132 | 36 ("0 both, /177777711097777. |... 401 17177116 | gav of control B. 101.9 | ..yuЙ996 2 yushЦs | 1011 | 7000 "were analyzed for 2 weeks.

Table 78

The number of cells of P. 5iiiiii5 and 9o cells (cells x 108/ml) (number/number in the control) x 100

R 1771717111278 | 771100 | 806 2 2 7-(| 7480 1 RE | 7 2lo | 7 7720 | 609 |

RO | --(юЙ033 2 юю | 863 2 щЩщщ | 96 2 щЩХ | b2 641117 771111000 |... 0256 | (bean | 72 7 с1Е | 000 | .-.ЮюЮюД036 2 2 щЩщ| 000 | 99 7 с5Е | 0007 77777008 | 000 |. 7 layers | 000 |... 585 .2.YUDDYJ| 2 Yuushch 000 | 1607 so | (ev | 660 | 574 | 888 2sh RGIG

ZPE 117

ZT5E

ZPOE

ZTZOE

ZTBOE

ЗТ1О0Е" 105.2 104.3 960 25juKkzh 104.0 103.0 111.3 "were analyzed for 2 weeks.

Generalization of the results of cellular toxicity of thorium 2 isotopes

As shown in Chart 1A, increased cell numbers (eg, more than control) were observed in samples containing P-132-10, (3-132-10 and MU5-132-10 at the 24 hour time point. Cell numbers in the presence of all other samples was comparable to the control This observation indicates that the substrates were not toxic to 72. toBbiih up to 24 hours after inoculation.

At the 36-hour time point, a decrease in cell number (eg, due to cell loss or cell death) was observed in all samples, including controls. The greatest decrease in the number of cells was observed for samples containing P-132-10, 4-132-10. A possible reason for this effect is common to all samples, including the control. Thus, the cause of this effect is the untested substrates, since they differ in each sample and are not present in the control. Possible explanations for this observation include inappropriate culture conditions (eg, temperature, medium composition) or ethanol concentrations in the sample.

Diagram 1A. Cell centers 2. toiiNh

OVO - schu m Fo l yuyu M l o v 5 Ya oo i kyu M KM i

And a YE V V ZK SHE in "SHY SUSHI zho so i Zatod, Shov

As shown in Figure 18, all cells produced comparable amounts of ethanol (eg, 5-10 g/L) at each time point, regardless of substrate. According to the cell count data presented in Chart 1A, the ethanol concentration in each sample peaked at the 24 hour time point. In contrast to the data on the number of cells, the concentration of ethanol did not decrease at further time points. This was expected as ethanol was not removed from the system. In addition, these data indicate that ethanol production in these samples may have resulted from glucose fermentation in the culture medium. It turned out that none of these substrates increased ethanol production.

Diagram 18. Ethanol concentrations for . comrade in c. not yet

Г2 ШЧе зи ши. Y B Z a INN VN NN NN g "Finding her V her a 3 MORE U sa x gz V y

KENE NISHI. ZE

ANN. '" And KE

There's still money.

Taken together, diagrams TA and 18 indicate that ethanol concentrations higher than about 6 g/L may be toxic to 7 Torbius5. These data are also presented as a percentage normalized relative to the control, as shown in Figure 1cC.

Diagram 1. Forest and ethanol production for L. tobine 00 250 150 shcha "VN poni be a v ren pe m ZU Z v ZHTN : і Eh t SCHO EZeN I v; "KI kp BO SHO s KI 5 m Shchi s and shchi shi shi neni I and A WYNEN VEN NO: shi I I zhi Z ZE I KS zh y UZH She VO YASCHI

E Me ay Z U eh shcha oy

KU AY Kok Kk ke Ki I KU i I he K m a i on no a MK DAMU i ee Z ozh KUDI oo SheKlynnysagodo SM Btanoloatodo ShbKlynny Zbtgodo (I Ethanol Zbtod,

Rispia virii

As shown in Figure 2A, cell numbers were comparable to controls. Moreover, although slightly reduced numbers of cells were present in samples containing -132 and M/5-132, for c-132-10, 2-132-100, A-132-10 or A- 132-100 no decrease in the number of cells was observed. Thus, it is unlikely that substrates C or A are toxic. Rather, the reduced cell numbers observed for 0-132 and MU5-132 are likely due to experimental error or the presence of unprocessed substrate that somehow interfered with cell growth. Overall, these data indicate that the glucose present in the control and experimental samples is likely sufficient to promote optimal growth of P. virii5 and that the presence of additional substrate in the sample does not increase this growth rate. These results also indicate that none of the samples is toxic to P. 5iiryi5.

Aiagram XA. Concentrations of cells of R. khyrikh o n 7: t

OO t | and - Z'SHH | ; | sh is sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh sh

EF «schu oyu s re EE is the same as E 7 ze Her still still m 4 goal. G148 h.

As shown in Figure 2B, despite the small numbers of cells represented in Figure 2B, significantly increased ethanol production was observed in all samples containing the experimental substrate. Ethanol concentrations increased over time for each of the three time points tested. The highest ethanol concentration was observed for A-132-10 at 48 hours (eg, approximately 26.0 g/L). By comparing the concentrations of substrates with the highest levels of ethanol production and the data on the number of cells presented in diagram 28, it can be seen that P. 5iiirii5 is apparently not sensitive to increasing concentrations of ethanol. Moreover, ethanol production is apparently not related to the number of cells, but rather related to the type of substrate present in the sample.

Diagram 2B. Ethanol concentrations for ?. "Prnih

For with her for g U V 29 ! in the city of Ko. 5 tires. oh shi shi shi no shi shi shi shi what 195 shi shi shi shi shi shi shi shE sh shne shke 'shi Shi shchi she. or: I'm not X and g EH Gu Z Z what. a Z I i: y. g. . And /',' is Inni y

NEO Z Kk i : 1 5 A E x se Z xh Z

VZ her shi shish y v shi zh i M. nyny s o vy v DA VA VSU rr nin a a a ON YN

Mn E ps i Dn I EK a Ku g Z | Kk su r ich their S . and 35 is Is te "And we

For 4 hours SaO hours KAB h.

Taken together, the results presented in Figures 2A and 28 indicate that the experimental substrates do not promote the increased growth of P. 5iiiri5, but they do significantly increase the amount of ethanol produced by this cell type. These data are also presented as a percentage normalized to control, as shown in Figure 2C.

Chart 2, because growth and production of ethanol for the R.

OO; I ; HE is 5

Zoo! shee shee shee shee shee shee shee shee shee:

MO she shi shi shi shi shi shen shi still

VISHNI NN and SHNM M SHE SHE.

I Ia EE NCHENI M V ZE I E V g z IS MORE Shh Ka ih Щ What Z KI de pi: y ni p ne A NA NO p v i V AN

F "in I-y s EE i se that g "EE "B he sh yin z her

Fo? bo ke in VN zi shk re - -

M Cells methodo SI Ethanol methol, VU Cells ZK tolo KA Evanoliv hot.

ZassNnagotusez segemisiae

As shown in diagram ZA, 5-132-100, A-132, A-132-10, A-132-100 and M/U5-132 provided significantly increased numbers of cells compared to the control. No significant decrease in the number of cells was observed for any of the samples. These results indicate that none of these samples are toxic to 5. segemiviae.

Aiagram ZA, Concepts of cellular. segeniae a !

M 35 m : : N Y A Z N i N N : : N x p nn nn kn pn nn zi pn Brzhovomeu i: ni a i a a Z NI U x KK ku zhk v S She

Z i a Fa a SHY sh i me her z y joyetodazyutka,

As shown in diagram 3B, increased ethanol production was observed in the treated cells in the case of each cell type compared to the control. A comparison of these samples containing the highest amount of ethanol with the cell count data presented in Chart 3A indicates that ethanol concentrations greater than 5 g/L can have an adverse effect on cell count. However, this observation is not the case for all samples.

Diagram of ZV. Concentrations of ethanol for 5, secekisin in B | | "SHE AND | and t | in schi shu ot in neck she ! eat MORE. s with the same tire i i i i i,

Katie 6

NOT: shi. Z Chi ZE

SHIV In ay ke ak i m V v s v V VI NI p Х t ia - a 7 t

Yao«F from the battle Zo In omomtodosiZOgod-Z Z6goya.

These data are also presented as a percentage normalized to the control, as shown in the ZS plot.

ZS diagram. 90 growth and production of ethanol for 5. segeuiviye 450: ; and

Zoo - E : 250: I p I I Same ; with

I I zya z i ZY: se vk 200 - y still g shk sho shi what V zh jah UVU Z: she she she shi kn NN EN RY EN I I

And it's not ШН or ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ ШШ

Not NK NNI in KIN with. x E

OK, NO, I'm Ku from Sha" WHAT ELSE?

Because I'm SCHI. in STILL MORE

N ZI o 1 vh ro Yu s o Z W a SHHI i V i M 8. i A "M. and I M. ЗB I Ya. what and, ZE with Zh. yah she Z.

How about 0 a and KV as an anna i m in VA I and in I Ra ch y s: vk and what -e I am t and - what I

J Cells PER goal E Ethanol h, SLOTS Collect h. K| Ethanol 36 vol.

In conclusion, none of the tested samples was found to be toxic in 7. tobiiiv5, R. 5iryi or 5. segemisiae. Moreover, it turned out that P. viiiryie is the most efficient among the three cell types in terms of ethanol production from the tested experimental substrates.

Example 32 - Production of alcohol using pretreatment in the form of irradiation - ultrasound treatment

The optimal size of biomass conversion plants is influenced by factors including economies of scale and the type and availability of biomass used as feedstock. Increasing plant size tends to increase the economies of scale associated with manufacturing processes. However, increasing plant size also tends to increase costs (e.g., transportation costs) per unit of biomass feedstock. Studies analyzing these factors indicate that an acceptable size of biomass conversion plant may be in the range of 2,000 to 10,000 tons of dry biomass feedstock per day. The installation described below is scaled to process 2,000 tons of dry biomass feedstock per day.

In FIG. 39 presents a technological diagram of a biomass conversion system configured for millet processing. The raw material preparation subsystem processes raw biomass raw materials to remove foreign objects and provides particles of constant size for further processing. The pretreatment subsystem changes the molecular structure (eg, reduces the average molecular weight and crystallinity) of the biomass feedstock by irradiating the biomass feedstock, mixing the irradiated biomass feedstock with water to form a slurry, and applying ultrasonic energy to the slurry. Irradiation and ultrasound treatment transform cellulosic and lignocellulosic components of biomass raw materials into fermentable materials. The primary processing subsystem ferments glucose and other low molecular weight sugars present after pretreatment to form alcohols.

Preparation of source material

The selected level of the given raw material for the enterprise is 2000 tons of dry millet biomass material per day. The specified starting material is chopped and/or crushed millet.

Biomass raw material, in the form of bundles of millet, is delivered to the installation on trucks with trailers. When trucks arrive, they are weighed and unloaded with forklifts. Some bundles are sent to local storage, while others are placed directly on conveyors. From there, the bundles are transported to an automatic unfolding system that cuts through the plastic packaging and/or netting surrounding the bundles. The biomass feedstock is then transported past a magnetic separator to remove foreign metals, after which it is fed into a slicer-shredder system where the material is reduced in size. Finally, the biomass feedstock is transported to the subsystem for pretreatment.

In some cases, the millet bundles are wrapped in plastic netting to ensure that they do not fall apart during transport, and they may also be wrapped in plastic film to protect them from the weather. Ties are either square or round. Bundles are delivered to installations from remote storage on large trucks with trailers.

Since millet is only available seasonally, long-term storage is required to supply the plant with raw material year-round. A long-term storage facility may consist of 400-500 acres of uncovered, clustered bonds in an area (or areas) reasonably close to the ethanol production facility. Local short-term storage, equivalent to 72 hours of production, is provided in open areas. Ties and surrounding driveways, as well as transport conveyors are on a concrete slab. The concrete slab is used due to the necessary volume of freight traffic for the delivery of the necessary large amount of biomass raw materials. The concrete slab minimizes the amount of standing water in the storage area and also reduces the impact of dirt on the biomass feedstock.

Stored material provides short-term supply during weekends, holidays and when normal direct delivery of material for processing is interrupted.

Bundles are unloaded by forklifts and placed directly on conveyors for transporting bundles or on a short-term storage area. Bundles are also pulled out of short-term storage by forklifts and loaded onto conveyors for bundle transportation.

Bundles are sent to one of two stations for deployment. The unfolded bundles are broken using a straightening bar and then unloaded onto a conveyor that passes a magnetic separator to remove metal before crushing. A doped iron magnet is provided to capture stray magnetic metal, and a receiving screen removes oversize and foreign material before a plurality of slicer-shredder systems reduce the size of the biomass feedstock to the proper size for pretreatment.

Slicing device-crushing device systems include slicing devices and rotary knife cutting devices. Slicing devices reduce the size of the raw biomass raw material and feed the resulting material into cutting devices with a rotating knife. Cutting devices with a rotating knife simultaneously crush raw biomass and sift the resulting material.

In order to limit the total downtime due to necessary maintenance and/or interruptions in the operation of the subsystem for the preparation of the raw material, three storage silos are provided. Each silo can hold approximately 55,000 cubic feet (1,560 m3) of biomass feedstock (43 plant hours).

Preliminary processing

A conveyor belt moves the biomass feedstock from the feedstock preparation subsystem 110 to the pretreatment subsystem 114. As shown in FIG. 40, in the pre-treatment subsystem 114, biomass raw materials are irradiated using electron beam emitters, mixed with water to form a suspension and exposed to ultrasonic energy. As discussed above, irradiation of biomass raw materials changes the molecular structure (for example, reduces the average molecular weight and crystallinity) of biomass raw materials. Mixing the irradiated biomass raw material into a suspension and applying ultrasonic energy to the suspension additionally changes the molecular structure of the biomass raw material. The use of radiation exposure and sonication sequentially may have a synergistic effect in that the combination of methods appears to provide more significant changes in molecular structure (e.g., lower average molecular weight and crystallinity) compared to changes that can be effectively provided by either method alone. independently Without regard to theory, in addition to reducing the polymerization of the biomass feedstock by breaking the intramolecular bonds between the segments of the cellulosic and lignocellulosic components of the biomass feedstock, irradiation can make the overall physical structure of the biomass feedstock more fragile. After mixing the fragile biomass raw material into a suspension, the application of ultrasonic energy additionally changes the molecular structure (for example, reduces the average molecular weight and crystallinity), and can also reduce the particle size of the biomass raw material.

Irradiation with an electron beam

A conveyor belt 491 that carries the biomass feedstock to the pretreatment subsystem distributes the biomass feedstock into a plurality of feed streams (eg, 50 feed streams) each leading to separate electron beam emitters 492. In this embodiment, the biomass feedstock is irradiated in dry state Each stream of source material is transported on a separate conveyor belt to its associated electron beam emitter.

Each conveyor belt for irradiation of raw materials can be approximately one meter wide. Before reaching the electron beam emitter, a localized vibration is induced in each conveyor belt to evenly distribute the dry biomass raw material across the width of the conveyor belt.

An electron beam emitter 492 (eg, electron beam irradiators commercially available from Tyap Sogrogayop, Zap Oiedo, CA) is configured to deliver an electron dose of 100 kilograys applied at a power of 300 kW. Electron beam emitters are devices with a scanning beam with a sector width of 1 meter, corresponding to the width of the conveyor belt. In some embodiments, electron beam emitters with a large fixed beam width are used. Factors including belt/beam width, desired dose, biomass feedstock density, and applied power govern the number of electron beam emitters required for a facility processing 2,000 tons of dry feedstock per day.

Ultrasound treatment

Before applying ultrasonic energy, the irradiated raw biomass is mixed with water to form a suspension. There may be a separate sonication system associated with each electron beam feed stream, or multiple electron beam streams may be combined as a feed for a single sonication system.

In each sonication system, irradiated biomass feedstock is fed into vessel 1214 through a first inlet 1232, and water is fed into vessel 1214 through a second inlet 1234. Appropriate valves (manual or automatic) regulate the flow of biomass feedstock and water flow to obtain the desired feedstock ratio. biomass and water (for example, 1095 cellulosic material, mass by volume). Each container 1214 includes a mixer 1240 for mixing the contents of the volume 1236 and dispersing the raw biomass in water.

In each sonication system, the slurry is pumped (eg, using a vortex impeller pump 1218) from a vessel 1214 and through a flow cell 1224 that includes an ultrasonic transducer 1226. In some embodiments, the pump 1218 is adapted to agitate the slurry 1216 so that the mixture of biomass feedstock and water was essentially homogeneous in the inlet channel 1220 of the flow cell 1224. For example, the pump 1218 may agitate the slurry 1216, creating a turbulent flow that is maintained throughout the conduit between the first pump and the inlet channel 1220 of the flow cell 1224.

In flow cell 1224, an ultrasonic transducer 1226 transmits ultrasonic energy to slurry 1216 as the slurry flows through flow cell 1224. Ultrasonic transducer 1226 converts electrical energy into high-frequency mechanical energy (eg, ultrasonic energy), which is then delivered to the slurry via booster 48 .Ultrasonic transducers are commercially available (e.g., by Nieizspeg OA, Ips. oi Kipjmood, Mem Uegzeu) capable of delivering a constant power of 16 kilowatts.

Ultrasonic energy passing through the booster 1248 in the reactor volume 1244 creates a series of compressions and vacuums in the process stream 1216 with an intensity sufficient to cause cavitation in the process stream 1216. The cavitation disaggregates the components of the biomass feedstock, including, for example, cellulosic and lignocellulosic material dispersed in the process stream 1216 (eg, suspensions). Cavitation also leads to the formation of free radicals in process water 1216 (eg, slurry). These free radicals act to further destroy cellulosic material in process stream 1216. Typically, approximately 250 MJ/m3 of ultrasonic energy is applied to process stream 1216 containing fragments of poplar sawdust. Different levels of ultrasonic energy (from about 5 to about 4000 MJ/m3, such as 10, 25, 50, 100, 250, 500, 750, 1000, 2000 or 3000 MJ/m3) can be applied to other biomass feedstocks. After exposure to ultrasonic energy in the reactor volume 1244, the process flow 1216 leaves the flow cell 24 through the outlet channel 1222.

The flow cell 1224 also includes a heat exchanger 1246 in thermal contact with at least a portion of the reactor volume 1244. A cooling fluid 1248 (eg, water) enters the heat exchanger 1246 and absorbs the heat generated when the process flow 1216

(e.g., slurry) is irradiated in the reactor volume 1244. In some embodiments, the flow of cooling fluid 1248 entering the heat exchanger 1246 is regulated to maintain an approximately constant temperature in the reactor volume 1244. Additionally or alternatively, the temperature of the cooling fluid 1248 entering the heat exchanger 1246 is regulated to maintain an approximately constant temperature in the reactor volume 1244.

The outlet channel 1242 of the flow cell 1224 is located near the bottom of the container 1214 to induce gravity flow of the process flow 1216 (e.g., slurry) from the container 1214 toward the inlet channel of the second pump 1230, which pumps the process flow 1216 (e.g., slurry) toward the subsystem basic processing.

Sonication systems may include a single flow path (as described above) or multiple parallel flow paths, each associated with an individual sonication element. Multiple sonication elements can also be arranged in series to increase the amount of irradiation energy applied to the slurry.

Basic processing

Before fermentation, a vacuum filter with a rotating drum removes solid particles from the suspension. The liquid from the filter is pumped out in a cooled state before entering the fermenters.

The filtered solids pass through the downstream processing subsystem for additional processing.

Fermentation tanks are large stainless steel tanks under low pressure, with conical bottoms and slow mixers. Several containers for the first stage of fermentation can be arranged in series. The temperature in the containers for the first stage of fermentation is adjusted to

With the use of external heat exchangers. Yeast is added to the container for the first stage of fermentation at the beginning of each series of containers, and it passes through other successively located containers.

The second stage of fermentation consists of two consecutive continuous fermenters. Both fermenters are constantly stirred by slow mechanical mixers. The temperature is regulated by chilled water in external heat exchangers with reverse recirculation.

Recirculation pumps are screw-type pumps because the concentration of solid particles is high.

Gases leaving the fermentation tanks and fermenters are combined and washed in a column with a counterflow of water before being released into the atmosphere. Off-gases are scrubbed to extract ethanol, not to control emissions to the atmosphere.

Further processing

Distillation

Distillation and adsorption on molecular sieves are used to extract ethanol from raw fermentation broth and obtain 99.5% ethanol. Distillation is carried out in two columns - the first, called the fermentation column, removes dissolved CO" and most of the water, and the second concentrates ethanol almost to an azeotropic mixture.

All water from the practically azeotropic mixture is removed by adsorption on vapor phase molecular sieves.

Regeneration of adsorption columns requires that the ethanol-water mixture be returned to the distillation process for extraction.

Off-gases from fermentation (which contain mainly CO2, but also some ethanol) as well as off-gas from the fermentation column are cleaned in a water scrubber, which extracts almost all the ethanol. The gas leaving the scrubber is fed to the first distillation column together with the fermentation broth.

The sediment after the first distillation contains all unconverted insoluble and dissolved solids.

Insoluble solids are dewatered by a pressure filter and sent to the combustion chamber. Liquid from the pressure filter, which is not reused, is concentrated in a multibody evaporator using waste heat from distillation.

The concentrated syrup from the evaporator is mixed with the solids sent to the combustor, and the evaporator condensate is used as relatively clean recycle water for processing.

Since the amount of water for distillation that can be reused is limited, an evaporator is included in the process. The total amount of water from the pressure filter that is directly returned to the process is set at 2595. Organic salts such as ammonium acetate or ammonium lactate, accumulated liquid components not disposed of by the body, or inorganic compounds in the biomass are detected in this stream. Reuse of very large amounts of this material can lead to levels of ionic strength and osmotic pressure that can be detrimental to the efficiency of the fermenting organism. For water that is not reused, the evaporator concentrates the dissolved solids into a syrup that can be sent to the combustion chamber, minimizing the burden on waste water treatment.

Treatment of waste water

The waste water treatment department processes process water for reuse in order to reduce the plant's need for additional water. Waste water is first filtered to remove large particles, which are collected in a waste container and sent to a landfill. After filtration, anaerobic digestion and aerobic digestion are performed to break down the organic material in the stream. Anaerobic digestion results in a methane-rich biogas stream that is fed into the combustion chamber. Aerobic digestion results in a relatively clean water stream for reuse in the process, as well as a bottom that is primarily composed of cell mass. The sludge is also burned in the combustion chamber. This filtration/anaerobic digestion/aerobic digestion scheme is standard in today's ethanol industry, and 1-5 million gallons per day equipment can be obtained from suppliers as "off-the-shelf" units.

Combustion chamber, steam boiler and turbogenerator

The purpose of the combustion chamber, boiler and turbogenerator subsystem is to burn various by-product streams to generate steam and electricity. For example, some part of lignin, cellulose and hemicellulose remains unconverted during pretreatment and basic processes. Most of the waste water from the process is concentrated into a syrup with a high content of soluble solids. Anaerobic digestion of the remaining waste water leads to the formation of biogas with a high methane content. Aerobic digestion leads to a small amount of waste biomass (bottom). Combustion of these by-product streams to generate steam and electricity allows the plant to be energy self-sufficient, reduces the cost of solid waste disposal, and generates additional revenue through the sale of excess electricity.

Three streams of primary fuel (solids after distillation, biogas and evaporator syrup) are fed into the circulating fluidized bed combustion chamber. A small amount of waste biomass (sludge) after wastewater treatment is also sent to the combustion chamber.

The fan supplies air to the combustion chamber. Treated water enters the circuit of the heat exchanger in the combustion chamber and evaporates and is subjected to excessive heating to steam at 510 "С (950 "Р) and 86 atm. (1265 psi abs. (8.7 MPa)). Furnace gases from the combustion chamber preheat the air entering the combustion chamber and then penetrate a fabric filter to remove particles that are sent to landfill. The gas exits through the chimney.

A multi-stage turbine is used to generate electricity. Steam comes out of the turbine in three different states for injection into the reactor for pretreatment and heat exchange during distillation and evaporation. Other steam is condensed with cooling water and returned to the boiler feedwater system along with condensate from the various heat exchangers in the process. Water from a well is used as additional water to replace the flow used in direct injection.

Other implementation options

A number of variants of implementation of the invention are described. However, it is clear that various modifications can be made without departing from the essence and scope of the invention.

In some embodiments, relatively low doses of radiation, optionally in combination with sonic energy, such as ultrasonic energy, are used to cross-link, graft, or otherwise increase the molecular weight of a natural or synthetic hydrocarbon-containing material, such as any of the materials in in any form (e.g., fibrous form) described herein, such as shredded or unshredded cellulosic or lignocellulosic materials such as cellulose. Cross-linking, grafting, or otherwise increasing the molecular weight of natural or synthetic hydrocarbon-containing material can be accomplished in a controlled or predetermined manner by selecting the type or types of radiation used (eg, e-beam and ultraviolet radiation or e-beam and gamma radiation) and/or dose or number of doses of applied radiation. Such a material having an increased molecular weight may be useful in the manufacture of a composite, such as a fiber-resin composite, having improved mechanical properties such as wear resistance, compressive strength, fracture strength, impact strength, flexural strength, modulus of elasticity at stretching, modulus of elasticity at bending and elongation at break. Cross-linking, grafting or otherwise increasing the molecular weight of the selected material can increase the thermal stability of the material relative to the untreated material.

Increasing the thermal stability of the selected material can provide the ability to process at higher temperatures without degradation. In addition, radiation treatment of materials can sterilize materials, which can reduce their tendency to decay, for example, in a composite. Cross-linking, grafting, or otherwise increasing the molecular weight of a natural or synthetic hydrocarbon-containing material can be carried out in a controlled and predetermined manner for a particular application in order to provide optimal properties, such as strength, by selecting the type or types of radiation used and/or the dose or doses of radiation used. radiation.

When a combination of radiation, such as low-dose radiation, and sonic energy, such as sonic or ultrasonic energy, is used, material yield can be increased and/or energy consumption can be minimized.

The resin can be any thermoplastic plastic, thermoset plastic, elastomer, adhesive, or mixtures of these resins. Suitable resins include any resin or mixture of resins described herein.

In addition to the resin alone, the high molecular weight material can be combined, mixed with other materials, or added to other materials such as metals, metal alloys, ceramics (e.g., cement), lignin, organic or inorganic additives, elastomers, bitumens, glass or mixtures of any of these materials and/or resins. When added to cement, fiber-reinforced cements can be obtained that have improved mechanical properties, such as those described herein, such as compressive strength and/or fracture toughness.

Cross-linking, grafting, or otherwise increasing the molecular weight of a natural or synthetic hydrocarbon-containing material using radiation can provide useful materials in many forms and for many applications. For example, the hydrocarbon-containing material may be in the form of a paper product such as paper, pulp or paper waste, compressed wood, laminated wood laminates such as veneer or plywood, lumber such as pine, poplar, oak or even cork lumber. Treatment of paper, pressed wood, laminates or lumber can increase their mechanical properties, such as their strength. For example, radiation treatment of pine lumber can give high strength to the structural material.

If the paper is produced using radiation, then radiation can be used at any time during its production. For example, you can irradiate paper pulp, you can irradiate a preform of pressed fibers, or you can irradiate the finished paper itself. In some embodiments, radiation is applied more than once during the manufacturing process.

For example, a fibrous material that includes a first cellulosic and/or lignocellulosic material having a first molecular weight can be irradiated to produce a second cellulosic and/or lignocellulosic material having a second molecular weight greater than the first molecular weight. For example, if gamma radiation is used as the radiation source, a dose of from about 0.2 Mrad to about 10 Mrad can be used, for example from about 0.5 Mrad to about 7.5 Mrad or from about 2.0 Mrad to about 5, 0 Mrad. If e-beam irradiation is used, a lower dose (relative to gamma radiation) can be used, such as from about 0.1 Mrad to about 5 Mrad, for example from about 0.2 Mrad to about 3 Mrad or from about 0.25 Mrad to about 2.5 Mrad. After a relatively low dose of radiation, the second cellulosic and/or lignocellulosic material can be combined with a material such as resin and formed into a composite, for example, by compression molding, injection molding, or extrusion.

Formation of resin-fiber composites is described in MMO 2006/102543. After forming the composites, they can be irradiated to further increase the molecular weight of the hydrocarbon-containing material in the composite.

Alternatively, a fibrous material comprising a first cellulosic and/or lignocellulosic material having a first molecular weight can be combined with a material such as a resin to form a composite, and the composite can then be irradiated with a relatively low dose of radiation to form a second cellulosic and /or lignocellulosic material having a second molecular weight that exceeds the first molecular weight. For example, if gamma radiation is used as a source of radiation, a dose of about 1 Mrad to about 10 Mrad can be used. Using this approach increases the molecular weight of the material when it is in conjunction with a matrix such as a resin matrix. In some embodiments, the resin is a cross-linkable resin and, in fact, is cross-linked as the molecular weight of the hydrocarbon-containing material increases, which can create a synergistic effect to provide maximum mechanical properties of the composite.

For example, such composites may have excellent low temperature performance, such as a reduced tendency to fracture and/or crack at low temperatures, such as below 0 "C, such as below -10, -20, -40, -50, -60 "C or even below -100 "C, and/or excellent high temperature performance, such as the ability to maintain their useful mechanical properties at relatively high temperatures, such as above 100 "C, such as above 125, 150, 200, 250, 300, 400 "C or even above 500 "C. In addition, such composites can have excellent chemical resistance, such as resistance to swelling in a solvent such as a hydrocarbon solvent, resistance to chemical attack such as strong acids, strong bases, strong oxidizing agents (such as chlorine or bleach) or reducing agents (such as active metals such as sodium and potassium).

In some embodiments, the resin or other matrix material is not cross-linked during irradiation. In some embodiments, additional radiation is applied when the carbohydrate-containing material is in the matrix to further increase the molecular weight of the carbohydrate-containing material. In some embodiments, the radiation leads to the formation of bonds between the matrix and the hydrocarbon-containing material.

In some embodiments, the carbohydrate-containing material is in the form of fibers. In such embodiments, when the fibers are used in a composite, the fibers may be randomly oriented in the matrix. In other embodiments, the fibers may be substantially oriented, for example, in one, two, three, or four directions. If desired, the fibers may be continuous or discrete.

One of the following additives may be added to fibrous materials, compacted fibrous materials, or any other materials and composites described in this document.

Additives, for example in the form of a solid, liquid or gas, can be added, for example, to a combination of fibrous material and resin. Additives include fillers such as calcium carbonate, graphite, wollastonite, mica, glass, fiberglass, silicon dioxide, and talc; inorganic flame retardants such as aluminum oxide trihydrate or magnesium hydroxide; organic flame retardants, such as chlorinated and brominated organic compounds; crushed construction waste; crushed tire rubber; carbon fiber or metal fiber or powders (e.g. aluminum, stainless steel). These additives can enhance, extend or change the electrical, mechanical or compatibility properties. Other additives include lignin, fragrances, binders, compatibilizers, such as maleated polypropylene, process additives, lubricants, such as fluorinated polyethylene, plasticizers, antioxidants, opacifiers, heat stabilizers, coloring agents, foaming agents, impact modifiers strength, polymers, such as degradable polymers, photostabilizers, biocides, antistatic agents, such as stearates or ethoxylated fatty acid amines. Suitable antistatic compounds include conductive carbons, carbon fibers, metallic fillers, cationic compounds such as quaternary ammonium compounds such as M-(3-chloro-2-hydroxypropyl/utrimethylammonium chloride), alkanolamides and amines. Typical examples of degradable polymers include polyhydroxy acids such as polylactides , polyglycolides and copolymers of lactic acid and glycolic acid, poly(hydroxybutyric acid), poly(hydroxyvaleric acid), copolymer of lactide and e-caprolactone, copolymer of glycolide and e-caprolactone, polycarbonates, poly(amino acids), poly(hydroxyalkanoates), polyanhydrides , complex polyorthoesters and mixtures of these polymers.

When the described additives are included, they may be present in amounts calculated on a dry weight basis ranging from less than 1 95 to 80 95 based on the total weight of the fibrous material. More typically, amounts range from about 0.5 wt. 95 to about 50 wt. 95 such as 5, 10, 20, 30 95 or more such as 40 95.

Any additives described herein may be encapsulated, such as spray-dried, or microencapsulated, such as to protect the additives from heat or moisture during storage.

Fibrous materials, compacted fibrous materials, resins or additives may be colored. For example, the fibrous material may be colored before being combined with resin and mixed to form composites. In some embodiments, this coloring may be useful in masking or concealing fibrous material, especially large agglomerates of fibrous material, in molded or extruded parts when desired. Such large agglomerates, when present in relatively high concentrations, may appear as grains on the surfaces of molded or extruded parts.

For example, the desired fibrous material can be dyed using an acid dye, a direct dye, or a reactive dye. Such dyes are available from Zresig Ruev,

Keagpu, MU or Keusiope Apiiipe Sogrogayop, Spisado, IS. Specific examples of dyes include

ZRESTNA"M ISHNT ME OMU 20, 5RESTVASIUTM MB OMU 40Ii SOMO 200, 5RESTAVAMUI M

ANORAMIME 8, 5RESTAVAMMU! M MEOTKAI KEO V, 5RESTKAMIMEM VEM2OREVRYVIME,

ZRESTAAVIA7O"M VI ASK OV, 5BRESTAAMIME M TOKOTSOYIIZE o and BRESTKAMIME "M SVEM I MI. 200 oo, each of which is available from Zresig Yuuev.

In some embodiments, concentrates of dyes in resins containing pigments are mixed with dyes. When such mixtures are then mixed with the desired amount of fibrous material, the fibrous material may be colored by the mixing process. Dye concentrates are available from

Siapapi

It may be useful to add a flavoring or perfuming agent to the fibrous material, compacted fibrous material, or composites. For example, it may be preferable for composites to have an odor and to be similar to natural wood, such as cedar. For example, fragrances, such as those with the aroma of natural wood, can be introduced into the resin used to make the composite. In some embodiments, the fragrance is injected directly into the resin as an oil.

For example, oil can be introduced into the resin using a roller mill, such as a mixer

Vaprigufb or extruder, for example, a twin-screw extruder with counter-rotating screws. An example of a VapriguF mixer is the E-bZegie5 Vapriguye mixer manufactured by RaiteI. An example of a twin-screw extruder is the MUR 75K 50 MESAsotripaeg"M manufactured by Ktyrr Umegpeg 5 RiPeidegeg. After introduction, the flavored resin can be added to the fibrous material and extruded or molded.

Alternatively, flavored resin mother blends are commercially available from Ipiegpabopai

Eiamog5 apa Rgadgapseh5 under the trade name RoiuiM or from KTR Sotrapu. In some embodiments, the amount of fragrance in the composite is from about 0.005 wt. 95 to about 10 wt. 95, such as from about 0.1 95 to about 5 95 or from about 0.25 95 to about 2.5 95.

Other natural wood fragrances include conifer or mahogany fragrances. Other fragrances include peppermint, cherry, strawberry, peach, lime, spearmint, cinnamon, anise, cornflower, bergamot, black pepper, camphor, chamomile, citronella, eucalyptus, pine, fir, geranium, ginger , grapefruit, jasmine, juniper berry, lavender, lemon, mandarin, marjoram, musk, myrrh, orange, patchouli, rose, rosemary, sage, sandalwood, tea tree, thyme, pear, ylang-ylang, vanilla, "new car " or mixtures of these fragrances. In some embodiments, the amount of fragrance in the fibrous material-fragrance combination is from about 0.005 wt. 95 to about 20 wt. 95, such as from about 0.1 95 to about 5 95 or from about 0.25 95 to about 2.5 95.

Although fibrous material such as cellulosic and lignocellulosic fibrous materials have been described, other fillers can be used to produce composites. For example, inorganic fillers can be used, such as calcium carbonate (eg, precipitated calcium carbonate or natural calcium carbonate), aragonite clay, orthorhombic clay, calcite clay, rhombohedral clay, kaolin, clay, bentonite clay, dicalcium phosphate, tricalcium phosphate, calcium pyrophosphate, insoluble sodium metaphosphate, precipitated calcium carbonate, magnesium orthophosphate, trimagnesium phosphate, hydroxyapatites, synthetic apatites, aluminum oxide, xerogel based on silicon dioxide, aluminosilicate metal complexes, sodium aluminosilicates, zirconium silicate, silicon dioxide, or combinations of inorganic additives. The fillers may have, for example, a particle size greater than 1 micrometer, such as greater than 2, 5, 10 micrometers, 25 micrometers, or even greater than 35 micrometers.

It is also possible to use fillers in the nanometer range alone or in combination with fibrous materials of any size or shape. Fillers can be in the form of, for example, particles, plates or fibers. For example, nanometer-sized clays, silicon and carbon nanotubes, and silicon and carbon nanowires can be used. The filler may have a transverse dimension of less than 1000 nm, for example less than 900, 800, 750, 600, 500, 350, 300, 250, 200 nm, less than 100 nm, or even less than 50 nm.

In some embodiments, the nanoclay is montmorillonite. Such clays are available from

Maposog, Ips. and Siau Rhodysov, and are described in US Patent Nos. 6,849,680 and 6,737,464. Clays may be surface treated prior to incorporation into, for example, a resin or fibrous material. For example, clay can be surface treated so that its surface has ionic properties, such as cationic or anionic.

It is also possible to use aggregated or agglomerated fillers in the nanometer range or fillers in the nanometer range that assemble into supramolecular structures, such as self-aggregating supramolecular structures. Aggregate fillers or fillers with a supramolecular structure can have an open or closed structure, and they can have different shapes, for example lattice, tubular or spherical.

Thus, other variants of implementation are included in the scope of the claims presented below.

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Fig. 33

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Fig. 34

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Claims (24)

1. A method of fermenting a low-molecular-weight sugar into a product that includes: mixing the low-molecular-weight sugar, one or more fermenting microorganisms, and modified biomass, wherein the modified biomass has a bulk density of less than about 0.5 g/cm" and contains cellulosic fibers, which have essentially been irradiated and contain carboxylic acid groups; and fermentation of the low molecular weight sugar under conditions suitable for converting the sugar into a product. 2. The method according to claim 1, where in the fermentation process at least part of the low molecular weight sugar is converted into ethanol. 3. The method according to claim 2, where the microorganism includes yeast. 4. The method according to claim 3, where the yeast is selected from the group consisting of 5. segeuiviaye 1 R. vypriyv. 5. The method according to claim 2, where the microorganism includes bacteria. 6. The method according to claim 5, wherein the bacteria include Lutotopav tobii. 7. The method according to claim 1, where the modified biomass is obtained by irradiation with ionizing radiation. 8. The method according to claim 1, where the modified biomass is obtained by irradiation with a beam of particles. 9. The method according to claim 1, where the modified biomass is obtained by irradiation under conditions selected to reduce the molecular weight of the biomass. 10. The method according to claim 1, where the modified biomass is obtained by impacting the biomass with ionizing radiation, the total dose of which is approximately 5 Mrad. 11. The method according to claim 1, which additionally includes physical preparation of biomass. 12. The method according to claim 11, wherein the preparation includes crushing. 13. The method according to claim 11, wherein the preparation includes reducing the size of the biomass by mill grinding, mechanical loosening or tearing, grinding on a rod mill, grinding or grinding by air grinding. 14. The method according to claim 1, where the biomass has a VET surface area greater than 0.25 m"/g. 15. The method according to claim 1, where the biomass has a ratio of length to diameter equal to at least 5. 16. The method according to claim 1, where the modified biomass is selected from the group consisting of paper, paper products, paper waste, wood, compressed wood, wood sawdust, agricultural waste, sewage, silage, grasses, rice husk, cake, cotton, jute, hemp, flax, bamboo, sisal, abaca, straw, corncob cores, corn straw, millet, alfalfa, hay, coconut fiber, sea grass, seaweed and mixtures thereof. 17. The method according to claim 1, where the biomass has a porosity greater than 50 95. 18. The method according to claim 1, which additionally includes the effect on biomass by enzymatic hydrolysis. 19. The method according to claim 4, where in the fermentation process ethanol is obtained with a yield equal to at least 140 90, which is determined by the equation: o yield - (ethanol in the sample taken from the medium containing the modified biomass/ethanol in the control taken from the medium without modified biomass) x 100. 20. The method according to claim 19, where the fermentation is characterized by a yield equal to at least 170 to. 21. The method according to claim 19, where the first product is formed in the fermentation process, while the method additionally includes the stage of converting the first product into a second product that differs from the first product. 22. The method according to claim 1, where the medium contains water. 23. The method of claim 1, wherein the bulk density is less than about 0.4 g/cm". 24. The method of claim 1, wherein the bulk density is less than about 0.35 g/cm".