BR122013007240B1 - BIOMASS PROCESSING METHOD - Google Patents

Description

(54) Title: METHOD FOR PROCESSING BIOMASS

(73) Holder: XYLECO, INC .. Address: 271 Salem St., Unit L Woburn, Massachusetts, 01801, UNITED STATES OF AMERICA (US) (72) Inventor: MARSHALL MEDOFF; THOMAS MASTERMAN; HARRISON MEDOFF

Validity Term: 20 (twenty) years from 05/18/2010, subject to legal conditions

Issued on: 10/02/2018

Digitally signed by:

Liane Elizabeth Caldeira Lage

Director of Patents, Computer Programs and Topographies of Integrated Circuits

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Descriptive Report of the Invention Patent for METHOD FOR PROCESSING BIOMASS.

Split order from PI1010672-3, deposited on 18.05.2010. RELATED REQUESTS

This application claims priority to US Provisional Order Serial No. 12 / 417,840, filed on April 4, 2009, US Provisional Order Serial No. 61 / 180,032, filed on May 20, 2009 and US Provisional Order No. Series 61 / 252.293, filed on October 16, 2009. The full disclosure of each of these provisional applications is hereby incorporated by reference.

BACKGROUND

Cellulosic and lignocellulosic materials are produced, processed and used in large quantities in a number of applications. Often, such materials are used once and then discarded as waste or are simply considered to be waste materials, for example, wastewater, bagasse, sawdust and straw.

Various cellulosic and lignocellulosic materials, their uses and applications have been described in U.S. Patent Nos. 7,074,918, 6,448,307, 6,258,876, 6,207,729, 5,973,035 and 5,952,105; and in several patent applications, including FIBROUS MATERIALS AND COMPOSITES, PCT / US2006 / 010648, filed March 23, 2006 and FIBROUS MATERIALS AND COMPOSITES, U.S. Patent Application Publication No. 2007/0045456.

SUMMARY

In some cases, the presence of biomass in a process, for example, fermentation, facilitates the conversion of a low molecular weight sugar to an intermediate or a product. The inventors have found that inclusion of biomass in a mixture with a low molecular weight sugar, a medium, for example, a solvent or solvent system and a microorganism can improve the yield and production rate of an intermediate or a product obtained by converting sugar, for example, an alcohol, such as ethanol or butanol. Inclusion of biomass can

2/117 also prevent incomplete, slow or compromised product conversion, for example, through fermentation.

Biomass itself cannot be converted to the product (such as an alcohol) or it can be partially or totally converted to the product together with low molecular weight sugar.

In cases where the biomass is partially converted, the surface area and porosity of the biomass are increased in relation to the surface area and porosity of the initial biomass which can, advantageously, increase the conversion rate of low molecular weight sugar to the product.

In some cases, biomass may be the remains of a cellulosic or lignocellulosic material that has been saccharified, for example, lignin and / or other materials that are left over after cellulose has been converted into sugar.

In one aspect, the invention is characterized by a method that includes the use of a microorganism and / or enzyme that is immobilized on a biomass material, for example, functionalized biomass fibers, to convert a carbohydrate, for example, a sugar low molecular weight, to a product. By immobilized it is understood that the microorganism and / or enzyme is linked, directly or indirectly (for example, through a chemical ligand) to the fibers by means of covalent, hydrogen, ionic and / or equivalent and / or through of mechanical interaction, for example, between the microorganism and pores of the biomass material, for example, fibers. Bonding can be created, for example, through electrical polarization of the biomass material. The interaction can be permanent, semi-permanent or transient. Mechanical interaction may include the microorganism or enzyme being in or attaching itself to the pores or other locations of the biomass material.

Some implementations include one or more of the following characteristics.

Conversion may include allowing the microorganism to convert at least part of the low molecular weight sugar to an alcohol, for example

3/117 example, ethanol or butanol or to a hydrocarbon or hydrogen. Conversion can include fermentation. The microorganism can comprise a yeast, for example, S. cerevisiae and / or P. stipitis or a bacterium, for example, Zymomonas mobilis. The method may also include irradiation of the biomass fibers, for example, with ionizing radiation, for example, using a particle beam. Biomass fibers can have a BET surface area of more than 0.25 m ² / g and / or a porosity of at least 70%. Biomass fibers can be derived from a biomass material that has internal fibers and that has been sheared to a point where its internal fibers are substantially exposed.

In another aspect, the invention is characterized by a mixture that includes a biomass material having polar functional groups, a microorganism having attractive functional groups and a liquid medium.

In another aspect, the invention is characterized by a composition comprising biomass fibers having functional groups and a microorganism having complementary attractive functional groups, the microorganism being immobilized on the biomass fibers.

The invention is also characterized by a method that includes conversion of a low molecular weight sugar or a material that includes a low molecular weight sugar, into a mixture with a biomass, a microorganism and a solvent or a solvent system, for example, water or a mixture of water and an organic solvent, to a product. Examples of solvents or solvent systems include water, hexane, hexadecane, glycerol, chloroform, toluene, ethyl acetate, petroleum ether, liquefied petroleum gas (LPG), ionic liquids and mixtures thereof. The solvent or solvent system can be in the form of a single phase or two or more phases. The biomass can be, for example, in fibrous form.

In some cases, having a biomass material (for example, treated by any method described here or untreated) present during the production of a product, can increase the product's production rate. Without wishing to be bound by any particular theory, it is believed4 / 117 that having a solid present, 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 constituting a substrate on which reactions can occur.

In some embodiments, a biomass material that has been irradiated, oxidized, chemically treated, mechanically treated, subjected to ultrasound, steam explosion and / or pyrolysis, can be added to a low molecular weight sugar fermentation process, for example, to intensify the fermentation rate and production.

For example, an irradiated or non-irradiated biomass material, for example, a paper fiber, can be added to a fermentation process, such as during a corn-ethanol fermentation or a sugarcane extract fermentation, to increase the production rate by at least 10, 15, 20, 30, 40, 50, 75, 100 percent or more, for example, at least 150 percent or even up to 1000 percent. Conversion, for example, fermentation, can exhibit a percentage performance, as defined in the Examples here, of at least 140%, in some cases at least 170%.

The biomass material can have a high surface area, high porosity and / or low volumetric density. In some embodiments, biomass is present in the mixture from about 0.5 percent to about 50 percent by weight, such as between about 1 percent and about 25 percent by weight or between about 2 percent and about 12.5 weight percent. In other embodiments, biomass is present in amounts greater than about 0.5 weight percent, such as more than about 1.2, 3, 4, 5, 6, 7, 8, 9 or even more than about 10 percent by weight.

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

Some implementations include one or more of the following characteristics.

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The method may include irradiation of the fibrous biomass before mixing, for example, with ionizing radiation, for example, in a total dosage of at least 5 Mrad. Irradiation can be carried out using a particle beam. Irradiation can be conducted under selected conditions to reduce the molecular weight of the biomass. Irradiation can be performed using multiple applications of radiation. Ionizing radiation can include electron beam radiation. For example, radiation can be applied at a total dose of between about 10 Mrad and about 150 Mrad, such as at a dose rate of about 0.5 to about 10 Mrad / day or about 1 Mrad / s about 10 Mrad / s. In some embodiments, irradiation includes the application of two or more radiation sources, such as gamma rays and an electron beam.

In some modalities, irradiation is carried out on the raw material of biomass while the raw material of biomass is exposed to air, nitrogen, oxygen, helium or argon. In some modalities, pretreatment may include pre-treatment of the biomass raw material with steam explosion.

In some embodiments, the method includes mechanical treatment of the biomass, for example, by reducing one or more dimensions of individual parts of the biomass, for example, by shearing, using an emery stone, mechanical cutting or tearing, pin crushing , wet or dry grinding, air friction grinding, cutting, tightening, compression or combinations of any of these processes. In some cases, after mechanical treatment, biomass includes fibers having an average length-to-diameter ratio of more than 5/1. In some embodiments, the prepared biomass may have a BET surface area of more than 0.25 m ² / g. The mechanically treated biomass can have a volumetric density of less than about 0.5 g / cm ³ , for example, less than 0.35 g / cm ³ .

In any of the methods disclosed here, radiation can be applied from a device that is in a dome.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly en6 / 117 tended by those versed in the field to which the present invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned here or attached thereto, are incorporated by reference in full by everything they contain. In case of conflict, this specification, including definitions, will prevail. 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 be apparent from the following detailed description and claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a block diagram illustrating the treatment of biomass and the use of treated biomass in a fermentation process.

FIG. 2 is a schematic representation of functionalized biomass that interacts with a microorganism.

FIG. 3 is an infrared spectrum of Kraft paper sheared over a rotary knife cutter.

FIG. 4 is an infrared spectrum of the Kraft paper of FIG. 3 after irradiation with 100 Mrad of radiation range.

FIGS. 5A-5I are ¹ H-NMR spectra of samples P132, P132-10, P132-100, P-1e, P-5e, P-10e, P-30e, P-70e and P-100e in Example 13. A FIG. 5J is a comparison of the ~ 16 ppm exchangeable proton of FIGS. 5A-5I. FIG. 5K is a ¹³ C-NMR from the P-100e sample. FIGS. 5L-5M is ¹³ C-NMR of sample P-100e with a delay time of 10 seconds. FIG. 5N is a ¹ H-NMR at a concentration of 10% by weight / weight of the sample P-100e.

Figure 6 is a graph of the glucose concentration (g / L) by time (hour) of the 4 best producers.

Figure 7A is a graph of cell concentrations (x 108 cells per ml) for Z. mobilis in 24 or 36 hours.

Figure 7B is a graph of ethanol concentrations (g / L) for

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Z. mobilis in 24, 30 or 36 hours.

Figure 7C is a graph of percent growth and ethanol production for Z. mobilis.

Figure 8A is a graph of cell concentrations (x 108 cells per ml) for P. stipitis in 24 or 48 hours.

Figure 8B is a graph of ethanol concentrations (g / L) for P. stipitis at 24, 36 or 48 hours.

Figure 9C is a graph of the percent growth and ethanol production for P. stipitis.

Figure 9A is a graph of cell concentrations (x 108 cells per ml) for S. cerevisiae in 24 or 36 hours.

Figure 9B is a graph of ethanol concentrations (g / L) for S. cerevisiae over 24, 30 or 36 hours.

Figure 9C is a graph of the percentage growth and ethanol production for S. cerevisiae.

DETAILED DESCRIPTION

Functionalized biomass materials having desired types and amounts of functionality, such as carboxylic acid groups, enol groups, aldehyde groups, ketone groups, nitrile groups, nitro groups or nitrous groups, can be prepared using the methods described here. Such functionalized materials can facilitate the conversion of low molecular weight sugar to a product, for example, during a fermentation process. TYPES OF BIOMASS

Preferred biomass materials for use in the process described here contain fibers which can be functionalized with functional groups that are complementary with functional groups on an agent to be used in converting sugar, for example, a microorganism, such as yeast.

Fiber sources include cellulosic fiber sources, including paper and paper products (for example, poly-coated paper and Kraft paper) and lignocellulosic fiber sources, including wood and wood-related materials, for example, particleboard. Other suitable fiber sources

8/117 include sources of natural fiber, for example, grasses, rice husks, bagasse, cotton, jute, hemp, flax, bamboo, sisal, abaca, straw, corn cobs, rice husks, coconut fur; fiber sources with a high content of acellulose, for example, cotton; and synthetic fiber sources, for example, extruded yarn (oriented yarn or non-oriented yarn). Sources of natural or synthetic fiber can be obtained from cutouts of virgin textile materials, for example, remaining textile materials or they can be a post-consumer waste, for example, rags. When paper products are used as a fiber source, they can be virgin materials, for example, cutouts of virgin materials or they can be post-consumer waste. In addition to virgin raw materials, post-consumer, industrial (eg, refuse) and processing (eg, paper processing effluent) waste can also be used as fiber sources. Also, the fiber source can be obtained from or derived from the waste of a human (e.g., sewage), animal or plant. Additional sources of fiber have been described in U.S. Patent Nos. 6,448,307, 6,258,876, 6,207,729, 5,973,035 and 5,952,105.

In some embodiments, the biomass material includes a carbohydrate that is or includes a material having one or more β-1,4-bonds and having an average numerical molecular weight between about 3,000 and 50,000. Such carbohydrate is or includes cellulose (I), which is derived from (β-glucose 1) through condensation of β (1 -> 4) -glycosidic bonds. This bond contrasts, in itself, with that for bonds to (1 - + 4) -glycosides present in starch and other carbohydrates.

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Starch materials include starch itself, for example, corn starch, wheat starch, rice starch or starch, a starch derivative, or a material that includes starch, such as an edible food product or a crop. For example, the starch material can be squid, wild wheat, banana, barley, cassava, kudzu, hollow, sago, sorghum, regular domestic potatoes, sweet potatoes, taro, yams or one or more beans, such as broad beans, lentils or peas. Mixtures of any two or more starch materials are also starch materials.

In some cases, biomass is a microbial material. Microbial sources include, but are not limited to, any naturally occurring or genetically modified microorganism or organism that contains or is capable of providing a source of carbohydrates (eg cellulose), eg protist animals (eg protozoa , such as flagellates, amoeboids, ciliates and sporozoans) and protist plants (for example, algae such as honeycombs, chloroachnophytes, cryptophytes, euglenoids, glaucophytes, haptophytes, red algae, stramenopils and viridaeplantae). Other examples include seaweed, plankton (e.g., macroplankton, mesoplankton, microplankton, nanoplankton, picoplankton and femptoplankton), phytoplankton, bacteria (e.g. gram positive bacteria, gram negative and extremophilic bacteria), yeasts and / or mixtures thereof. In some cases, microbial biomass can be obtained from natural sources, for example, oceans, lakes, bodies of water, for example, salt water or fresh water or on land. Alternatively or in addition, microbial biomass can be obtained from culture systems, for example, wet and dry large-scale culture systems.

Mixtures of any biomass materials described here can be used to produce any of the intermediates or products described here. For example, mixtures of cellulosic materials and starch materials can be used to produce any product described here.

SYSTEMS FOR TREATING BIOMASS AND USE OF BIOMASS

TREATMENT IN FERMENTATION

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The figure shows a system 100 for converting biomass, particularly fibrous biomass and then using the treated biomass to intensify a fermentation process. System 100 includes a module 102 in which a biomass raw material is mechanically treated, for example, exposing the internal fibers of the raw material. Examples of mechanical treatments will be described in detail below. The system 100 also includes a module 104 in which the mechanically treated raw material is functionalized, for example, by means of irradiation. After functionalization, the functionalized fibers are distributed to a fermentation system 106 through a distribution module 108.

Functionalized fibers are then present during fermentation and intensify the fermentation process by providing a substrate that can interact with the microorganisms used in fermentation, for example, yeast cells. This interaction is shown schematically in figure 2, which represents a functionalized polar fiber 10 and a yeast cell 12 having a complementary polar functional group. Due to the polarity of the fibers and the yeast cell, the cell can become immobilized on one or more of the fibers. Bonding of the yeast cell (or other microorganism) to the fibers can be through hydrogen bonding or through covalent or ionic bonding. In some cases, the functional groups on the fibers may react with those on the microorganism, forming a covalent bond. The increased surface area and porosity of the biomass material that results from mechanical treatment (for example, in module 102) constitutes a greater surface area for fiber and microorganism interaction and, thus, intensifies this interaction. Immobilized cells are more productive, increasing the efficiency and yield of the fermentation process and preventing the process from becoming prematurely compromised.

It should be noted that, if mixing is carried out during fermentation, the mixing is preferably relatively mild (low shear) in order to minimize disruption of the interaction between microorganisms and fibers. In some embodiments, jet blending is used, as described

11/117 in USSN 61 / 218,832 and USSN 61 / 179,995, the full disclosures of which are incorporated herein by reference.

Referring again to figure 1, fermentation produces a mixture of crude ethanol, which flows into a 110 containment tank. Water or other solvent and other non-ethanolic components are extracted from the crude ethanol mixture using an extraction column 112 and ethanol it is then distilled using a distillation unit 114, for example, a rectifier. Finally, the ethanol can be dried using a 116 molecular sieve, denatured if necessary and sent to a desired transport method.

In some cases, the systems described here or components thereof may be portable, so that the system can be transported (for example, by train, truck or ship) from one location to another. The method steps described here can be performed in one or more locations, and in some cases, one or more of the steps can be performed in transit. Such mobile processing is described in U.S. Serial No. 12 / 374,549 and International Application No. WO 2008/011598, the full disclosures of which are incorporated herein by reference.

Any or all of the steps in the method described here can be performed at room temperature. If desired, cooling and / or heating can be employed during certain stages. For example, the raw material can be cooled during mechanical treatment to increase its fragility. In some modalities, cooling is employed before, during or after the initial mechanical treatment and / or subsequent mechanical treatment. Cooling can be performed as described in U.S. Document Serial No. 12 / 502,629, the full disclosure of which is incorporated herein by reference. In addition, the temperature in the fermentation system 106 can be controlled to intensify fermentation.

PHYSICAL TREATMENT

Physical treatment processes that can be used to change the morphology of the biomass material and / or functionalize the po12 / 117 material may include one or more of any of those described here, such as mechanical treatment, chemical treatment, irradiation, ultrasound , oxidation, pyrolysis or vapor explosion. Treatment methods can be used in combinations of two, three, four or even all of these technologies (in any order). When more than one treatment method is used, the methods can be applied at the same time or at different times. Other processes that functionalize a biomass raw material and / or alter its morphology can also be used, alone or in combination with the processes disclosed here.

Mechanical Treatments

In some cases, methods may include mechanical treatment of the biomass raw material. Mechanical treatments include, for example, cutting, grinding, pressing, grinding, shearing and shearing. Grinding can include, for example, ball grinding, hammer grinding, dry or wet grinding with a rotor / stator or other types of grinding. Other mechanical treatments include, for example, grinding emery stone, fracturing, cutting or tearing, pin grinding or grinding by friction with air.

Mechanical treatment can be advantageous for opening, tensioning, breaking and dispersing cellulosic or lignocellulosic materials, making the cellulose of the materials more susceptible to chain splitting and / or reduced crystallinity. Open materials can also be more susceptible to oxidation when irradiated.

In some cases, mechanical treatment may include an initial preparation of the raw material as received, for example, size reduction of the materials, such as by cutting, grinding, shearing, spraying or chipping. For example, in some cases, loose raw material (for example, recycled paper, starch materials or switchgrass) is prepared by shearing or shearing.

Alternatively or in addition, the raw material material can first be physically treated using one or more of the other physical treatment methods, for example, chemical treatment, radiation, ultrasound, oxidation, pyrolysis or vapor explosion and then , mechanically treated. This sequence can be advantageous, since materials treated by one or more of the other treatments, for example, irradiation or pyrolysis, tend to be more fragile and, therefore, it may be easier to further change the molecular structure of the material through treatment mechanical.

In some embodiments, the biomass material is fibrous and mechanical treatment includes shear to expose the fibers of the fibrous material. Shearing can be performed, for example, using a rotary knife cutter. Other methods of mechanical treatment of biomass include, for example, grinding or grinding. Milling can be carried out using, for example, a hammer mill, ball mill, colloidal mill, cone or conical mill, disc mill, wedge mill, Wiley mill or grain mill. Grinding can be carried out using, for example, a grinder with emery stone, pin grinder, coffee grinder or sludge grinder. Crushing can be checked, for example, by a reciprocating pin or another element, as is the case in a pin mill. Other methods of mechanical treatment include cutting or mechanical tearing, other methods that apply pressure to the material and grinding by friction with air. Adequate mechanical treatments include any other technique that alters the molecular structure or morphology of the biomass material.

If desired, the mechanically treated material can be passed through a sieve, for example, having an average opening size of 1.59 mm or less (1/16 inch, 0.0625 inch). In some modalities, shearing or other mechanical treatment and screening are carried out concurrently. For example, a rotary knife cutter can be used to cut and sieve the biomass material concurrently. Biomass is sheared between stationary blades and rotating blades to provide a sheared material that passes through a sieve and is captured in a container.

The biomass material can be mechanically treated in a dry state (for example, having little or no free water over its

14/117 surface), a hydrated state (for example, having up to ten percent by weight of water absorbed) or in a wet state, for example, having between about 10 percent and about 75 percent by weight of water . The biomass material can even be mechanically treated while partially or totally submerged under a liquid, such as water, ethanol or isopropanol. Biomass material can also be mechanically treated under a gas (such as a current or atmosphere other than air), for example, oxygen or nitrogen or steam.

Mechanical treatment systems can be configured to produce chains with specific morphological characteristics such as, for example, surface area, porosity, volumetric density and, in the case of fibrous feed stocks, fiber characteristics such as length-to-length ratio width.

In some embodiments, a BET surface area of the mechanically treated material is greater than 0.1 m ² / g, for example, greater than 0.25 m ² / g, greater than 0.5 m ² / g, greater than 1.0 m ² / g, greater than 1.5 m ² / g, greater than 1.75 m ² / g, greater than 5.0 m ² / g, greater than 10 m ² / g, greater than 25 m ² / g, greater than 35 m ² / g, greater than 50 m ² / g, greater than 60 m ² / g, greater than 75 m ² / g, greater than than 100 m ² / g, greater than 150 m ² / g, greater than 200 m ² / g or even greater than 250 m ² / g.

A porosity of mechanically treated material can be, for example, greater than 20 percent, greater than 25 percent, greater than 35 percent, greater than 50 percent, greater than 60 percent, greater than 70 percent, greater than 80 percent, greater than 85 percent, greater than 90 percent, greater than 92 percent, greater than 94 percent, greater than 95 percent, greater than 97 , 5 percent, greater than 99 percent or even greater than 99.5 percent.

In some embodiments, after mechanical treatment, the material has a volumetric density of less than 0.25 g / cm ³ , for example, 0.20 g / cm ³ , 0.15 g / cm ³ , 0.10 g / cm ³ , 0.05 g / cm ³ or less, for example, 0.025 g / cm ³ . Volumetric density is determined using ASTM D1895B. Briefly, the method involves filling a measuring cylinder

15/117 of known volume with a sample and obtaining a sample weight. The volumetric density is calculated by dividing the sample weight, in grams, by the known volume of the cylinder in cubic centimeters.

If biomass is a fibrous material, the fibers of the mechanically treated material may have a relatively large average length-to-diameter ratio (for example, greater than 20-to-1), even if they have been sheared more than once. In addition, the fibers of the fibrous materials described herein may have a relatively limited length-to-diameter ratio and / or length distribution.

As used here, average fiber widths (for example, diameters) are those determined optically by randomly selecting approximately 5,000 fibers. Average fiber lengths are weighted average corrected lengths. BET surface areas (Brunauer, Emmet and Teller) are multi-point surface areas and porosities are those determined by means of mercury porosimetry.

If biomass is a fibrous material, the average length-to-diameter ratio of the fibers in the mechanically treated material can be, for example, greater than 8/1, For example, greater than 10/1, greater than 15/1, greater than 20/1, greater than 25/1 or greater than 50/1. An average fiber length of the mechanically treated material can be, for example, between about 0.5 mm and 2.5 mm, for example, between about 0.75 mm and 1.0 mm and an average width (for example , diameter) of the second fibrous material 14 can be, for example, between about 5 pm and 50 pm, for example, between about 10 pm and 30 pm.

In some embodiments, if the biomass is a fibrous material, the standard deviation of the fiber length of the mechanically treated material may be less than 60 percent of an average fiber length of the mechanically treated material, for example, less than 50 percent of the average length, less than 40 percent of average length, less than 25 percent of average length, less than 10 percent of average length, less than 5 percent of average length or even less than 1 percent of average length.

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In some situations, it may be desirable to prepare a material with a low volumetric density, densify the material (for example, make it easier and less expensive to transport to another location) and then revert the material to a state of lower volumetric density. . Densified materials can be processed using any of the methods described here or any material processed using any of the methods described here can subsequently be densified, for example, as disclosed in US Serial No. 12 / 429.045 and WO 2008/073186 , the full disclosures of which are incorporated herein by reference.

Radiation Treatment

One or more irradiation processing sequences can be used to process the biomass, for example, to functionalize the material. Radiation can also sterilize the materials or any means necessary to bioprocess the material.

In some embodiments, the energy deposited in a material that releases an electron from its atomic orbital is used to irradiate the materials. Radiation can be provided by 1) heavily charged particles, such as alpha particles or protons, 2) electrons produced, for example, in electron beam accelerators or beta decline or 3) electromagnetic radiation, for example, gamma rays, x-rays or ultraviolet rays. In one approach, radiation produced by radioactive substances can be used to irradiate the raw material. In some modalities, any combination in any order or concurrently (1) to (3) can be used. The doses applied depend on the desired effect and the raw material in particular.

In some cases, when chain splitting is desirable and / or polymeric chain functionalization is desirable, particles heavier than electrons, such as protons, helium nuclei, argon ions, silicon ions, neon ions, carbon ions , phosphorus ions, oxygen ions or nitrogen ions can be used. When ring-split chain cleavage is desired, positively charged particles can be used for their Lewis acid properties for enhanced ring-split chain cleavage. For example, when maximum oxidation is desired, oxygen ions can be used and, when maximum nitration is desired, nitrogen ions can be used. The use of heavy particles and positively charged particles is described in U.S. Document Serial No. 12 / 417,699, the full disclosure of which is incorporated herein by reference.

In one method, a first material that is or includes cellulose having a first average numerical molecular weight (M _N i) is irradiated, for example, by treatment with ionizing radiation (for example, in the form of gamma radiation, x-ray radiation, ultra-violet (UV) light from 100 nm to 280 nm, an electron beam or other charged particles) to provide a second material that includes cellulose having a second average numerical molecular weight (M _N2 ) less than the first numerical molecular weight medium. The second material (or the first and second materials) can be combined with a microorganism (with or without enzymatic treatment) that can use the second and / or the first material or its constituent sugars or lignin to produce a product or intermediate, such as those described here.

Since the second material includes cellulose having a reduced molecular weight with respect to the first material and, in some cases, reduced crystallinity as well, the second material is, in general, more dispersible, swelling and / or soluble, for example, in a solution containing a microorganism and / or an enzyme. These properties make the second material easier to process and more susceptible to chemical, enzymatic and / or biological attack compared to the first material, which can greatly improve the production rate and / or production level of a desired product, for example , ethanol.

In some embodiments, the second average numerical molecular weight (M _N2 ) is less than the first average numerical molecular weight (M _N i) by more than about 10 percent, for example, 15, 20, 25, 30, 35 , 40, 50 percent, 60 percent or even more than about 75 percent.

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In some cases, the second material has cellulose which has a crystallinity (C ₂ ) that is less than the crystallinity (C-ι) of the cellulose of the first material. For example, (C ₂ ) can be less than (Ch) by more than about 10 percent, for example, 15, 20, 25, 30, 35, 40 or even more than about 50 percent.

In some embodiments, the initial crystallinity index (before irradiation) is about 40 to about 87.5 percent, for example, about 50 to about 75 percent or about 60 to about 70 percent and the crystallinity index after irradiation is about 10 to about 50 percent, for example, about 15 to about 45 percent or about 20 to about 40 percent. However, in some modalities, for example, after extensive irradiation, it is possible to have a crystallinity index of less than 5 percent. In some embodiments, the material after irradiation is substantially amorphous.

In some embodiments, the initial average numerical molecular weight (before irradiation) is from about 200,000 to about 3,200,000, for example, from about 250,000 to about 1,000,000 or from about 250,000 to about 700,000 and the weight average numerical molecular number after irradiation is from about 50,000 to about 200,000, for example, from about 60,000 to about 150,000 or from about 70,000 to about 125,000. However, in some embodiments, for example, after extensive irradiation, it is possible to have an average numerical molecular weight of less than about 10,000 or even less than about 5,000.

In some embodiments, the second material may have an oxidation level (O ₂ ) that is higher than the oxidation level (O ^ of the first material. A higher oxidation level of the material can aid in its dispersibility, swelling capacity and / or solubility, further intensifying the material's susceptibility to chemical, enzymatic or biological attack.In some modalities, to increase the oxidation level of the second material in relation to the first material, irradiation is carried out under an oxidation environment, for example, under a current of air or oxygen, producing a second material that is more oxidized than

19/117 the first material. For example, this second material can have more hydroxyl groups, aldehyde groups, ketone groups, ester groups or carboxylic acid groups, which can increase its hydrophilicity.

Ionizing radiation

Each form of radiation ionizes the carbon-containing material via particular interactions, as determined by the radiation energy. Heavily charged particles primarily ionize matter via Coulomb dispersion; in addition, these interactions produce energetic electrons that can further ionize matter. Alpha particles are identical to the nucleus of a helium atom and are produced by the alpha decline of several radioactive nuclei, such as isotopes of bismuth, polonium, astatin, radon, francium, radio, various actinides, such as actinium, thorium, uranium, neptunium , curium, californium, americium and plutonium.

When particles are used, they can be neutral (not charged), positively charged or negatively charged. When charged, the charged particles can carry a single positive or negative charge or multiple charges, for example, one, two, three or even four or more charges. In cases where chain splitting is desired, positively charged particles may be desirable, in part, because of their acidic nature. When particles are used, the particles can have the mass of an electron at rest or greater, for example, 500, 1000, 1500 or 2000 or even more times the mass of an electron at rest. For example, particles can have a mass of about 1 atomic unit to about 150 atomic units, for example, from about 1 atomic unit to about 50 atomic units or from about 1 to about 25, for example, 1, 2, 3, 4, 5, 10, 12 or 15 amu. Acceleration used to accelerate particles can be electrostatic DC, electrodynamic DC, linear RF, linear or continuous magnetic induction wave. For example, cyclotron-type accelerators are available from IBA, Belgium, as is the Rhodotron® system, while DC-type accelerators are available from RDI, now IBA Industrial, such as Dynamitron®. ions and ion accelerators are discussed in Introductory Nuclear Physics, Kenneth S. Krane, John Wiley & Sons, Inc.

20/117 (1988), Krsto Prelec, FIZIKA B 6 (1997) 4, 177-206, Chu, William T., OverView of Light-lon Beam Therapy Columbus-Ohio, ICRU-IAEA Meeting, 18-20 March 2006, Iwata, Y. et al., Alternating-Phase-Focused IH-DTL for Heavy-lon Medical Accelerators Proceedings of EPAC 2006, Edinburgh, Scotland and Leaner, CM et al., Status of the Superconducting ECR lon Source Venus Proceedings of EPAC 2000, Vienna, Austria.

Gamma radiation has the advantage of a significant penetration depth in a variety of materials. Gamma ray sources include radioactive nuclei, such as isotopes of cobalt, calcium, technetium, chromium, gallium, indium, iodine, iron, krypton, samarium, selenium, sodium, butchery and xenon.

X-ray sources include electron beam collision with metal targets, such as tungsten or molybdenum, or compact alloys or light sources, such as those commercially produced by Lyncean.

Sources for ultraviolet radiation include deuterium or cadmium lamps.

Sources for infrared radiation include sapphire, zinc or ceramic lamps with selenide elements.

Microwave sources include RF sources such as Slevin, Klystrons or atom beam sources that employ hydrogen, oxygen or nitrogen gases.

In some embodiments, an electron beam is used as the radiation source. An electron beam has the advantage of high dose rates (for example, 1, 5 or even 10 Mrad per second), high throughput, less containment and less containment equipment. Electrons can also be more efficient in causing chain fission. In addition, electrons having energies of 4-10 MeV can have a penetration depth of 5 to 30 mm or more, such as 40 mm.

Electron beams can be generated, for example, through electrostatic generators, cascade generators, transforming generators, low-energy accelerators with a scanning system, low-energy accelerators with a linear cathode, linear accelerators and accelerators

21/117 pulsed. Electrons as a source of ionizing radiation can be useful, for example, for relatively thin stacks of materials, for example, less than 0.5 inches, for example, less than 0.4 inches, 0.3 inches, 0.2 inches or less than 0.1 inch. In some embodiments, the energy of each electron in the electron beam is from about 0.3 MeV to about 2.0 MeV (millions of electron volts), for example, from about 0.5 MeV to about 1 , 5 MeV or from about 0.7 MeV to about 1.25 MeV.

Electron beam irradiation devices can be sought commercially from lon Beam Applications, Louvain-la-Neuve, Belgium or Titan Corporation, San Diego, CA. Typical electron energies can be 1 MeV, 2 MeV, 4.5 MeV, 7.5 MeV or 10 MeV. The typical energy of the electron beam irradiation device can be 1 kW, 5 kW, 10 kW, 20 kW , 50 kW, 100 kW, 250 kW or 500 kW. The efficacy of depolymerization of the raw material paste depends on the electron energy used and the dose applied, while the exposure time depends on the energy and dose. Typical doses can have values of 1 kGy, 5 kGy, 10 kGy, 20 kGy, 50 kGy, 100 kGy or 200 kGy.

Bundles of conical particles

Particles heavier than electrons can be used to irradiate the biomass materials described here. For example, protons, helium nuclei, argon ions, silicon ions, neon ions, carbon ions, phosphorus ions, oxygen ions or nitrogen ions can be used. In some embodiments, particles heavier than electrons can induce greater amounts of chain cleavage (compared to lighter particles). In some cases, positively charged particles can induce greater amounts of chain cleavage than negatively charged particles due to their acidity.

Bundles of heavier particles can be generated, for example, using linear accelerators or cyclotrons. In some embodiments, the energy of each particle in the beam is about 1.0 MeV / atomic unit to about 6,000 MeV / atomic unit, for example, from about 3 MeV / atomic unit to about 4,800 MeV / atomic unit or about

11/22

MeV / atomic unit at about 1,000 MeV / atomic unit.

In certain embodiments, ion beams used to radiate carbon-containing materials, for example, biomass materials, may include more than one different type of ions. For example, ion beams can include mixtures of two or more (for example, three or four or more) different types of ions. Exemplary mixtures can include carbon ions and protons, carbon ions and oxygen ions, nitrogen ions and protons and iron ions and protons. More generally, mixtures of any of the ions discussed above (or any other ions) can be used to form beams of irradiation ions. In particular, mixtures of relatively light and relatively heavier ions can be used in a single ion beam.

In some embodiments, ion beams for irradiation of materials include positively charged ions. Positively charged ions can include, for example, positively charged hydrogen ions (eg protons), noble gas ions (eg helium, neon, argon), carbon ions, nitrogen ions, oxygen ions, atoms silicon, phosphorus ions and metal ions, such as sodium ions, calcium ions and / or iron ions. Without wishing to be trapped by any theory, it is believed that such positively charged ions behave chemically like portions of Lewis acid when exposed to materials, initiating and sustaining chain splitting reactions to open cationic ring in an oxidative environment.

In certain embodiments, ion beams for irradiation of materials include negatively charged ions. negatively charged ions can include, for example, negatively charged hydrogen ions (for example, hydride ions) and negatively charged ions from several relatively electronegative nuclei (eg, oxygen ions, nitrogen ions, carbon ions, silicon ions and phosphorus ions). Without wishing to be trapped by any theory, it is believed that such negatively charged ions behave like base portions of Lewis when exposed to materials, causing chain splitting reactions to open

23/117 anionic ring in a reduction environment.

In some embodiments, beams for irradiating materials may include neutral atoms. For example, any one or more of hydrogen atoms, helium atoms, carbon atoms, nitrogen atoms, oxygen atoms, neon atoms, silicon atoms, phosphorus atoms and iron atoms can be included in beams that are used for irradiation of biomass materials. In general, mixtures of any two or more of the above atom types (for example, three or more, four or more or even more) may be present in the beams.

In certain embodiments, ion beams used to radiate biomass materials include simply charged ions, such as one or more of H ⁺ , H ', He ⁺ , Ne ⁺ , Ar ⁺ , C ⁺ , C', O ⁺ , Ο, N ⁺ , Ν ', Si ⁺ , Si', P ⁺ , P ', Na ⁺ , Ca ⁺ and Fe ⁺ . In some embodiments, ion beams may include multiply charged ions, such as one or more of C ²⁺ , C ³⁺ , C ⁴⁺ , N ³⁺ , N ⁵⁺ , N ³ ', O ²⁺ , O ² ' , O ₂ ² ', Si ²⁺ , Si ⁴⁺ , Si ² ' and Si ⁴ '. In general, ion beams can also include more complex polynuclear ions that carry multiple positive or negative charges. In certain embodiments, due to the structure of the polynuclear ion, positive or negative charges can be effectively distributed over substantially the entire structure of the ions. In some embodiments, the positive or negative charges may be somewhat located on portions of the ion structure.

Electromagnetic radiation

In modalities in which irradiation is carried out with electromagnetic radiation, electromagnetic radiation may have, for example, photon energy (in electron volts) of more than 10 ² eV, for example, more than 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ or even more than 10 ⁷ eV. In some embodiments, electromagnetic radiation has a photon energy of between 10 ⁴ and 10 ⁷ , for example, between 10 ⁵ and 10 ⁶ eV. Electromagnetic radiation may have a frequency, for example, greater than 10 ¹⁶ hz, greater than 10 ¹⁷ hz, 10 ¹⁸ , 10 ¹⁹ , 10 ² ° or even greater than 10 ²¹ hz. In some embodiments, electromagnetic radiation has a frequency of between 10 ¹⁸ and 10 ²² hz, for example, between 10 ¹⁹ to 10 ²¹ hz.

11/24

Controlled Biomass Dissipation and Functionalization

After treatment with ionizing radiation, any of the materials or mixtures described here become ionized; that is, they include radicals at levels that are detectable with an electron orbital resonance spectrometer. If the ionized biomass remains in the atmosphere, it will be oxidized, such as to a point where the carboxylic acid groups are generated by the reaction with atmospheric oxygen. In some cases, with some materials, such oxidation is desired because it can assist in the extra decomposition of the molecular weight of the biomass containing carbohydrate and oxidation groups, for example, carboxylic acid groups, can be useful for solubility and use of microorganisms in some cases. However, since radicals can live for some time after irradiation, for example, more than 1 day, 5 days, 30 days, 3 months, 6 months or even more than 1 year, the properties of the materials can continue to change with the weather which, in some cases, may be undesirable. Thus, it may be desirable to dissipate the ionized material.

After ionization, any biomass material that has been ionized can be dissipated to reduce the level of radicals in the ionized biomass, for example, so that the radicals are no longer detectable with the electron orbital resonance spectrometer. For example, radicals can be dissipated by applying sufficient pressure to the biomass and / or using a fluid in contact with the ionized biomass, such as a gas or liquid, which reacts with (dissipates) the radicals. Use of a gas or liquid to at least assist in the dissipation of radicals can be done to functionalize the ionized biomass with a desired number and type of functional groups, such as carboxylic acid groups, enol groups, aldehyde groups, nitro groups, nitrile groups, amino groups, alkyl amino groups, alkyl groups, chloroalkyl groups or chlorofluoroalkyl groups.

In some cases, such dissipation can improve the stability of some of the ionized biomass materials. For example, dissipation can improve the biomass's resistance to oxidation. Functionalization through dissipation can also improve the solubility of any bio25 / 117 mass described here, can improve its thermal stability and can improve the use of the material by various microorganisms. For example, the functional groups conferred to the biomass material by dissipation can act as receptor sites for fixation by microorganisms, for example, to intensify the hydrolysis of cellulose by various microorganisms.

In some embodiments, dissipation includes applying pressure to the biomass, such as through mechanical deformation of the biomass, for example, direct mechanical compression of the biomass in one, two or three dimensions or applying pressure to a fluid in which the biomass is immersed, for example, isostatic compression. In such cases, the deformation of the material itself maintains the radicals, which are often enclosed in crystalline domains, close enough that the radicals can recombine or react with another group. In some cases, pressure is applied along with the application of heat, such as a sufficient amount of heat to raise the temperature of the biomass above a melting point or softening point of a biomass component, such as lignin, cellulose or hemicellulose. Heat can improve molecular mobility in the polymeric material, which can assist in the dissipation of radicals. When pressure is used to dissipate, the pressure can be greater than about 1000 psi, such as greater than about 1250 psi, 1450 psi, 3625 psi, 5075 psi, 7250 psi, 10,000 psi or even greater than 15000 psi .

In some embodiments, dissipation includes contact of the biomass with a fluid, such as a liquid or gas, for example, a gas capable of reacting with radicals, such as acetylene or a mixture of acetylene in nitrogen, ethylene, chlorinated ethylene or chlorofluoroethylene, propylene or mixtures of these gases. In other particular embodiments, dissipation includes contact of the biomass with a liquid, for example, a soluble liquid or at least capable of penetrating the biomass and reaction with radicals, such as a diene, such as 1,5-cyclooctadiene. In some specific modalities, the dissipation includes contact of the biomass with an antioxidant, such as

26/117 as Vitamin E. If desired, the biomass raw material may include an antioxidant dispersed in it and the dissipation may arise from contact of the antioxidant dispersed in the biomass raw material with the radicals.

Functionalization can be enhanced using heavy charged ions, such as any of the heavier ions described here. For example, if it is desired to intensify 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. Likewise, if sulfur or phosphorus groups are desired, sulfur or phosphorus ions can be used in irradiation.

Doses

In some cases, irradiation is carried out at a dosage rate greater than about 0.25 Mrad per second, for example, more than about 0.5, 0.75, 1.0, 1.5, 2, 0 or even more than about 2.5 Mrad per second. In some modalities, irradiation is performed at a dose rate of between 5.0 and 1500.0 kilorads / hour, for example, between 10.0 and 750.0 kilorads / hour or between 50.0 and 350.0 kilorads / hours.

In some embodiments, irradiation (with any radiation source or a combination of sources) is carried out until the material receives a dose of at least 0.1 Mrad, at least 0.25, for example, at least 1.0 Mrad , at least 2.5 Mrad, at least 5.0 Mrad, at least 10.0 Mrad, at least 60 Mrad or at least 100 Mrad. In some embodiments, irradiation is carried out until the material receives a dose of about 1.0 Mrad to about 500 Mrad, from about 0.5 Mrad to about 200 Mrad, from about 1 Mrad to about 100 Mrad or about 5 Mrad to about 60 Mrad. In some embodiments, a relatively low dose of radiation is applied, for example, less than 60 Mrad.

Ultrasound

Ultrasound can reduce the molecular weight and / or crystallinity of materials, such as one or more of any of the materials described here, for example, one or more sources of carbohydrate, such as cellulosic or lignocellulosic materials or starch materials. Ultrasound can also be used to sterilize materials.

In one method, a first material that includes cellulose having a first numerical average molecular weight (M _N i) is dispersed in a medium, such as water and subjected to ultrasound and / or otherwise cavitation, in order to provide a second material that includes cellulose having a second average numerical molecular weight (M _N2 ) less than the first average numerical molecular weight. The second material (or the first and second materials in certain modalities) can be combined with a microorganism (with or without enzymatic treatment) that can use the second and / or first material to produce a product or intermediate.

Since the second material includes cellulose having a reduced molecular weight in relation to the first material and, in some cases, reduced crystallinity as well, the second material is, in general, more dispersible, swelling and / or soluble in a solution containing the microorganism.

In some embodiments, the second average numerical molecular weight (Mn2) is less than the first average numerical molecular weight (Mn-i) by more than about 10 percent, for example, more than about 15, 20, 25, 30, 35, 40, 50 percent, 60 percent or even more than about 75 percent.

In some cases, the second material has cellulose which has a crystallinity (C ₂ ) that is less than the crystallinity (C-ι) of the cellulose of the first material. For example, (C ₂ ) can be less than (Ci) by more than about 10 percent, for example, more than about 15, 20, 25, 30, 35, 40 or even more than about 50 per cent.

In some embodiments, the initial crystallinity index (before ultrasound) is about 40 to about 87.5 percent, for example, about 50 to about 75 percent or about 60 to about of 70 percent and a crystallinity index after ultrasound is about 10 to about 50 percent, for example, about 15 to about 45 percent or about 20 to about 40 percent. However, in certain modalities, for example, after extensive ultrasound, it is possible to have a cris28 / 117 talinity index of less than 5 percent. In some embodiments, the material, after ultrasound, is substantially amorphous.

In some embodiments, the initial average numerical molecular weight (before ultrasound) is from about 200,000 to about 3,200,000, for example, from about 250,000 to about 1,000,000 or from about 250,000 to about 700,000 and the average numerical molecular weight after ultrasound is about 50,000 to about 200,000, for example, from about 60,000 to about 150,000 or about 70,000 to about 125,000, however, in some embodiments, for example, after extensive ultrasound, it is possible to have an average numerical molecular weight of less than about 10,000 or even less than about 5,000.

In some embodiments, the second material may have an oxidation level (O ₂ ) that is higher than the oxidation level (Oi) of the first material. A higher level of oxidation of the material can aid in its dispersibility, swelling capacity and / or solubility, further intensifying the material's susceptibility to chemical, enzymatic or microbial attack. In some embodiments, to increase the level of oxidation of the second material in relation to the first material, ultrasound is performed in an oxidation medium, 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, which can increase its hydrophilicity.

In some embodiments, the ultrasound medium is an aqueous medium. If desired, the medium can include an oxidizer, such as a peroxide (for example, hydrogen peroxide), a dispersing agent and / or a buffer. Examples of dispersing agents include ionic dispersing agents, for example, sodium lauryl sulfate and non-ionic dispersing agents, for example, (poly) ethylene glycol.

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

11/29

Pyrolysis

One or more pyrolysis processing sequences can be used to physically treat the biomass material. Pyrolysis can also be used to sterilize the material.

In one example, a first material that includes cellulose having a first average numerical molecular weight (Mm), undergoes pyrolysis, for example, by heating the first material in a tubular oven (in the presence or absence of oxygen), in order to provide a second material that includes cellulose having a second average numerical molecular weight (Mn2) less than the first average numerical molecular weight.

Since the second material includes cellulose having a reduced molecular weight in relation to the first material and, in some cases, reduced crystallinity as well, the second material is, in general, more dispersible, swelling and / or soluble in a solution containing a microorganism.

In some embodiments, the second average numerical molecular weight (M _N2 ) is less than the first average numerical molecular weight (M _N i) by more than about 10 percent, for example, more than about 15, 20, 25 , 30, 35, 40, 50 percent, 60 percent or even more than about 75 percent.

In some cases, the second material has cellulose which has a crystallinity (C ₂ ) that is less than the crystallinity (C-ι) of the cellulose of the first material. For example, (C ₂ ) can be less than (Ci) by more than about 10 percent, for example, more than about 15, 20, 25, 30, 35, 40 or even more than about 50 per cent.

In some embodiments, the initial crystallinity (before pyrolysis) is about 40 to about 87.5 percent, for example, about 50 to about 75 percent or about 60 to about 70 percent and the crystallinity index, after pyrolysis, is about 10 to about 50 percent, for example, about 15 to about 45 percent, or about 20 to about 40 percent. However, in certain modalities, for example, after extensive pyrolysis, it is possible to have a crystallinity index of less than

11/30 percent. In some embodiments, the material, after pyrolysis, is substantially amorphous.

In some embodiments, the initial average numerical molecular weight (before pyrolysis) is from about 200,000 to about 3,200,000, for example, from about 250,000 to about 1,000,000 or from about 250,000 to about 700,000 and the weight average numerical molecular weight, after pyrolysis, is about 50,000 to about 200,000, for example, about 60,000 to about 150,000 or about 70,000 to about 125,000, however, in some embodiments, for example, after extensive pyrolysis , it is possible to have an average numerical molecular weight of less than about 10,000 or even less than about 5,000.

In some embodiments, the second material may have an oxidation level (O ₂ ) that is higher than the oxidation level (Oi) of the first material. A higher level of oxidation of the material can assist in its dispersibility, swelling capacity and / or solution, further intensifying the material's susceptibility to chemical, enzymatic or microbial attack. In some embodiments, to increase the level of oxidation of the second material with respect to the first material, pyrolysis is performed in an oxidation environment, 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, than the first material, thereby increasing the hydrophilicity of the material.

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

Oxidation

One or more oxidative processing sequences can be used to physically treat the biomass material. The oxidation conditions can be varied depending, for example, on the lignin content of the raw material, with a higher degree of oxidation being, in general, desired for food stocks with a higher lignin content.

In one method, a first material that includes cellulose having a first numerical average molecular weight (M _N1 ) and having a first oxygen content (Oi) is oxidized, for example, by heating the first material in a tubular oven in a stream of air or oxygen-enriched air, in order to provide a second material that includes cellulose having a second numerical average molecular weight (Mn2) θ having a second oxygen content (O ₂ ) greater than the first oxygen content (Cb) .

The second medical numerical molecular weight of the second material is generally less than the first average numerical molecular weight of the first material. For example, the molecular weight can be reduced to the same extent as discussed above with respect to other physical treatments. The crystallinity of the second material can also be reduced to the same extent as discussed above with respect to the other physical treatments.

In some modalities, the second oxygen content is at least about five percent higher than the first oxygen content, for example, 7.5 percent higher, 10.0 percent higher, 12.5 percent higher, 15.0 percent higher or 17.5 percent higher. In some preferred embodiments, the second oxygen content is at least about 20.0 percent greater than the oxygen content of the first material. The oxygen content is measured through elemental analysis through pyrolysis of the sample in an oven operating at 1300 ° C or higher. A suitable elemental analysis equipment is the LECO CHNS-932 analyzer with a VTF-900 high temperature pyrolysis oven.

In general, oxidation of the first material occurs in an oxidation environment. For example, oxidation can be performed or aided by pyrolysis in an oxidation environment, such as in air or air-enriched argon. To aid oxidation, various chemical agents, such as oxidants, acids or bases, can be added to the material before or during oxidation. For example, a peroxide (for example, benzoyl peroxide), can be added before oxidation.

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Some oxidative methods of reducing recalcitrance in a biomass raw material employ Fenton-type chemistry. Such methods are disclosed, for example, in U.S. Serial No. 12 / 639,289, the full disclosure of which is incorporated herein by reference.

Exemplary oxidants include peroxides, such as hydrogen peroxide and benzoyl peroxide, persulfates, such as ammonium persulfate, activated forms of oxygen, such as ozone, permanganates, such as potassium permanganate, perchlorates, such as sodium perchlorate and hypochlorites, such as sodium hypochlorite (household bleach).

In some situations, the pH is maintained at or below about 5.5 during contact, such as between 1 and 5, between 2 and 5, between 2.5 and 5, or between about 3 and 5. Oxidation conditions too they may include a contact period of between 2 and 12 hours, for example, between 4 and 10 hours or between 5 and 8 hours. In some cases, the temperature is maintained at or below 300 ° C, for example, at or below 250, 200, 150, 100 or 50 ° C. In some cases, the temperature remains substantially ambient, for example, at or around 20-25 ° C.

In some embodiments, the one or more oxidants are applied as a gas, such as by generating ozone in situ by irradiating the material through air with a beam of particles, such as electrons.

In some embodiments, the mixture further includes one or more hydroquinones, such as 2,5-dimethoxyhydroquinone (DMHQ) and / or one or more benzoquinones, such as 2,5-dimethoxy-1,4-benzoquinone (DMBQ), which can assist in electron transfer reactions.

In some embodiments, the one or more oxidants are electrochemically generated in situ. For example, hydrogen peroxide and / or ozone can be electrochemically produced inside a reaction or contact vessel.

Other Processes to Functionalize

Any of the processes in that paragraph can be used alone without any of the processes described here or in combination with any of the processes described here (in any order): vapor explosion, chemical treatment (for example, acid treatment ( including treatment with concentrated acid and diluted with mineral acids, such as sulfuric acid, hydrochloric acid and organic acids, such as trifluoroacetic acid) and / or base treatment (e.g. treatment with lime or sodium hydroxide), UV treatment, screw extruder treatment (see, for example, US Patent Application Serial No. 61 / 115,398, filed November 17, 2008), solvent treatment (eg treatment with ionic liquids) and freeze grinding (see , for example, US Serial No. 12 / 502,629).

FERMENTATION

Microorganisms can produce a series of intermediates and useful products, such as those described here, by fermenting a low molecular weight sugar in the presence of functionalized biomass materials. For example, fermentation or other bioprocesses can produce alcohols, organic acids, hydrocarbons, hydrogen, proteins or mixtures of any of these materials.

The microorganism can be a natural microorganism or a manipulated microorganism. For example, the microorganism can be a bacterium, for example, a cellulolytic bacterium, a fungus, for example, a yeast, a plant or a protist, for example, an algae, a protozoan, or a fungus-like protist, for example example, a mud mold. When the organisms are compatible, mixtures of organisms can be used.

Suitable fermentation microorganisms have the ability to convert carbohydrates, such as glucose, xylose, arabinose, mannose, galactose, oligosaccharides or polysaccharides, into fermentation products. Fermentation microorganisms include strains of the genera Sacchromyces spp., For example, Sacchromyces cerevisiae (baker's yeast), Saccharomyces distaticus, Saccharomyces uvarurrr, the genus Kluyveromyces, for example, species Kluyveromyces marxianus, Kluyveromyces fragidais, o example, Candida pseudotropicalis and Candida brassi34 / 117 cae, Pichia stípitis (a relative of Candida shehatae), the genus Clavispora, for example, species Clavispora lusitaniae and Clavispora opuntiae, the genus Pachysolen, for example, species Pachysolen tannophilus, the genus, Bret for example, Bretannomyces clausenii species (Philippidis, GP, 1996, Cellulose Bioconversion Technology in Handbook on Bioethanol: Production and Utilization, Wyman, CE, ed., Taylor & Francis, Washington, DC, 179-212).

Commercially available yeasts include, for example, Red Star® / Lesaffre Ethanol Red (available from Red Star / Lesaffre, USA), FALI® (available from Fleischmanris Yeast, a division of Burns Philip Food Inc., USA); SUPERSTART® (available from Alltech, now Lallemand), GERT STRAND® (available from Gert Strand AB, Sweden); and FERMOL® (available from DSM Specialties).

Bacteria can also be used in fermentation, for example, Zymomonas mobilis and Clostridium thermocellum (Philippidis, 1996, supra).

The optimal pH for yeast is a pH of about 4 to 5, while the optimal pH for Zymomonas is a pH of about 5 to 6. Typical fermentation times are about 24 to 96 hours, with temperatures in the range of 26 ° C to 40 ° C, however thermophilic microorganisms prefer higher temperatures.

In some embodiments, all or part of the fermentation process can be stopped before the low molecular weight sugar is completely converted into ethanol. Intermediate fermentation products include high concentrations of sugar and carbohydrates. These intermediate fermentation products can be used in the preparation of food for human or animal consumption. In addition or alternatively, intermediate fermentation products can be ground to a fine oc size in a stainless steel laboratory grinder to produce a flour-like substance.

Mobile fermenters can be used, as described in U.S. Provisional Patent Application Serial No. 60 / 832,735, now Order

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International Published No. WO 2008/011598.

POST-PROCESSING

Distillation

After fermentation, the resulting fluids can be distilled using, for example, a yeast column to separate ethanol and other alcohols from most water and residual solids. The steam leaving the yeast column can be, for example, 35% by weight of ethanol and can be fed to a rectifying column. A mixture of ethanol (92.5%) and quasi-azeotropic water from the rectification column can be purified to pure ethanol (99.5%) using molecular vapor sieves. The lower parts of the yeast column can be sent to the first stage of a three-stage evaporator. The reflux condenser in the rectification column can provide heat for this first stage. After the first stage, solids can be separated using a centrifuge and dried in a rotary dryer. A portion (25%) of the centrifuge's effluent can be recycled for fermentation and the rest sent to the second and third stages of the evaporator. Most of the condensate from the evaporator can be returned to the process as a reasonably clean condensate, with a small portion of the treated water diverted to prevent the development of low boiling compounds.

INTERMEDIARIES AND PRODUCTS

The processes described here can be used to produce one or more intermediates or products, such as energy, fuels, food and materials. Specific examples of products include, but are not limited to, hydrogen, alcohols (for example, monohydric alcohols or dihydric alcohols, such as ethanol, n-propanol or n-butanol), hydrated or hydric alcohols, for example, containing more than 10 %, 20%, 30% or even more than 40% water, xylitol, sugars, biodiesel, organic acids (eg acetic acid and / or lactic acid), hydrocarbons, co-products (eg proteins, such as cellulolytic proteins (enzymes) or proteins with a single cell) and mixtures of any of these in combination or relative concentration and optionally in combination with any

36/117 additives, for example, fuel additives. Other examples include carboxylic acids, such as acetic or butyric acid, salts of a carboxylic acid, a mixture of carboxylic acids and salts of carboxylic acids and esters of carboxylic acids (for example, methyl, ethyl and n-propyl esters), ketones (for example, acetone), aldehydes (for example, acetaldehyde), alpha, beta unsaturated acids, such as acrylic acid and olefins, such as ethylene. Other alcohols and alcohol derivatives include propanol, propylene glycol, 1,4butanediol, 1,3-propanediol, methyl or ethyl esters of any of these alcohols. Other products include methyl acrylate, methyl methacrylate, lactic acid, propionic acid, butyric acid, succinic acid, 3-hydroxypropionic acid, a salt of any of the acids and a mixture of any of the acids and their salts.

Other intermediates and products, including food and pharmaceuticals, are described in U.S. Serial No. 12 / 417,900, the full disclosure of which is incorporated herein by reference.

EXAMPLES

The following Examples are intended to illustrate and not to limit the teachings of the present disclosure.

Example 1 - Preparation of Fibrous Material from Poly-Coated Paper

A load of 680.39 kilograms (1500 pounds) of virgin half-gallon juice packs made of unprinted poly-coated white Kraft cardboard having a volumetric density of 320.37 kg / m ³ (20 lb / ft ³ ) was obtained International Paper. Each package was folded and then fed to a 3 hp Flinch Baugh chipper at a rate of approximately 6.80 to 9.07 kilograms per hour (15 to 20 pounds per hour). The chipper was equipped with two 30.48 cm (12 inch) rotating blades, two fixed blades and a 0.762 centimeter (0.30 inch) discharge sieve. The gap between the rotating and fixed blades has been adjusted to 0.254 cm (0.10 inches). The mincer product resembled confetti having a width of between 0.254 centimeter (0.1 inch) and 1.27 centimeter (0.5 inch), a length of between 0.635 centimeter (0.25 inch) and 2.54 centimeters (1 inch) and an equivalent thickness

37/117 to that of the starter material (about 0.19 centimeter (0.075 inch)).

The confetti-like material was fed to a Munson rotary knife cutter, Model SC30. The Model SC30 is equipped with four rotating blades, four fixed blades and a discharge sieve with 1/8 inch openings. The gap between the rotating and fixed blades has been set to approximately 0.020 inches. The rotary knife cutter slices the confetti-like pieces across the edges of the knives, tearing the pieces apart and releasing fibrous material at a rate of about a pound an hour. The fibrous material had a BET surface area of 0.9748 m ² / g +/- 0.0167 m ² / g, a porosity of 89.0437 percent and a volumetric density (@ 3.65 kPa gauge (0, 53 psia)) of 0.1260 g / ml. The average length of the fibers was 1.141 mm and the average width of the fibers was 0.027 mm, providing an average L / D of 42: 1. Example 2 - Preparation of Fibrous Material from Bleached Kraft Paper

A load of 680.39 kilograms (1500 pounds) of bleached virgin white Kraft paper having a volumetric density of 480.55 kg / m ³ (30 pounds / foot ³ ) was obtained from International Paper. The material was folded and then fed to a 3 hp Flinch Baugh chipper at a rate of approximately 6.80 to 9.07 kilograms per hour (15 to 20 pounds per hour). The chipper was equipped with two 30.48 cm (12 inch) rotating blades, two fixed blades and a 0.762 centimeter (0.30 inch) discharge sieve. The gap between the rotating and fixed blades has been adjusted to 0.254 cm (0.10 inches). The mincer product resembled confetti having a width of between 0.254 centimeter (0.1 inch) and 1.27 centimeter (0.5 inch), a length of between 0.635 centimeter (0.25 inch) and 2.54 centimeters (1 inch) and a thickness equivalent to that of the starting material (about 0.19 centimeter (0.075 inch)). The confetti-like material was fed to a Munson rotary knife cutter, Model SC30. The discharge screen had 0.32 centimeter (1/8 inch) openings. The gap between the rotating and fixed blades has been set to approximately 0.05 cm (0.020 inches). The rotary knife cutter shears the confetti-like pieces, releasing a

38/117 fibrous material at a rate of about a pound an hour. The fibrous material had a BET surface area of 1.1316 m ² / g +/- 0.0103 m ² / g, a porosity of 88.3285 percent and a volumetric density (@, 65 kPa gauge (0.53 psia)) of 0.1497 g / ml. The average length of the fibers was 1.063 mm and the average width of the fibers was 0.0245 mm, providing an average L / D of 43: 1.

Example 3 - Preparation of Sheared Fibrous Material Twice From

Bleached Kraft Paper

A load of 680, 39 kilograms (1500lbs) of bleached virgin white Kraft paper having a volumetric density of 480.55 kg / m ³ (30 lb / ft ³ ) was obtained from International Paper. The material was folded and then fed to a 3 hp Flinch Baugh chipper at a rate of approximately 6.80 to 9.07 kilograms per hour (15 to 20 pounds per hour). The chipper was equipped with two 30.48 cm (12 inch) rotating blades, two fixed blades and a 0.762 centimeter (0.30 inch) discharge sieve. The gap between the rotating and fixed blades has been adjusted to 0.254 cm (0.10 inches). The mincer product resembled confetti (as above). The confetti-like material was fed to a Munson rotary knife cutter, Model SC30. The discharge screen had openings of 0.16 centimeter (1/16 inch). The gap between the rotating and fixed blades has been set to approximately 0.05 cm (0.020 inches). The rotary knife cutter confetti-like pieces, releasing a fibrous material at a rate of about a pound an hour. The material resulting from the first shear was re-fed in the same configuration described above and sheared again. The resulting fibrous material had a BET surface area of 1.4408 m ² / g +/- 0.0156 m ² / g, a porosity of 90.8998 percent and a volumetric density (@ 65 kPa gauge (0.53 psia)) of 0.1298 g / ml. The average fiber length was 0.891 mm and the average fiber width was 0.026 mm, providing an average L / D of 34: 1.

Example 4 - Preparation of sheared fibrous material three times from

Bleached Kraft Paper

11/39

A load of 680, 39 kilograms (1500lbs) of bleached virgin white Kraft paper having a volumetric density of 480.55 kg / m ³ (30 lb / ft ³ ) was obtained from International Paper. The material was folded and then fed to a 3 hp Flinch Baugh chipper at a rate of approximately 6.80 to 9.07 kilograms per hour (15 to 20 pounds per hour). The chipper was equipped with two 30.48 cm (12 inch) rotating blades, two fixed blades and a 0.762 centimeter (0.30 inch) discharge sieve. The gap between the rotating and fixed blades has been adjusted to 0.254 cm (0.10 inches). The mincer product resembled confetti (as above). The confetti-like material was fed to a Munson rotary knife cutter, Model SC30. The discharge screen had 0.32 centimeter (1/8 inch) openings. The gap between the rotating and fixed blades was set to approximately 0.05 centimeter (0.020 inch). The rotary knife cutter shears the confetti-like pieces across the edges of the knives. The material resulting from the first shear was re-fed in the same configuration and the sieve was replaced by a 0.16 centimeter (1/16 inch) sieve. This material was sheared. The material resulting from the second shear was fed back in the same configuration and the sieve was replaced by a 0.08 centimeter (1/32 inch) sieve. This material was sheared. The resulting fibrous material had a BET surface area of 1.6897 m ² / g +/- 0.0155 m ² / g, a porosity of 87.7163 percent and a volumetric density (@ 0.53 psia) of 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, providing an average L / D of 32: 1.

Example 5 - Electron Beam Processing

The samples were treated with an electron beam using a Rhodotron® TT200 continuous wave accelerator under a vault that distributes electrons from 5 MeV to 80 kW of output power. Table 1 describes the parameters used. Table 2 reports the nominal dose used for the sample ID (in Mrad) and the corresponding dose distributed to the sample (in kgy).

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Table 2. Dosages Distributed to the Samples

Total Dosage (Mrad) (Number associated with the Sample ID) Distributed dose (kgy) ¹ 1 9.9 3 29.0 5 50.4 7 69.2 10 100.0 15 150.3 20 198.3 30 330.9 50 529.0 70 695.9 100 993.6

¹ For example, 9.9 kgy were distributed over 11 seconds at a beam current of 5 mA and a line speed of 12.9 feet / minute. The time to cool down between treatments was around 2 minutes.

Example 6 - Methods for determining the molecular weight of cellulosic and lignocellulosic materials using gel permeation chromatography

Cellulosic and lignocellulosic materials for analysis were treated according to Example 4. Sample materials presented in the following tables include Kraft paper (P), wheat straw (WS), alfalfa (A), cellu10 lose (C), switchgrass ( SG), grasses (G) and starch (ST) and sucrose (S). Sample ID number 132 refers to the particle size of the material after shearing through a 1/32 inch sieve. The number after the dash refers to the radiation dosage (MRad) and US refers to ultrasonic treatment. For example, a sample ID P132-10 refers to Kraft paper that has been sheared to a particle size of 132 mesh and has been irradiated with 10 MRad.

For samples that were irradiated with e-beam, the number after the dash refers to the amount of energy distributed to the sample. For example, a sample ID P-100e refers to Kraft paper to which an energy dose of about 100 MRad or about 1000 kgy has been delivered (Table 2).

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Table 3. Average peak molecular weight of irradiated Kraft paper

Source of Sample ID sample Dosage ¹ (Mrad) Ultrasound ² Average MW ± Detour Standard P132 0 Not 32853 ± 10006 Paper P132-10 10 II 61398 ± 2468 ** P132-100 100 II 8444 ± 580 Kraft P132-181 181 II 6668 ± 77 P132-US 0 Yes 3095 ± 1013

** Low radiation doses appear to increase the molecular weight of some materials ¹ Dosage rate = 1MRad / hour ² Treatment for 30 minutes with 20 kHz ultrasound using a 1000W horn antenna under conditions of recirculation with the material dispersed in water.

Table 4. Average molecular weight of peak Kraft paper irradiated with Efeixe

Source of Sample ID sample Dosage ¹ (Mrad) Ultrasound ² Kraft paper P-1e 1 63489 ± 595 P-5e 5 56587 ± 536 P-10e 10 53610 ± 327 P-30e 30 38231 ± 124 P-70e 70 12011 ± 158 P-100e 100 9770 ± 2

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Table 5. Average peak molecular weight of irradiated gamma materials

Sample ID Peak # Dosage ¹ (Mrad) Ultrasound ² Average MW ± Detour Standard WS132 1 0 Not 1407411 ± 175191 2 II II 39145 ± 3425 3 II 2886 ± 177 WS132-10 * 1 10 II 26040 ± 3240 WS132-100 * 1 100 II 23620 ± 453 A132 1 0 II 1604886 ± 151701 2 II II 37525 ± 3751 3 II II 2853 ± 490 A132-10 * 1 10 II 50853 ± 1665 2 II II 2461 ± 17 A132-100 * 1 100 II 38291 ± 2235 2 II II 2487 ± 15 SG132 1 0 II 1557360 ± 83693 2 II II 42594 ± 4414 3 II II 3268 ± 249 SG132-10 * 1 10 II 60888 ± 9131

Table 5. Continuation

Sample ID Peak # Dosage ¹ (Mrad) Ultrasound ² Average MW ± Detour Standard SG132-100 * 1 100 II 22345 ± 3797 SG132-10-US 1 10 Yes 86086 ± 43518 2 II II 2247 ± 468 SG132-100-US 1 100 II 4696 ± 1465

Coalescent peaks after treatment

44/117 ** Low doses of radiation appear to increase the molecular weight of some materials ¹ Dosage rate = 1MRad / hour ² Treatment for 30 minutes with 20 kHz ultrasound using a 1000W horn antenna under conditions of recirculation with the material dispersed in water.

Table 6. Average molecular weight of material irradiated with E-beam

Sample ID Peak # Dosage Average MW ± Standard Deviation A-1e 1 1 1004783 ± 97518 2 34499 ± 482 3 2235 ± 1 A-5e 1 5 38245 ± 346 2 2286 ± 35 A-10e 1 10 44326 ± 33 2 2333 ± 18 A-30e 1 30 47366 ± 583 2 2377 ± 7 A-50e 1 50 32761 ± 168 2 2435 ± 6 G-1e 1 1 447362 ± 38817 2 32165 ± 779 3 3004 ± 25 G-5e 1 5 62167 ± 6418 2 2444 ± 33 G-10e 1 10 72636 ± 4075 2 3065 ± 34 G-30e 1 30 17159 ± 390 G-50e 1 50 18960 ± 142 ST 1 0 923336 ± 1883 2 150265 ± 4033 ST-1e 1 1 846081 ± 5180 2 131222 ± 1687

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Sample ID Peak # Dosage Average MW ± Standard Deviation ST-5e 1 5 90664 ± 1370 ST-10e 1 10 98050 ± 255 ST-30e 1 30 41884 ± 223 ST-70e 1 70 9699 ± 31 ST-100e 1 100 8705 ± 38

Gel Permeation Chromatography (GPC) is used to determine the molecular weight distribution of polymers. During GPC analysis, a solution of the polymer sample is passed through a column packed with small porous gel containment molecules. The sample is separated based on the molecular size, with larger molecules eluting before small molecules. The retention time of each component is most often detected by the refractive index (RI), evaporative light scattering (Evaporative Light Scattering - ELS) or Ultraviolet (UV) and compared with a calibration curve. The resulting data is then used to calculate the molecular weight distribution for the sample.

A molecular weight distribution, instead of a single molecular weight, is used to characterize synthetic polymers. To characterize this distribution, statistical averages are used. The most common of these averages are the average numerical molecular weight (M _n ) and the average gravimetric molecular weight (M _w ).

Methods for calculating these values are described in Example 9 of WO 2008/073186.

The polydispersity index or PI (Polydispersivity Index) is defined as the ratio M _w / M _n . The larger the PI, the wider or more dispersed the distribution. The least value that a PI can have is 1. This represents a monodisperse sample; that is, a polymer with all molecules in the distribution having the same molecular weight.

The peak molecular weight (M _P ) value is another descriptor defined as the mode of molecular weight distribution. It means the molecular weight that is most abundant in the distribution. This value also provides a

46/117 insight into molecular weight distribution.

Most GPC measurements are made against a different polymeric standard. The accuracy of the results depends on how close the characteristics of the polymer being analyzed are to those of the standard used. The expected error in reproducibility between different series of determinations, calibrated separately, is approximately 5-10% and is characteristic of the limited precision of determinations by GPC. Therefore, the GPC results are useful mainly when the comparison between the molecular weight distributions of different samples is made during the same series of determinations.

Lignocellulosic samples require sample preparation before analysis by GPC. First, a saturated solution (8.4% by weight) of lithium chloride (LiCl) was prepared in dimethyl acetamide (DMAc). Approximately 100 mg of each sample was added to approximately 10 g of a freshly prepared saturated LiCI / DMAc solution and each mixture was heated to approximately 150 ° C-170 ° C with stirring for 1 hour. The resulting solutions were generally light to dark yellow in color. The temperature of the solutions was lowered to approximately 100 ° C and the solutions were heated for an additional 2 hours. The temperature of the solutions was then lowered to approximately 50 ° C and each sample solution was heated for approximately 48 to 60 hours. Of note, samples irradiated at 100 MRad were more easily solubilized when compared to their untreated counterpart. In addition, the shear samples (denoted by the number 132) had slightly lower average molecular weights when compared to uncut samples.

The resulting sample solutions were diluted 1: 1 using DMAc as a solvent and were filtered through a 0.45 pm PTFE filter. The filtered sample solutions were then analyzed using GPC using the parameters described in Table 7. The average peak molecular weight (Mp) of the samples, as determined by Gel Permeation Chromatography (GPC), is summarized in the Tables 3-6. Each sample

47/117 was prepared in duplicate and each sample preparation was analyzed in duplicate (two injections) for a total of four injections per sample. The EasiCal® PS1A and PS1B polystyrene standards were used to generate a calibration curve for the molecular weight scale from about 580 to

7,500.00 Daltons.

Table 7. Analysis conditions by GPC

Instrument: Waters Alliance GPC 2000 Plgel 10μ Mixed -B S / N’s: 10M-MB-148-83; 10M-MB-148- Columns (3): 84; 10M-MB-174-129 Mobile phase (solvent): Column / Detector temperature: 0.5% LiCI in DMAc (1.0 mL / min.) 70 ° C Injector temperature: 70 ° C Sample loop size: 323.5 μΐ Example 7. Surface analysis by ion mass spectrometry Secondaries by Time-Of-Fliqht (ToF-SIMS)

Time-Of10 Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is a surface-sensitive spectroscopy that uses a pulsed ion beam (Cs or microfocalized Ga) to remove molecules from the outermost surface of the sample. The 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 in which they reach the detector (that is, time-offlight). ToF-SIMS provides detailed elemental and molecular information about the surface, thin layers, sample interfaces and constitutes a complete three-dimensional analysis. The use is widespread, including in semiconductors, polymers, paint, coatings, glass, paper, metals, ceramics, biomaterials, pharmaceuticals and organic tissue. Since ToF-SIMS is an inspection technique, all elements in the periodic table, including H, are detected. ToF-SIMS data are shown in Tables 8-11.

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Parameters used are reported in Table 12.

Table 8. Average intensities of several positive ions of interest (normalized ion counts with respect to the total x 10,000)

m / z Species P132 Average σ P132-10 Average σ Ρ132-100 Average σ 23 At 257 28 276 54 193 36 27 Al 647 43 821 399 297 44 28 Si 76 45.9 197 89 81.7 10.7 15 ch ₃ 77.9 7.8 161 26 133 12 27 C ₂ H ₃ 448 28 720 65 718 82 39 c ₃ h ₃ 333 10 463 37 474 26 41 C3H5 703 19 820 127 900 63 Table 8. . continuation 0 P132 P132-10 P132-10I 3 m / z Species Average σ Average σ Average σ 43 C ₃ H ₇ 657 11 757 162 924 118 115 c ₉ h ₇ 73 13.4 40.3 4.5 42.5 15.7 128 CioH ₈ 55.5 11.6 26.8 4.8 27.7 6.9 73 C ₃ H ₉ Sí * 181 77 65.1 18.4 81.7 7.5 147 C ₅ H ₁₅ OSi ₂ * 72.2 33.1 24.9 10.9 38.5 4 207 C ₅ Hi ₅ O ₃ Sí ₃ * 17.2 7.8 6.26 3.05 7.49 1.77 647 C42H64PO3 3.63 1.05 1.43 1.41 10.7 7.2

Table 9. Average intensities of various negative ions of interest (ion counts normalized to the total x 10,000)

m / z Species P132 Average σ P132-10 Average σ P132-100 Average σ 19 F 15.3 2.1 42.4 37.8 19.2 1.9 35 Cl 63.8 2.8 107 33 74.1 5.5 13 CH 1900 91 1970 26 1500 6 25 C ₂ H 247 127 220 99 540 7 26 CN 18.1 2.1 48.6 30.8 43.9 1.4 42 CNO 1.16 0.71 0.743 0.711 10.8 0.9 46 no ₂ 1.87 0.38 1.66 1.65 12.8 1.8

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Table 10. Average normalized intensities of several positive ions of interest (normalized ion counts with respect to the total x 10,000)

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Table 11. Average normalized intensities of several negative ions of interest (normalized ion counts with respect to the total x 10,000)

B CM O σ> •ç- CM CM O O co oo CM O O φ ns O 05 O τ- Τ3 .φ O CM xr m 05 CM OO CO xr LO ι CM CM 0- 2 CM CM CM CO Χ- O 05 ΙΟ 05 CO CM 05 05 oo LO CM B χΤ CM cm CM O O Φ re O 1 τ O O O 05 CO «0 LO LC5 ~ CO K 1 ^ - co CM 05 CL 2 CM CM CM xt LO X- x ^- 05 CM 05 U5 H- M CM 05 CM x ~ _ B CO x— co CM O O Φ ns -30 ledi O CO CO O CO CO_ C5 CO l · - CM xr 05 CM Μ ^- xr 0. 2 CM xr co co x ^- χ ^- 05 05 CO \ - 05 xr oo xr O T— B O) M LO x— O O φ ns 00 O Tf O H- 00 1 ^ - LO CO l · - t— CM CO T .φ xT co oo CO co 0. 2 T— r- xr CM O O H- in CO - H- LC5 LO CO xt LC5 co O CM B CO CO <o cm O O φ Π5 C0 CO τ O co CO r- co - 05 LC5 1 Φ O oo xT oo CM CO LO 0. 2 05 CM co co O O co r- LO O5_ oo O CM H- co oo T— O B H- xt O O Φ to CM τ O LO σ>  1 1 M CM O5_ H- co 1 -φ > 4 LD io 05 ç- LO co_ Q_ 2 τ— CM CO r- O O φ Ό spé I X CM z O z CM O LU O O UL O O O z _N CO tn 05 LC5 to CM co AND CM v— CO CM xT

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Table 12. ToF-SIMS parameters

Instrument Conditions: Instrument: PHI TRIFT II Primary ion source: ⁶⁹ Ga Potential of the primary ion beam: 12 kV + ions 18 kV - ions Primary ion current (DC): 2 na for P # E samples 600 pA for P132 samples Power filter / CD: Output / Output Global masses: none Load compensation: On

ToF-SIMS uses a pulsed, focused particle beam (typically Cs or Ga) to move chemical species over the surface of materials. Particles produced closer to the impact site tend to be dissociated ions (positive or negative). Secondary particles generated farther from the impact site tend to be molecular compounds, typically fragments of much larger organic macromolecules. The particles are then accelerated on a flight path on their way towards a detector. Due to the fact that it is possible to measure the time-of10 flight of the particles from the moment of impact to the detector on a nanosecond scale, it is possible to produce a mass resolution as refined as 0.00X atomic mass units ( that is, one part in a million of the mass of a proton). Under typical operating conditions, the results of analysis by ToF-SIMS include: a mass spectrum that inspect all atomic masses over a range of 0-10,000 amu, the tracked beam produces maps of any mass of interest on a scale of Sub-microns and depth profiles are produced by removing surface layers through deposition under the ion beam. Negative ion analysis showed that the polymer had increasing amounts of CNO, CN and NO2 groups.

Example 8 - Porosimetry analysis of irradiated materials

Analysis of pore volume and pore size by mercury (Ta52 / 117 bela 21) is based on forcing mercury (a non-wetting liquid) into a porous structure under hermetically controlled pressures. Since mercury does not wet most substances and will not spontaneously penetrate the pores through capillary action, it must be forced into the sample voids by applying external pressure. The pressure required to fill the voids is inversely to provide pore size. Only a small amount of force or pressure is required to fill large voids, while greater pressure is required to fill very small pore voids.

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Table 21. Pore size and volumetric distribution through mercury porosimetry ID Sample Total volume Total area of Average diameter- Diameter- Average diameter- Density ap- PorosiΦ □

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Table 21. continued

Sample ID Total volume of total area of average diameter- diameter- medium diameter- density Density ap- Porosintrusion (mL / g) pore (m ² / g) in the pore (volumetric pore volume volumetric @ rente (skeletal) heat (%) fire) (um) ro (area) (um) (4V / A) (um) 0.50 psia (g / mL) (g / mL)

O O O 00 00 CM χ— 00 CD CD O LO oo X— CD 'M ’Μ’ r- 00 00 co CO O O co CD r- CD C3O 5— CO CD co 10 χ— M ^- CM H- CD x—- r * - N- x— r- x— LO 10 00 x— x— CO co LO 00 l · - OO O CO oo σί ç — Γ H- χ— ” χ— ” CD σ> x— ~ O CD 00 M * CO CO co Xj- Xt LXO LO LO * d · LO LO xr

00 LO O CM CD LO CM LO O LO co u- 00 CM ’Μ’ σ> LO co OO • ^ r O r ^ - LO CD O x— OO σ> r- CO co LO co χ— 00 r- σ> r- xf LO LO co LO LO CD LO LO LO LO Li) LO LO LO LO LO CO > - > - > - · - x— > - x— · «- x— x—

00 M CM co CD LO X— OO r- X— ’Μ * CM LO H- CD co r- LO CD CD CM l · - LO LO CD LO H- co CM CD co r- 00 O LO r- CD r- CD CD CM X— CD CD CD CD CD 00 CD r- r- r- H- H- l · - l · - O O θ ' O O The ~ CD O O θ “ O CD θ co CD

CO CD O CD § ° CD CO oo CD > - O CM CD co 00 s § co O u- CD χ— CD 00 O CD CM LO X— xtf · 00 -sr CD co Μ ^- xr co LO 'M ^- X— cp. r- co CM CM X— x ^- x ^- 5— x— X ~ • Έ ^- H- in θ ' O co O O O θ ' O CD θ '

O CO CM CM CO CO CM CD O ’Μ 00 co r- Xt LO LO LO LO LO LO LO LO s ° O O CO O CO O O CO CO O CO O t '' * - *P. O CD O O C0_ O O O co CO O_ O CD O 3rd CD θ O <D CD O CD O O CD CD O

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r- CM co M- <D CD r- l · - co O 00 CM CM x- O CD co X— U- χ— X— X— CD CM CO co CD CD r ^ - LO CO CD CD X— CD CM co LO H- co CM OO CD xr co ’Φ CO CO ’Μ’ ’Μ’ LO 00 CD CD CD CD CD_ CD CD LO O O CD O O O O O O CD O CD co O

φ Φ φ Φ O φ φ φ O Φ O Φ O Φ O O O O co 1 O ID 1 O V " ιο ç? r- 1 1 4- · 4- * 4- » 4- * 4-4 4-4 4-1 CC cc co cc co ω cc CC CC CC CC

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The AutoPore® 9520 can reach a maximum pressure of 414 MPa or 413, 69 Mpa gauge (60,000 psia). There were four low pressure stations for sample preparation and data collection of macropore 1.38 kPa gauge (0.2 psia) to 344.74 gauge MP gauge (50 psia) converter. There were two high pressure chambers which collect data from 172.37 kPa gauge (25 psia) to 413, 69 Mpa gauge (60,000 psia). The sample is placed in a tub-like device called a penetrometer, which is connected to a glass capillary rod with a metal coating. As the mercury invades the voids in and around the sample, it moves down the capillary rod. The loss of mercury from the hair shaft results in a change in electrical capacitance. The change in capacitance during the experiment is converted to a volume of mercury knowing the volume of the rod of the penetrometer in use. A variety of penetrometers with different tub sizes (sample) and capillaries are available to accommodate most sample sizes and configurations. Table 22 below defines some of the key parameters calculated for each sample.

Table 22. Definition of Parameters_

Parameter Description

Total intrusion volume

Total pore area:

Median pore diameter (volume):

Median pore diameter (area):

Average pore diameter:

Volumetric density:

Apparently density:

Porosity:

The total volume of mercury that penetrates during an experiment. This can include interstitial filling between small particles, sample porosity and sample compression volume.

The total volume of intrusion converted to an area admitting pores of cylindrical shape.

The size in the 50th percentile on a cumulative volume chart.

The size in the 50th percentile on the cumulative area chart.

The total pore volume divided by the total pore area (4V / A).

The mass of the sample divided by the dense volume. The dense volume is determined at the filling pressure, typically 0.5 psia.

The sample mass divided by the sample volume measured at the highest pressure, typically 60,000 psia. (Volumetric density / Bulk density) x 100%

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Example 9 - Particle size analysis of irradiated materials

The particle sizing technique using static light scattering is based on Mie's theory (which also covers Fraunhofer's theory). Mie's theory predicts the relationship of intensity vs. Angle as a function of size for spherical dispersion particles, as long as other system variables are known and kept constant. These variables are the wavelength of the incident light and the relative shrinkage index of the sample material. Application of Mie's theory provides detailed information about particle size. Table 23 summarizes the particle size using the median diameter, average diameter and modal diameter as parameters.

Table 23. Particle size through laser light scattering (dry sample scattering)

Sample ID Diameter me- Average diameter- Modal diameter diano (pm) dio (pm) (pm)

A132 380,695 418,778 442,258 A132-10 321,742 366,231 410,156 A132-100 301,786 348,633 444,169 SG132 369,400 411,790 455.508 SG132-10 278,793 325,497 426,717 SG132-100 242,757 298,686 390,097 WS132 407,335 445,618 467,978 WS 132-10 194,237 226,604 297,941 WS132-100 201,975 236,037 307,304

The particle size was determined using Dispersion of

Laser Light (Dry Sample Dispersion) using a Malvern Mastersizer 2000 using the following conditions:

Feed rate: 35%

Disperser pressure: 400 kPa (4 Bar)

Optical model: (2,610, 1,000i), 1,000

An appropriate amount of sample was placed on a vibrating tray. The feed rate and air pressure was adjusted to ensure that the particles were properly dispersed. The key component is selecting an air pressure that will break up agglomerations, but will not compromise the integrity of the sample. The amount of

57/117 sample required varies depending on the size of the particles. In general, samples with fine particles require less material than samples with thick particles.

Example 10 - Surface area analysis of irradiated materials

The surface area of each sample was analyzed using a Micromeritics ASAP 2420 Acelerated Surface Area and Porosimetry system. The samples were first prepared by degassing for 16 hours at 40 ° C. Then, the free space (hot and cold) with helium is calculated and then the sample tube is evacuated again to remove the helium. Data collection begins after this second evacuation and consists of defining target pressures, which controls how much gas is metered into the sample. At each target pressure, the amount of gas adsorbed and the actual pressure are determined and recorded. The pressure inside the sample tube is measured with a pressure transducer. Additional doses of gas will continue until the target pressure is obtained and allowed to equilibrate. The amount of adsorbed gas is determined by adding multiple doses over the sample. Pressure and quantity define a gas adsorption isotherm and are used to calculate a series of parameters, including BET surface area (Table 24).

Table 24. Summary of the surface area by means of gas adsorption

Sample ID Surface area about (m ² / g) a single point BET surface area (m ² / g) P132 @ P / Po = 0.250387771 1.5253 1.6897 P132-10 @ P / Po = 0.239496722 1.0212 1.2782 P132-100 @ P / Po = 0.240538100 1.0338 1.2622 P132-181 @ P / Po = 0.239166091 0.5102 0.6448 P132-US @ P / Po = 0.217359072 1.0983 1.6793 A132 @ P / Po = 0.240040610 0.5400 0.7614 A132-10 @ P / Po = 0.211218936 0.5383 0.7212 A132-100 @ P / Po = 0.238791097 0.4258 0.5538 SG132 @ P / Po = 0.23789893 0.6359 0.8350 SG132-10 @ P / Po = 0.238576905 0.6794 0.8689 SG132-100 @ P / Po = 0.241960361 0.5518 0.7034 SG132-10-US @ P / Po = 0.225692889 0.5693 0.7510 SG132-100-US @ P / Po = 0.225935246 1.0983 1.4963 G-10-US 0.751 G100-US 1,496 G132-US 1,679 WS132 @ P / Po = 0.23823664 0.6582 0.8663 WS132-10 @ P / Po = 0.2381212476 0.6191 0.7912 WS132-100 @ P / Po = 0.238398832 0.6255 0.8143 A-1e @ P / Po = 0.238098138 0.6518 0.8368 A-5e @ P / Po = 0.243184477 0.6263 0.7865

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Table 24. continued

Sample ID Surface area about one (m ² / g) single point BET surface area (m ² / g) A-10e @ P / Po = 0.243163236 0.4899 0.6170 A-50e @ P / Po = 0.243225512 0.4489 0.5730 G-1e @ P / Po = 0.238496102 0.5489 0.7038 G-5e @ P / Po = 0.242792602 0.5621 0.7086 G-10e @ P / Po = 0.243066031 0.5021 0.6363 G-50e @ P / Po = 0.238291132 0.4913 0.6333 P-1e @ P / Po = 0.240842223 1.1413 1.4442 P-5e @ P / Po = 0.240789274 1.0187 1.3288 P-10e @ P / Po = 0.240116967 1.1015 1.3657 P-50e @ P / Po = 0.240072114 1.0089 1.2593 P-100e @ P / Po = 0.236541386 0.9116 1.1677 s @ P / Po = 0.225335038 0.0147 0.0279 S-1e @ P / Po = 0.217142291 0.0193 0.0372 S-5e @ P / Po = 0.1333107838 0.0201 0.0485 S-10e @ P / Po = 0.244886517 0.0236 0.0317 S-30e @ P / Po = 0.23729400 0.0309 0.0428 S-50e @ P / Po = 0.245494494 0.0262 0.0365 S-100e @ P / Po = 0.224698551 0.0368 0.0506 St @ P / Po = 0.23824949 0.3126 0.4013 St-1e @ P / Po = 0.23832726 0.3254 0.4223 St-5e @ P / Po = 0.238363587 0.3106 0.4071 St-10e @ P / Po = 0.238341099 0.3205 0.4268 St-30e @ P / Po = 0.238629889 0.3118 0.4189 St-50e @ P / Po = 0.244430980 0.3119 0.3969 St-100e @ P / Po = 0.238421621 0.2932 0.3677

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

Example 11 - Determination of the fiber length of Irradiated Materials

Fiber length distribution testing was performed in triplicate on samples sent using the Techpap MorFi LB01 system. The average length and width of fiber are reported in Table 25.

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Table 25. Summary of lignocellulosic fiber width and length data

Sample ID Arithmetic mean (mm) Weighted average length (mm) Statistically corrected weighted average length (mm) Width (microme- tros) (pm) P132-10 0.484 0.615 0.773 24.7 P132-100 0.369 0.423 0.496 23.8 P132-181 0.312 0.342 0.392 24.4 A132-10 0.382 0.423 0.650 43.2 A132-100 0.362 0.435 0.592 29.9 SG132-10 0.328 0.363 0.521 44.0 SG132-100 0.325 0.351 0.466 43.8 WS132-10 0.353 0.381 0.565 44.7 WS132-100 0.354 0.371 0.536 45.4

Example 12 - Fourier Transformation Infrared Spectrum (FT-IR) of irradiated and non-irradiated Kraft paper

FT-IR analysis was performed on a Nicolet / lmpact 400. The results indicate that samples P132, P132-10, P132-100, P-1e, P-5e, P-10e, P-30e, P-70e and P-100e are consistent with a cellulose-based material.

Figure 3 is an infrared spectrum of Kraft paper scraped according to Example 4, while Figure 4 is an infrared spectrum of Kraft paper of Figure 3 after irradiation with 100 Mrad of radiation range. The irradiated sample shows an additional peak in region A (centered around 1730 cm ' ¹ ) that is not found in the non-irradiated material. Of note, an increase in the amount of carbon absorption at -1650 cm ^-1 was detected when going from P132 to P132-10 to P132100. Similar results were observed for samples P-1e, P-5e, P-10e, P-30e, P-70e and P-100e.

Example 13 - Proton and carbon-13 nuclear magnetic resonance spectra ( ¹ H-NMR and ¹³ C-NMR) of irradiated and non-irradiated Kraft paper

Sample Preparation

Samples P132, P132-10, P132-100, P-1e, P-5e, P-10e, P30e, P-70e and P-100e were prepared for analysis by dissolving with DMSO-d ₆ with fluoride trihydrate 2% tetrabutyl ammonium. The samples 60/117 that had undergone lower levels of irradiation were significantly less soluble than samples with higher irradiation. Non-irradiated samples formed a gel in this solvent mixture, but heating to 60 ° C decomposed the peaks in the NMR spectra. The samples that underwent higher levels of irradiation were soluble in a concentration of 10% weight / weight.

Analyze

¹ H-NMR spectra of the samples at 15 mg / mL showed a distinct very wide resonance peak centered at 16 ppm (FIGS. 5A-5J). This peak is characterized by a proton -OH interchangeable with an enol and was confirmed by d ₂ O shake. Model compounds (acetylacetone, glucuronic acid and keto-gulonic acid) were analyzed and became a convinced case that this peak was, in fact, an exchangeable enol proton. This proposed enol peak was very sensitive to the effects of concentration and we were unable to conclude whether this resonance was due to an enol or possibly a carboxylic acid.

The proton resonances of carboxylic acid of the model compounds were similar to those observed for the treated cellulose samples. These model compounds were shifted to ~ 5-6 ppm. Preparation of P-100e in higher concentrations (-10% w / w) led to a dramatic field shift where the carboxylic acid resonances of the model compounds were found (-6 ppm) (FIG. 5N). These results led to the conclusion that this resonance is not reliable for characterizing this functional group, however, the data suggest that the number of exchangeable hydrogens increases with increasing sample irradiation. Also, no vinyl protons were detected.

The ¹³ C NMR spectra of the samples confirm the presence of a carboxylic acid carbonyl or a carboxylic acid derivative. This new peak (at 168 ppm) is not present in the untreated samples (FIG. 5K). A ¹³ C NMR spectrum with a long decline allowed signal quantification for P-100e (FIGS. 5L-5M). Comparison of the integration of carbonyl resonance with resonances at approximately

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100 ppm (the C1 signals) suggests that the carbonyl to C1 carbon ratio is 1: 13.8 or approximately 1 carbonyl for every 14 glucose units. The chemical shift at 100 ppm correlates well with glucuronic acid.

Titration

Samples P-100e and P132-100 (1 g) were suspended in deionized water (25 ml). The yellow alizarin indicator was added to each sample with agitation. P-100e was more difficult to moisten. Both samples were titrated with a 0.2M NaOH solution. The endpoint was very subtle and was confirmed using a pH paper. The initial pH of the samples was ~ 4 for both samples. P132-100 required 0.4 milliequivalents of hydroxide, which provides a molecular weight for the carboxylic acid of 2500 amu. If 180 amu are used for a monomer, this suggests that there is a carboxylic acid group for 3.9 monomer units. Likewise, P-100e required 3.2 milliequivalents of hydroxide, which was calculated to be a carboxylic acid group for each 17.4 monomer units.

Conclusions

The C-6 carbon in cellulose appears to be oxidized to carboxylic acid (a derivative of glucuronic acid) in that oxidation is surprisingly specific. This oxidation is in agreement with the IR band, which grows with irradiation at ~ 1740 cm ¹ , which corresponds to an aliphatic carboxylic acid. The titration results are in accordance with ¹³ C quantitative NMR. The increased solubility of the sample with the highest levels of irradiation correlates well with the increasing number of protons of carboxylic acid. A proposed mechanism for the degradation of oxidized C-6 cellulose is provided below in Scheme 1.

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Layout 1

Example 14 - Pre-treated biomass microbial testing

Specific lignocellulosic materials pretreated as described here are analyzed for toxicity by common strains of mild and bacteria used in the biofuel industry for the fermentation stage in ethanol production. In addition, the sugar content and compatibility with cellulase enzymes are examined to determine the viability of the treatment process. Testing of pre-treated materials is carried out in two stages, as follows.

Phase 1: Toxicity and Sugar Content

The toxicity of pre-treated grass and paper feed stocks is measured in the yeast Saccharomyces cerevisiae (grape yeast) and Pichia stipitis (ATCC 66278), as well as in the bacteria Zymomonas mobilis (ATCC 31821) and Clostridium thermocellum (ATCC 31924). A growth study is carried out with each of the organisms to determine the optimal incubation and sampling time.

Each feed stock is then incubated in duplicate with S. cerevisiae, P. stipitis, Z. mobilis and C. thermocellum in a

63/117 standard microbiological medium for each organism. YM broth is used for the two strains of yeast, S. cerevisiae and P. stípitis. RM medium is used for Z. mobilis and CM4 medium for C. thermocellum. A positive control, with pure sugar added, but without raw material, is used for comparison. During incubation, a total of five samples are taken every 12 hours at times 0, 3, 6, 9 and 12 hours and analyzed for viability (plate counts for Z. mobilis and direct counts for S. cerevisiae) and concentration of ethanol.

The sugar content of feed stocks is measured using High Performance Liquid Chromatography (HPLC) equipped with a Shodex® SP0810 or Biorad Aminax® HPX-87P sugar column. Each of the feed stocks (approx. 5 g) is mixed with water subjected to reverse osmosis (RO) for 1 hour. The liquid portion of the mixture is removed and analyzed for glucose, galactose, xylose, mannose, arabinose and cellobiosis. The analysis is performed according to the protocol of the National Bioenergy Center Determination of Structural Carbohydrates and Lignin in Biomass.

Phase 2. Cellulase Compatibility

Food stocks are tested, in duplicate, with a commercially available Accellerase® 1000 enzyme complex, which contains an enzyme complex that reduces lignocellulosic biomass in fermentable sugars, at the recommended temperature and concentration in an Erlenmeyer flask. The flasks are incubated with moderate shaking at around 200 rpm for 12 hours. During this time, samples are taken every three hours at times 0, 3, 6, 9 and 12 hours to determine the concentration of reducing sugars (Hope and Dean, Biotech J., 1974, 144: 403) in the liquid portion of the bottles.

Example 15 - Analysis of sugar concentration using HPLC samples were analyzed for sugar concentration (HPLC) and toxicity against 3 microorganisms (Pichia stípitis, Saccharomyces cerevisiae and Zymomonas mobilis. Table 26 lists the equipment used for these experiments. Tables 27 and 28 provide an

64/117 list of sugars (including vendor and batch numbers) used to prepare the HPLC standard and the protocol used to prepare the HPLC standard, respectively.

Table 26. Equipment used in the experiments

Equipment Manufacturer, Name PH meter Orion Agitators (2) B. Braun Biotech, Certomat BS-1 HPLC Waters, 2690 HPLC Module Spectrophotometer Unicam, UV300 YSI Biochem Analyzer Interscience, YSI

Table 27. Sugars used in HPLC analysis

Sugar Manufacturer Ref # Lot # glucose 49140 1284892 xylose 95731 1304473 51707231 cellobiosis BioChemika 22150 1303157 14806191 arabinose 10840 1188979 24105272 mannose 63582 363063/1 22097 galactose 48259 46032/1 33197

Table 28. Preparation of HPLC standards

Concentration desired Solution volume Water flow Total volume jada (mg / mL) of sugar nanopure (mL) (mL) 4 50 mL of 4 mg / mL 0 50 2 25 mL of 4 mg / mL 25 50 1 25 mL of 2 mg / mL 25 50 0.5 25 mL of 1 mg / mL 25 50 0.1 5 ml of 1 mg / ml 20 25 Check pattern 18.75 mL of 4 mg / mL 31.25 50 1.5 mg / mL

Analyze

Each sample (1 gram) was mixed with water that underwent reverse osmosis at 200 rpm and 50 ° C overnight. The pH of the sample was adjusted to between 5 and 6 and filtered through a 0.2 μιτι syringe filter. The samples were stored at -20 ° C before analysis to maintain the integrity of the samples. The observations made during sample preparation are shown in Table 29.

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Table 29. Observations during sample preparation for HPLC

Sample Quantity used (g) Water added (mL) pH Comments P132 1 30 5.38 Cute, hard to mix P132-10 1 25 6.77 Cute, hard to mix P132-100 1 20 3.19 pH is low, difficult to bring to pH 5.0, 10 N NaOH used P132-US 0.3 5 6.14 A132 1 15 5.52 A132-10 1 15 4.9 A132-100 1 15 5.39 SG132 1 15 5.59 SG132-10 1 15 5.16 SG132-100 1 15 4.7 SG132-10-US 0.3 5 5.12 SG132-100-US 0.3 5 4.97 WS 132 1 15 5.63 WS132-10 1 15 5.43 WS132-100 1 15 5.02

* pH of these samples was adjusted to pH using 1N NaOH.

Standards were prepared fresh from a stock solution of 4 mg / ml of the 6 combined sugars, glucose, xylose, cellobiose, arabinose, mannose and galactose. The stock solution was prepared by dissolving 0.400 grams of each sugar in 75 mL of nanopure water (filtered through a 0.3 micron filter). Once dissolved, the stock solution was diluted to 100 mL using a volumetric flask and stored at -20 ° C. Standard working solutions of 0.1, 0.5, 1, 2 and 4 mg / mL were prepared by serial dilution of the stock solution with nanopure water. In addition, a check pattern of 1.5 mg / mL was also prepared from the stock solution.

Sugar concentrations were analyzed according to the Determination of Structural Carbohydrates in Biomass protocol (NREL Bio15 mass Program, 2006) and this protocol is incorporated here by reference in its entirety. A SHODEX SUGAR SP0810 column with an Evaporative Light Scattering detector was used. A verification standard (1.5 mg / mL of standard) was analyzed every 8 injections to ensure that the integrity of the column and the detector were maintained during the experiment. The standard curve coefficient of variation (R ² value) was at least 0.989 and the concentration of the verification standards was within 10% of the actual concentration. The HPLC conditions were as follows:

Table 30. HPLC parameters

Injection volume: 20 pL Mobile phase: Nanopure * water, filtered through a 0.45 pm filter and degassed Flow rate: 0.5 mL / min Column temperature: 85 ° C Detector Temperature: Evaporator temperature 110 ° C, nebulizer temperature 90 ° C

* Initial tests noted that better separation was observed when using nanopure water instead of acetonitrile: 15/85 water in the mobile phase (manufacturer did not recommend using more than 20% acetonitrile with this column).

Results

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

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Table 31. Sugar concentration expressed as mg / mL and mg / g of extract

(D o v 0 s? O> X TO φ> S.ÇO O E O Q mg / g O O O 8.13 | I 7.20 I Ο ο Ο Ο ο θ ' Ο ο θ ' ! 6.71 | Ο σ ο | 6.45 I ! οο'ο | I οο'ο | I 12.2 | | οο’ο | σ ο_ σ ' I 5.25 | and is O O the ~ 00.33 | 0.36 | ο Ο go‘o ΟΟ'Ο LO ο ο ο ο 0.39 οσΌΙ ο ο ο I 0.81 οο'ο | σ σ ο I 0.35 Mannose (see glycine) mg / mL: mg / g mg / g O O θ ' 0.00 | 0.00 | 0.00 I 13.76 I 14.66 I 8.28 I 0.00 I | 6.70 I 1 16.02 I 5.52 Ο ο σ ' Ο σ ο I 7.55 I 5.33 and is O O θ ' O O O" 0.00 | I 00’0 0.92 0.98 0.55 ΟΟ'Ο σ χτ θ ' [1.07 ! 0.37 ο ο σ 00Ό j I 0.50 I 0.36 Galactose (see glycine) mg / mL: mg / g mg / g ο, οο I O O O" ο, οο | ο ο θ ' 5.04 | 5.19 I Ο ο θ ' ο ο ο C0 LO 5.84) I ΟΟΌΙ σ ο σ ' ΟΟ'Ο j I 5.07 οο'ο | and is O O O' O O O O O θ ' I ΟΟ'Ο I0J4 I 0.35 | [οο'ο σ σ σ ' 0.33 j I 0.39] ί ο, οο ο ο σ ' οο’ο I I 0.34 σ ο ο d oY ίΐξδ 0 Ε O 2 mg / g O O O 8.60 | I 6.14 | 5.62 | I 12.66 | I 16.04 I 6.14 | C0 LO I 5.90 I 16.22 ΟΟ'Ο I I 5.18 [5.90 | 10.95 I 5.36 mg / mL ο, οο | CO c> 0.34 0.34 | 0.84 I 1.07 ί 0.41 I 0.33 0.35 I 1.08 Ο ο σ ' I 0.35 I 0.39 I 0.73 I 0.36 <D o 8i £ q · § <° o <Ε O 2 mg / g O O O | 00Ό | | 00Ό | V ^- I 5.73 6.18 I 5.54 6.45 I I 6.22 I ΟΟ'Ο | ο ο ο Ο ο ο I 6.15 I 5.99 I 5.51 AND IS O O O O O O Ο ο θ ’ 0.43 '| 0.38 I 0.41 I 0.37 0.39 | 0.37 | οο’ο | ο ο ο ο ο ο I 0.41 σ σ [0.37 to O O ΞΕ IO O O to Sj o O:> O> <E 2 mg / g 0.00 | O O O 7.04! 5.80 I 5.88 | 7.50 Ο ο_ σ ' 5.73 Ο ο ο σ> CO ο ~ 04 17.87 <ο ! 7.41 σ CO I 6.39 mg / mL 0.00 I I 00’0 0.35 | I 0.35 | I 0.39 Π ί 0.50 | Γο, οο Π I 0.34 | ο σ ' I 1.36 j σ> I 1.07 I 0.49 | θ [57 I 0.43 Sample ID 0. I P-132 I I P-132-10 I | Ρ-132-100 | | P-132-BR I 0 I G-132 I | G-132-10 I G-132-100 I G-132-10-US I G-132-100-US <- I Α-132 I Α-132-10 I Α-132-100 SM | | WS-132 | WS-132-10 | WS-132-100

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Example 16 - Toxicity Study

Twelve samples were analyzed for toxicity against a panel of cultures that produce ethanol. In this study, glucose was added to the samples in order to distinguish between deprivation of cultures and toxicity of the samples. A thirteenth sample was tested for toxicity against Pichia stipitis. A summary of the protocol used is listed in Table 32. A description of the chemicals and equipment used for toxicity testing is reported in Tables 34-36.

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Table 34. Conditions for toxicity testing

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Table 35. Reagents used for toxicity testing

Middle component Manufacturer Reference # Lot # Urea ScholAR Chemistry 9472706 AD-7284-43 Nitrogen base for yeast Becton Dickinson 291940 7128171 Peptone Becton Dickinson 211677 4303198 Xylose Fluka 95731 1304473 51707231 Glucose Sigma G-5400 107H0245 Yeast extract (used for S. cerevisiae) Becton Dickinson 288620 4026828 Yeast extract (used for P. stipitis and Z. mobilis) Becton Dickinson 212750 7165593 MgSO ₄ 7H ₂ O Sigma M5921 034K0066 (NH ₄ ) ₂ SO ₄ Sigma A4418 117K5421 kh ₂ po ₄ Sigma P5379 074K0160 YM Broth Becton Dickinson 271120 6278265

Table 36. YSI components used in shaking bottle study_

Component Catalog # Lot # YSI ethanol membrane 2786 07L100153 YSI ethanol standard (3.2 g / L) 2790 012711040 YSI ethanol buffer 2787 07M1000053, 07100215 Testing was performed using the three microorganisms,

as described below.

Saccharomyces cerevisiae ATCC 24858 (American Type Culture Collection)

An inclined culture medium of S. cerevisiae was prepared from a rehydrated lyophilized culture obtained from the ATCC. A portion of the slant culture material was placed on a YM Broth + 20 g / L agar (pH 5.0) and incubated at 30 ° C for 2 days. 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 of 5.0) was inoculated with a colony of slide with YM and incubated for 24 hours at 25 ° C and 200 rpm. After 23 hours of growth, a sample was taken and analyzed for optical density (600 nm in a UV spectrophotometer) and purity (Gram staining). Based on these results, a flask (called the Culture Flask) with an OD of 14.8 and Gram staining was chosen to inoculate all test flasks.

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The test containers were 500 ml Erlenmeyer flasks containing 100 ml of the sterile medium described above. All vials were autoclaved at 121 ° C and 103.42 kPa (15psi) before adding the test materials. The test materials were not sterilized, since being subjected to the autoclave will change the content of the samples. Test samples were added at the time of inoculation (rather than before) to reduce the possibility of contamination. In addition to the test samples, 1 mL (1% v / v) of material from the culture flask was added to each flask. The flasks were incubated as described above for 36 hours.

Pichia stípitis NRRL Y-7124 (ARS Culture Collection)

An inclined culture medium of P. stípitis was prepared from a rehydrated lyophilized culture obtained from the ARS Culture Collection. A portion of the slant culture material was placed on a YM Broth + 20 g / L agar (pH 5.0) and incubated at 30 ° C for 2 days. One 250 mL Erlenmeyer flask containing 100 mL of medium (40 g / L of glucose, 1.7 g / L of nitrogen base for yeast, 2.27 g / L of urea, 6.56 g / L of peptone , 40 g / L xylose, pH 5.0) was inoculated with a small amount of slide material and incubated for 24 hours at 25 ° C and 125 rpm. After 23 hours of growth, a sample was taken and analyzed for optical density (600 nm in a UV spectrophotometer) and purity (Gram staining). Based on these results, a flask (called the Culture Flask) at an optical density of 5.23 and with a clear Gram stain was chosen to inoculate all test flasks.

The test containers were 250 ml Erlenmeyer flasks containing 100 ml of the sterile medium described above. All vials were autoclaved empty at 121 ° C and 103.42 kPa (15psi) and sterilized filter medium (0.22 pm filter) added to the vials before adding the test materials. The test materials were not sterilized, since being subjected to the autoclave will alter the content of the samples and filter sterilization is not suitable for sterilizing solids. Test samples were added at the time of inoculation (rather than before) to reduce the possibility of contamination. In addition to the test samples, 1

74/117 mL (1% v / v) of material from the culture flask was added to each flask. The flasks were incubated as described above for 48 hours. Zymomonas mobilis ATCC 31821 (American Type Culture)

An inclined culture medium of Z. mobilis was prepared from a rehydrated lyophilized culture obtained from ATTC. A portion of the slant culture material was placed on DYE slides (glucose 20 g / L, Yeast Extract 10 g / L, Agar 20 g / L, pH 5.4) and incubated at 30 ° C and 5% CO ₂ for 2 days. A 20 mL screw cap test tube containing 15 mL of medium (25 g / L of glucose, 10 g / L of yeast extract, 1 g / L of MgSO ₄ 7H ₂ O, 1 g / L of ( NH ₄ ) ₂ SO ₄ , 2 g / L KH ₂ PO ₄ , pH 5.4) was inoculated with a colony and incubated for 24 hours at 30 ° C without shaking. After 23 hours of growth, a sample was taken and analyzed for optical density (600 nm in a UV spectrophotometer) and purity (Gram staining). Based on these results, a tube (OD 1.96) was chosen to inoculate the second culture flask. The second culture flask was a 125 ml flask containing 70 ml of the medium described above and was inoculated with 700 pL (1% v / v) and incubated for 24 hours at 30 ° C without shaking. After 23 hours of growth, a sample was taken and analyzed for optical density (600 nm in a UV spectrophotometer) and purity (Gram staining). Based on these results, a flask (called the Culture Flask) with an OD of 3.72 was chosen to inoculate all test flasks.

The test containers were 250 ml Erlenmeyer flasks containing 100 ml of the sterile medium described above, except the yeast extract at 5 g / L. All vials were autoclaved empty at 121 ° C and 103.42 kPa (15psi) and sterile filter medium (0.22 pm filter) added to the vials before adding the test materials. The test materials were not sterilized, since being subjected to the autoclave will alter the content of the samples and filter sterilization is not suitable for sterilizing solids. Test samples were added at the time of inoculation to reduce the possibility of contamination. In addition to the test samples, 1 mL (1% v / v) of material from the culture flask was added to each flask. The

75/117 flasks were incubated as described above for 36 hours

Analyze

Two samples were analyzed for cell concentration (using slide dispersion for Z. mobilis and direct counts (hemocytometer and microscope) for S. cerevisiae and P. stipitis). Properly diluted samples of Z. mobilis were dispersed on slides with Dextrose Yeast Extract (glucose 20 g / L, Yeast Extract 10 g / L, Agar 20 g / L, pH 5.4), incubated at 30 ° C 5% CO ₂ for 2 days and the number of colonies counted. Appropriately diluted samples of S. cerevisiae and P. stipitis were mixed with 0.05% Trypan blue, loaded on a Neubauer hemocytometer. The cells were counted under 40 X magnification.

Three samples were analyzed for ethanol concentration using the YSI Biochem Analyzer based on the alcohol dehydrogenase assay (YSI, Interscience). The samples were centrifuged at 14,000 rpm for 20 minutes and the supernatant stored at -20 ° C to preserve integrity. The samples were diluted to between 0-3.2 g / L of ethanol before analysis. A standard of 3.2 g / L of ethanol was analyzed approximately every 30 samples to ensure that the integrity of the membrane was maintained during analysis. The optical density (600 nm) of the samples is not reported because the solid test samples interfered with the measurement of absorbance by increasing the turbidity of the samples and are inaccurate.

Ethanol Analysis Results

Performance was used to compare each sample to the control for each microorganism (Tables 37-39). However,% performance cannot be used to compare between strains. When comparing strains, the total ethanol concentration should be used. When analyzing data, a performance% of less than 80% can indicate toxicity when accompanied by a low number of cells. The equation used to determine performance% is:

Performance% = (ethanol in the sample / ethanol in the control) x 100

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Table 39. Ethanol concentration and% performance using Zymomonas mobilis

36 hours Performance % 93 00 00 CO CM 109 | 93 | CD I ⁰⁶ I 102! I ⁶⁶ I I ⁰⁶ Ί C \ J O v— O O Ethanol concentration _ 7.5 | 6.8 T " r-7 σί I 8.8] J r- I 7.3 I 8.3 oo I 7.3 I 8.3 ro Ui Π5 ι- Ο _ç O CO Performance % ’Μ 00 59 \ Tl 103 in O v CD CO 00 | 85 CO 00 co oo | 88 | 98 100 Ethanol concentration _ 6.8] 4.8 6.2 8.3 I 8.4 1 7.6 t < I 6.8 I 6.9 l < I 7.0 I 7.8 8.0 24 hours I Performance son % Lf> CO 85] 83 | 60Í I I 105 CO CO 68 I | 93 86 I 66 | 00 00 00 CD 100 Ethanol concentration í 7.5 | 7.5. J 7.3) I 9.6 I 9.2 l CM 00 I 7.9 I 8.2 I 8.7 I 8.7 00 l < I 8.6 i 8.8 Sample # P132 I P132-10 P132-100 CM CO < O 1 CM CO < I A132-100 I WS132 | WS132-10 I WS132-100 CM CO 0 I G132-10 | G132-100 Control

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Cell Concentration Analysis Results

The% of cells is used to compare each sample to the control for each organism (Tables 40-42). However, the% of cells cannot be used to compare between strains. When comparing strains, total cell concentration should be used. When analyzing the data, a performance of less than 70% may indicate toxicity when accompanied by a low concentration of ethanol. The equation used to determine performance% is:

% of cells = (number of cells in the sample / number of cells in the control) x 100

Table 40. Results of cell concentration analysis for Saccharomyces cerevisiae

Sample # 24 hours 36 hours Cell concentration (x 10 ⁸ / ml) % cells Cell concentration (x 10 ⁸ / ml) % cells P132 1.99 166 2.51 83 P132-10 2.51 209 1.91 63 P132-100 1.35 113 1.99 66 A132 3.80 316 2.59 85 A132-10 1.73 144 3.90 129 A132-100 3.98 331 2.51 83 G132 2.14 178 3.12 103 G132-10 2.33 194 2.59 85 G132-100 3.57 298 2.66 88 WS132 4.10 341 2.66 88 WS132-10 2.63 219 2.81 93 WS132-100 2.29 191 2.40 79 Control 1.20 100 3.03 100

Table 41. Results of cell concentration analysis for Pichia stipitis

Sample # 24 hours 48 hours Cell concentration (x 10 ⁸ / ml) % cells Cell concentration (x 10 ⁸ / mL) % cells P132 16.4 108 20.3 87 P132-10 11.5 76 9.5 41 P132-100 6.5 43 17.8 76 A132 7.1 47 10.2 44 A132-10 12.7 84 9.3 40 A132-100 11.8 78 18.3 78

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Continuation

G132 4.5 30 4.8 21 G132-10 22.8 151 9.8 42 G132-100 10.1 67 21.7 93 WS 132 17.6 117 8.2 35 WS132-10 5.3 35 10.8 46 WS132-100 9.3 62 10.7 46 Control 15.1 100 23.4 100

Table 42. Results of cell concentration analysis for Zymomonas mobilis

Sample # 24 hours 36 hours Cell concentration (x 10 ⁸ / ml) % cells Cell concentration (x 10 ⁸ / mL) % cells P132 7.08 86 2.97 66 P132-10 21.80 264 4.37 98 P132-100 4.50 54 3.35 75 A132 6.95 84 1.99 44 A132-10 6.13 74 4.05 91 A132-100 9.60 116 4.20 94 G132 7.48 90 3.84 86 G132-10 14.75 178 2.89 65 G132-100 6.00 72 2.55 57 WS132 9.70 117 4.55 102 WS132-10 13.20 160 4.32 97 WS132-100 5.15 62 2.89 65 Control 8.27 100 4.47 100

Example 17- Fermentation in a shake flask of celu5 lose samples using P. stipitis

summary

Thirteen samples were tested for ethanol production in P. stipitis without sugar added. They were tested in the presence and absence of cellulase (Accellerase 1000® enzyme complex,

Genencor). The equipment and reagents used for the experiment are listed below in Tables 43-45.

Table 43. Equipment and maintenance frequency

Equipment Manufacturer Maintenance frequency Agitators (2) B. Braun Biotech, Certomat BS-1 Every four months Spectrophotometer Unicam, UV300 Every two years YSI Biochem Analyzer Interscience, YSI Monthly

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Table 44. YSI components used in shaking bottle study

Component Catalog # Lot # YSI ethanol membrane 2786 07L100153 YSI ethanol standard (3.2 g / L) 2790 012711040 YSI ethanol buffer 2787 07M1000053, 07100215 Table 45. Chemicals used for fermentation in a flask agitation

Middle components Manufacturer Reference # Lot # Urea ScholAR Chemistry 9472706 AD-7284-43 Nitrogen base for yeast Becton Dickinson 291940 7128171 Peptone Becton Dickinson 211677 4303198 YM Broth Becton Dickinson 271120 6278265 Ac- enzyme complex cellerase® Genencor Accellerase® 1000 1600794133 Xylose BioChemika 95731 1304473 51707231 Glucose Sigma G-5400 107H0245

An inclined culture medium of P. stipitis NRRL Y-7124 was prepared from a rehydrated lyophilized culture obtained from the ARS Culture Collection. A part of the material from the inclined culture was placed on a Yeast Mold Broth (Yeast Mold - YM) + 20 g / L agar (pH 5.0) and incubated at 30 ° C for 2 days. A 250 mL Erlenmeyer flask containing 10 mL containing 100 mL of medium (40 g / L of glucose, 1.7 g / L of nitrogen base for yeast, 2.27 g / L of urea, 6.56 g / L of peptone, 40 g / L xylose, pH 5.0) was inoculated with a colony and incubated for 24 hours at 25 ° C and 100 rpm. After 23 hours of growth, a sample was taken and analyzed for optical density (600 nm in a UV spectrophotometer) and purity (Gram staining). Based on these results, a flask (called the Culture Flask) with an optical density of 6.79 and a light Gram stain was chosen to inoculate all test flasks.

The test containers were 250 mL Erlenmeyer flasks containing 100 mL of medium (1.7 g / L of nitrogen base for yeast, 2.27 g / L of urea and 6.56 g / L of peptone). No sugar (glucose or xylose) was added to the medium in the growth flask. All vials were autoclaved empty at 121 ° C and 103.42 kPa (15psi) and sterile filter medium (0.22 pm filter) added to the vials before adding the

82/117 test materials. The test materials were not sterilized, since being subjected to the autoclave will alter the content of the samples and filter sterilization is not suitable for sterilizing solids. Test samples (listed in Table 46) were added at the time of inoculation (rather than before) to reduce the possibility of contamination. In addition to the test samples, 1 mL (1% v / v) of material from the culture flask was added to each flask. Flasks containing P132-100 sample required the addition of 0.4 mL of 1 M NaOH to bring the pH to 5.0. The flasks were incubated at 30 ° C and 150 rpm for 96 hours.

A set of bottles in duplicate per raw material contained an Accellerase® enzyme complex (1.25 mL per bottle, the highest recommended dosage is 0.25 mL per gram of biomass, Genencor) to attempt simultaneous saccharification and fermentation (SSF). The other set of duplicate vials does not contain an Accellerase® enzyme complex. A total of 52 bottles were analyzed.

Six control bottles were also analyzed. Positive control flasks contained 200 NF SolkaFloc Cellulose Powder (lot # UA158072, International Fiber Corporation) at a concentration of 2.5 grams per 100 mL flask (25 grams per L) with and without the addition of 20 pellets of Accellerase enzyme ®. In addition, a control containing sugars (glucose and xylose) was only used.

Table 46. The amount of each raw material added to each bottle

Xyleco Number Quantity added to the vial (g / 100 mL) P132 2.5 P132-10 2.5 P132-100 2.5 A132 5 A132-10 5 A132-100 5 G132 5 G132-10 5 G132-100 5 WS 132 5 WS132-10 5 WS132-100 5 Sample A 5

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Analyze

The samples were analyzed for ethanol concentration (Tables 47, 48 and 49) using the YSI Biochem Analyzer based on the alcohol dehydrogenase assay (YSI, Interscience). Samples were centrifuged at 14,000 rpm for 20 minutes and the supernatant stored at -20 ° C. The samples were diluted to between 0-3.2 g / L of ethanol before analysis. A standard of 2.0 g / L of ethanol was analyzed approximately every 30 samples to ensure that the integrity of the membrane was maintained during analysis.

Results

Table 47. Control Bottle Results

Control Ethanol concentration (g / L) 24 hours 36 hours 48 hours 96 hours Containing glucose, no cellulose, no enzyme 13.20 19.00 20.60 21.60 Containing crystalline cellulose (Solka Floc), without sugar, without enzyme 0.00 0.00 0.00 0.00 Containing crystalline cellulose (Solka Floc) at 25 g / L, without sugar, Accellerase® added 6.56 7.88 9.80 8.65

Table 48. Results from shake flasks without enzyme complex

Accellerase® 1000

Sample number Ethanol concentration (g / L) 24 hours 36 hours 48 hours 96 hours P132 0.09 0.00 0.00 0.12 P132-10 0.02 0.01 0.02 0.17 P132-100 0.09 0.01 0.00 0.02 A132 1.74 1.94 2.59 3.70 A132-10 1.82 2.36 2.30 2.96 A132-100 0.30 0.73 1.31 2.38 G132 0.40 0.09 0.24 0.42 G132-10 0.69 0.42 0.22 0.24 G132-100 0.19 0.05 0.05 0.21 WS 132 0.47 0.50 0.68 0.65 WS132-10 0.47 0.49 0.34 0.92 WS132-100 0.14 0.07 0.08 0.22 Sample A 1.88 1.89 2.30 3.28

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Table 49. Results of shake bottles with enzyme complex

Accellerase® 1000

Sample number Ethanol concentration (g / L) 24 hours 36 hours 48 hours 96 hours P132 7.04 8.72 9.30 5.80 P132-10 4.22 4.48 4.49 1.24 P132-100 3.18 4.28 4.70 3.35 A132 2.79 2.91 2.03 4.30 A132-10 3.31 1.62 2.11 2.71 A132-100 2.06 1.92 1.02 1.47 G132 0.87 0.40 0.32 0.44 G132-10 1.38 1.04 0.63 0.07 G132-100 2.21 2.56 2.34 0.12 WS 132 1.59 1.47 1.07 0.99 WS132-10 1.92 1.18 0.73 0.23 WS132-100 2.90 3.69 3.39 0.27 Sample A 2.21 2.35 3.39 2.98

Example 18 - Cellulase assay

summary

Thirteen samples were tested for cellulose susceptibility using an industrial cellulase (Accellerase® 1000, Genencor) under optimal conditions of temperature and pH.

Protocol

The protocol is a modification of the NREL Laboratory Analytical 10 Procedure LAP-009 Enzymatic Saccharification of Lignocellulosic Biomass. A sample of material was added to 10 mL of 0.1 M sodium citrate buffer (pH 4.8) and 40 mg / mL tetracycline (to prevent bacterial growth) in a 50 mL duplicate tube. The amount of sample added to each tube is listed in Table 50. Some samples were difficult to mix (P132, P132-10, P132-100), so they were added in a lower concentration. A positive control of 0.2 grams of Cellulose Powder SolkaFloc 200 NF (lot # UA158072, International Fiber Corporation) and a negative control (without sample) were also included. Water submitted to reverse osmosis (Reverse Osmosis - RO) takes the volume to a total of 20 mL and was added to the tubes. The sodium citrate buffer and water were heated to 50 ° C before use.

Accellerase® 1000 enzyme was added to each tube in a

85/117 dosage of 0.25 mL per gram of biomass (highest dosage recommended by Genencor). The tubes were incubated at an angle of 45 ° at 150 rpm and 50 degrees C (recommended by Genencor) for 72 hours. Samples were taken at 0, 3, 6, 9, 12, 18, 24, 48 and 72 hours (Tables 52 and 53), cen5 trifuged at 14,000 rpm for 20 minutes and the supernatant frozen at 20 ° C. The glucose concentration in the samples was analyzed using the YSI Biochem Analyzer (Interscience) using the conditions described in Table 51. A standard 2.5 g / L glucose solution was prepared by dissolving 2,500 grams of glucose (Sigma Cat # G7528-5KG , Lot #: 107H0245) in distilled water. Once dissolved, the total volume was brought to 1 L with distilled water in a volumetric flask. The standard was prepared fresh weekly and stored at 4 ° C.

Table 50. Quantity of each sample added _

Xyleco Number Quantity added to the tube (g / 20 mL) P132 0.5 P132-10 0.5 P132-100 0.5 A132 0.75 A132-10 0.75 A132-100 0.75 G132 0.75 G132-10 0.75 G132-100 0.75 WS 132 0.75 WS132-10 0.75 WS132-100 0.75 Sample A 0.75 SolkaFloc 200NF (Control) 0.2 Negative control 0

Table 51. YSI components used in a shake bottle study

Component Catalog # Lot# YSI glucose membrane 2365 07D100124 YSI glucose buffer 2357 014614A

Results

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Table 52. Results of the cellulose test

Sample number Glucose concentration (mg / mL) in incubation time (hours) 0 3 6 9 12 18 24 48 72 P132 0.59 4.19 7.00 8.72 9.70 10.95 12.19 15.10 15.65 P132-10 0.36 3.37 5.08 6.39 6.98 7.51 8.99 11.25 11.65 P132-100 0.91 3.86 5.67 7.31 8.08 9.47 10.70 12.70 13.80 A132 0.39 1.51 1.92 2.40 2.64 3.04 3.30 3.90 4.06 A132-10 0.42 1.80 2.27 2.63 2.86 3.16 3.43 4.02 4.14 A132-100 0.46 2.09 2.72 3.16 3.43 3.78 4.09 4.84 5.26 G132 0.40 1.16 1.35 1.52 1.60 1.67 1.85 2.10 2.21 G132-10 0.34 1.34 1.64 1.95 2.03 2.09 2.36 2.77 3.02 G132-100 0.61 1.84 2.32 2.89 3.14 3.52 3.97 4.81 5.44 WS132 0.35 1.48 1.81 2.14 2.26 2.50 2.70 3.18 3.26 WS132-10 0.44 1.77 2.22 2.60 2.76 2.61 3.15 3.62 3.82 WS132-100 0.70 2.76 3.63 4.59 4.78 5.29 5.96 6.99 7.43 Sample A 0.42 1.09 1.34 1.55 1.69 1.66 2.17 2.96 3.71 Negative control (no sample) 0.03 0.03 0.01 0.01 0.02 0.01 0.02 0.02 0.02 Positive control (SolkaFloc) 0.17 2.38 3.65 4.71 5.25 5.98 7.19 9.26 9.86

The amount of cellulose digested in the tube was calculated as follows:

g / mL glucose x 20 mL (sample volume) x 0.9 (to 5 correct the water molecule added when cellulose hydrolysis)

The percentage of the total sample released as glucose (in Table 53 below) was calculated as follows:

g of digested cellulose / g of added sample (see Table 5 for 10 sizes) * 100

Table 53. Results of the cellulose test

Sample number Percentage of the total sample released as glucose (%) in the incubation time (h) 0 3 6 9 12 18 24 48 72 P132 2.02 14.98 25.16 31.36 34.85 39.38 43.81 54.29 56.27 P132-10 1.19 12.02 18.25 22.97 25.06 27.00 32.29 40.43 41.87 P132-100 3.17 13.79 20.38 26.28 29.02 34.06 38.45 45.65 49.61 A132 0.86 3.55 4.58 5.74 6.29 7.27 7.87 9.31 9.70 A132-10 0.94 4.25 5.42 6.29 6.82 7.56 8.18 9.60 9.89 A132-100 1.03 4.94 6.50 7.56 8.18 9.05 9.77 11.57 12.58 G132 0.89 2.71 3.22 3.62 3.79 3.98 4.39 4.99 5.26

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Sample number Percentage of the total sample released as glucose (%) in the incubation time (h) 0 3 6 9 12 18 24 48 72 G132-10 0.74 3.14 3.91 4.66 4.82 4.99 5.62 6.60 7.20 G132-100 1.39 4.34 5.54 6.91 7.49 8.42 9.48 11.50 13.01 WS132 0.77 3.48 4.32 5.11 5.38 5.98 6.43 7.58 7.78 WS 132-10 0.98 4.18 5.30 6.22 6.58 6.24 7.51 8.64 9.12 WS132-100 1.61 6.55 8.69 10.99 11.42 12.67 14.26 16.73 17.78 Sample A 0.94 2.54 3.19 3.70 4.01 3.96 5.16 7.06 8.86 Control positive (SolkaFloc) 1.29 21.15 32.72 42.30 47.07 53.73 64.53 83.16 88.56

Example 19 - Shaking bottle fermentation using Pichia stipitis

summary

Shaking bottle fermentation using Pichia stipitis was performed using four cellulosic materials having the highest performance% from Table 36.

Protocol

Experiments were carried out under the parameters outlined in Tables 54-56.

Table 54. Equipment and maintenance frequency

Equipment Agitators (2) YSI Biochem Analyzer Spectrophotometer Manufacturer, Name B. Braun Biotech, Certomat BS-1 Unicam, UV300 Interscience, YSI Maintenance frequency Every four months Every two years Monthly Table 55. YSi components used in a shaking bottle study tation Component Catalog # Lot# YSI ethanol membrane 2786 07M100361 YSI ethanol standard (3.2 g / L) 2790 1271040 YSI ethanol buffer 2787 07J100215

Table 56. Chemicals used for shaking bottle fermentation

Middle components Manufacturer Reference # Lot # Urea ScholAR Chemistry 9472706 AD-7284-43 Nitrogen base for Becton Dickinson 291940 7128171 yeast Peptone Becton Dickinson 211677 4303198 YM Broth Becton Dickinson 271120 6278265 Xylose Alpha Aesar A10643 10130919 Glucose Fisher Scientific BP350-1 030064

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Culture development

For all of the following shake flask experiments, the culture flasks were prepared using the following procedure.

A working cell bank of P. stipitis NRRL Y-7124 was prepared from a rehydrated lyophilized culture obtained from the ARS Culture Collection. Cryoprasks containing P. stipitis culture in 15% v / v glycerol were stored at -75 ° C. A part of the material from the frozen working cell bank was placed on a Yeast Mold Broth (Yeast Mold - YM) + 20 g / L agar (pH 5.0) and incubated at 30 ° C for 2 days. The slides were kept for 2 days at 4 ° C before use. One 250 mL Erlenmeyer flask containing 100 mL of medium (40 g / L of glucose, 1.7 g / L of nitrogen base for yeast, 2.27 g / L of urea, 6.56 g / L of peptone , 40 g / L xylose, pH 5.0) was inoculated with a colony and incubated for 24 hours at 25 ° C and 100 rpm. After 23 hours of growth, a sample was taken and analyzed for optical density (600 nm in a UV spectrophotometer) and purity (Gram staining). Based on these results, a flask (called the Culture Flask) at an optical density of between 4 and 8 and with a light Gram stain was used to inoculate all test flasks.

Three experiments were performed using samples A132-10, A132-100, G132-10 and G132-100. Experiment # 1 tested these four samples for ethanol concentration at varying concentrations of xylose and at constant concentrations of glucose. Experiment # 2 tested these four samples for the concentration of ethanol in the double concentration of raw material used in the experiments in Table 36. Finally, experiment # 3 tested these four samples for the concentration of ethanol while varying the xylose concentrations and glucose, simultaneously.

Experiment # 1 - Variation of Xylose Concentration

Four cellulosic samples (A132-10, A132-100, G132-10 and G132-100) were tested at varying concentrations of xylose as listed in Table 57 below.

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Table 57. Composition of vial medium in Experiment # 1

Treatment Xylose concentration (g / L) Glucose concentration (g / L) 100% xylose 40.0 40.0 50% xylose 20.0 40.0 25% xylose 10.0 40.0 10% xylose 4.0 40.0 0% xylose 0.0 40.0

The test vessels (a total of 40 250 ml Erlenmeyer flasks) contained 100 ml of medium. Five different types of medium were prepared with the amount of xylose and glucose outlined in Table 57. In addition, the medium contained 1.7 g / L of nitrogen base for yeast (Becton Dickinson # 291940) 2.27 g / L of urea (ScholAR Chemistry # 9472706) and 6.56 g / L of peptone (Becton Dickinson # 211677). All flasks were autoclaved empty at 121 ° C and 103.42 kPa (15psi)) and sterile filter medium (0.22 pm filter) was added to the flasks before adding the test materials. The bottles were kept at room temperature for 4 days and checked for contamination (turbidity) before use. The test materials were not sterilized, since being subjected to the autoclave will alter the content of the samples and filter sterilization is not suitable for sterilizing solids. Test samples (A132-10, A132-100, G132-10 and G132-100 at 5 g per 100 mL) were added at the time of inoculation (rather than before) to reduce the possibility of contamination. In addition to the test samples, 1 mL (1% v / v) of material from the culture flask was added to each flask. The flasks were incubated at 30 ° C and 150 rpm for 72 hours.

Unfortunately, a vial (sample A132-100 with 100% xylose) was broken during testing. Therefore, all results after 24 hours of incubation are reported as a single bottle. After 72 hours of incubation, 100% of the original amount of cellulosic material (5.0 g) was added to the flasks with 100% xylose (7 flasks in total, a flask containing A132-100 sample broke) and incubated as above for another 48 hours.

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Table 58. Addition of raw material to flasks with 100% xylose in the 72-hour incubation time

Feedstock

A132-10 A132-100 G132-10 G132-100

Added to 72 hours (grams)

Analyze

Samples were taken from 40 test flasks at incubation times of 0, 6, 12, 24, 36, 48 and 72 hours. In addition, samples were taken 24 and 48 hours after adding the second quantity of raw material to the vials with 100% xylose (see Table 58).

A total of 292 samples were analyzed for ethanol concentration using a YSI Biochem Analyzer based on the alcohol dehydrogenase assay (YSI, Interscience). Samples were centrifuged at 14,000 rpm for 20 minutes and the supernatant stored at 20 ° C. Of note, samples at time 0 required filtration through a 0.45 pm syringe filter. The samples should be diluted to between 0-3.2 g / L of ethanol before analysis. A standard of 2.0 g / L of ethanol was analyzed approximately every 30 samples to ensure that the integrity of the membrane was maintained.

A total of 47 samples were analyzed for cell count. Samples will be taken at the incubation time of 72 hours and 48 hours after adding more cellulosic material. Properly diluted samples were mixed with 0.05% Trypan blue and loaded into a Neubauer hemocytometer. The cells were counted at 40 X magnification. Experiment # 2 - Analysis of 2 X Concentration of raw material

The 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 base for yeast (Becton Dickinson # 291940) 2.27 g / L of urea (ScholAR Chemistry # 9472706) and 6.56 g / L of peptone (Becton Dickinson # 211677). The bottles were prepared according to Experiment # 1. The test samples (A132-10, A132-100, G132-10 and G132-100 at 10 g per 100 mL) were added at the time of inoculation (rather than before) to reduce the possibility of contamination. In addition to the test samples, 1 mL (1% v / v) of material from the culture flask was added to each flask. The flasks were incubated at 30 ° C and 150 rpm for 72 hours.

Analyze

Samples were from 8 test flasks at an incubation time of 0, 6, 12, 24, 36, 48 and 72 hours. Ethanol analyzes of the 56 samples were performed as per Experiment # 1 and are reported in Table 59. A cell count was performed on the sample for 72 hours as per Experiment # 1 and is shown in Table 60.

Table 59. Concentration of ethanol in bottles with double raw material

Sample time Ethanol concentration (g / L) A132-10 A132-100 G132-10 G132-100 0 1.38 0.26 0.12 0.11 6 1.75 0.21 0.20 0.10 12 2.16 0.73 0.69 0.31 24 19.05 15.35 16.55 12.60 36 21.75 17.55 18.00 15.30 48 26.35 23.95 24.65 20.65 72 26.95 27.35 28.90 27.40

Table 60. Concentration of cells in the 72-hour incubation time in flasks with double raw material

Sample A132-10 A132-100 G132-10 G132-100

Cell concentration

4.06

5.37

5.18

4.47 (x10 ⁸ / ml)

Experiment # 3 - Varied concentrations of xylose and glucose

Four cellulosic samples (A132-10, A132-100, G132-10 and

G132-100) were tested at varying concentrations of xylose and glucose as listed in the table below (Table 60).

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Table 61. Flask medium composition in Experiment # 3

Treatment Xylose concentration (g / L) Glucose concentration (g / L) 50% sugar 20.0 20.0 25% sugar 10.0 10.0 10% sugar 4.0 4.0 0% sugar 0.0 0

The 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 outlined in Table 61. In addition, the medium contained 1.7 g / L of nitrogen base for yeast (Becton Dickinson # 291940) 2.27 g / L of urea (ScholAR Chemistry # 9472706) and 6.56 g / L of peptone (Becton Dickinson # 211677). The bottles were prepared according to Experiment # 1. Test samples (A132-10, A132-100, G132-10 and G132-100) were added at the time of inoculation (rather than before) to reduce the possibility of contamination. In addition to the test samples, 1 mL (1% v / v) of material from the culture flask was added to each flask. The flasks were incubated at 30 ° C and 150 rpm for 72 hours.

Analyze

Samples were taken from 32 test flasks at an incubation time 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 YSi Biochem Analyzer based on the alcohol dehydrogenase assay (YSI, Interscience). Samples were centrifuged at 14,000 rpm for 20 minutes and the supernatant stored at -20 ° C. Of note, some of the samples required centrifugation and then filtration through a 0.45 pm syringe filter. The samples were diluted to between 03.2 g / L of ethanol before analysis. A standard of 2.0 g / L of ethanol was analyzed approximately every 30 samples to ensure that the integrity of the YSI membrane was maintained.

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Table 62. Ethanol results in Sample A132-10

ethanol (g / L) 50% sugars * 0.56 I 00 co O CM CO cm 13.35 IO U) r- T- 16.60 14.20 1 1 25% sugars * 0.56 I 0.83 2.47 r- <n 9.99] 8.33 I 5.08 i ! 10% sugar * í 0.57 | v- cn O CO V CM oo CO 1 ^ - CM 't 3.03 1.52 I 1 1 0% sugars * I 0.53 j 0.93 1 CM Ϊ 1.02! <J> CM ’Ί t— I 1.14 Ί ! 1 100% xylose í 0.39 1.12 I 1.90 I 16.95 20.25 26.80 [28.00 23.15 21.55 Concentration 50% xylose 0.41 j CO σ> r- T " 17.05 21.35 23.00 21.85 ! 1 25% xylose í 0.42 | 1.15 | I 1.71 I 17.05 | IO CM O CM 19.70] | 19.25 1 1 1 10% xylose 0.42 j I 9ΪΛ to OO 15.90! 17.40 17.05 15.55 1 ! 0% xylose 0.43 | CO I 1.72 I | 15.55 | 17.10 | 16.40 I 15.15 1 1 1 1 Time gives sample O CO CM -m- CM I 36 I 48 I 72 24 hours post- addition 48 hours post- addition

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Table 65. Results of ethanol in Sample G132-100

tanol (g / L) 50% sugars * 0.06 | co O d 1.44 | 10.55 | 16.10 IO co d x— 14.30 | 1 1 1 1 25% sugars * 0.05 | 0.05 j co x " 7.62 | 8.73 7.76 co O d 1 ! 10% sugar * 0.05 | 0.05 I 1.37 | 3.67 | 3.09 1, θ1 I O d 1 ! 0% sugars * 0.05 | O d 0.13 | 0.03 | T- CD d O d l 0.03 | 1 1 & Concentration Xylose at 100 % 0.05 | 0.07 | r ~ - d 12.05 19.25 24.45 27.55 25.20 24.60 50% xylose xt O d co O O co I Q d 13.95 I l 18.20 22.55 j I 22.10 1 1 25% xylose 0.04 I co O d 0.67 I 14.90 ! 18.25 ^! i 19.35 l 18.45 1 1 1 The 10% xylose Tt O d 0.07 | 0.56 | U) xt I 17.10 | O cq d I 15.40 l 1 0% xylose 0.04 I 0.07 0.60 I 13.05 15.10 14.40 14.70 1 ! Sample time O co CM xt CM co co 00 xt CM I- 24 hours post-addition 48 hours post-addition

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Samples were taken at 72 hours of incubation for cell counts (see Tables 66-67). Properly diluted samples were mixed with 0.05% Trypan blue and loaded into a Neubauer hemocytometer. The cells were counted at 40 X magnification.

Results

A culture flask was used to inoculate all test flasks from Experiments # 1 and # 2. The optical density (600 nm) of the culture flask was measured to be 5.14 and the cell concentration was 4.65 χ 10 ⁸ cells / mL (Tables 65-66). Therefore, the initial cell concentration in the test flasks was approximately 4.65 χ 10 ⁶ cells / mL.

A second flask of culture was used to inoculate the flasks of Experiment # 3. The optical density (600 nm) of the culture flask was 5.78 and the cell concentration was 3.75 χ 10 ⁸ cells / mL. Therefore, the initial cell concentration in the test flasks was approximately

3.75 χ 10 ⁶ cells / mL.

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Table 66. Cell counts at 72 hour incubation time

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Table 67. Cell counts at 48-hour post-dition incubation time (100% xylose and glucose) __

Sample Cell concentration (x 10 ⁸ / mL)

A132-10 10.17

A132-100 3.38

G132-10 3.94

G132-100 6.53

Example 20 - Toxicity testing of lignocellulosic samples against P. stipitis and S. cerevisiae 5 Summary

Thirty-seven samples were analyzed for toxicity against two cultures that produce ethanol, Saccharomyces cerevesiae and Pichia stipitis. In this study, glucose was added to the samples in order to distinguish between deprivation of cultures and toxicity of the samples.

Table 68. Conditions for testing for toxicity_

Variable Body Saccharomyces cerevisiae ATCC 24858 Pichia stipitis NRRL Y-7124 Inoculation volume (mL) 0.5-1 (target 6-7 x 10 ⁵ cells / mL) 1 (target 3-4 x 10 ⁶ cells / mL) Repeating the test Single bottles Incubation temperature (± 1 ° C) 25 ° C 25 ° C Agitator speed (rpm) 200 125 Container type 500 mL Erlenmeyer Flask 250 mL Erlenmeyer Flask Volume of medium 100ml 100ml Total incubation time (hours) 72 72 Ethanol analysis (hours) 0, 6, 12, 24, 36, 48, 72 0, 6, 12, 24, 36, 48, 72 Cell counts (hours) 24, 72 24, 72 PH 0 hours 0 hours

Protocol

A summary of the protocol used is listed in Table 68. A description of the chemicals used in toxicity testing is listed 100/177 in Table 69. Two control flasks (no sample added) were taken for each microorganism for each week of testing. A total of 82 bottles were analyzed.

During the experiments, no ethanol or cells appeared 5 in the flasks with P. stipitis containing samples C, C-1e, C-5e and C-10e in the first 24 hours of incubation. In order to confirm the results, the test was repeated. The second test confirmed some growth inhibition of P. stipitis when samples C, C1E, C5E and C10E were added to the flasks.

Table 69. Chemicals and materials used for toxicity testing

Component of middle Manufacturer Reference # Lot# Urea ScholAR Chemistry 9472706 AD-7284-43 Nitrogen base for yeast Becton Dickinson 291940 7128171 Peptone Becton Dickinson 211677 4303198 Xylose Alpha Aesar A10643 10130919 Glucose Sigma G-5400 107H0245 Yeast extract Becton Dickinson 288620 4026828 YM Broth Becton Dickinson 271120 6278265

Table 70. YSI components used in a toxicity study Component Catalog #

YSI 2786 ethanol membrane

YSI ethanol standard (3.2 g / L) 2790

YSI_2787 ethanol buffer

Test samples

Seven test samples (all with designation C) were ground using a small sample coffee grinder. The samples were ground to a consistent particle size (between samples) with the naked eye. Sample number C-100e was easily ground to a small particle size.

All samples were added to the flasks in a concentration of 50 grams per liter, except for the six samples P (25

101/117 grams per liter). These samples were white to grayish in color and were visually fluffy and the bottles did not mix properly (without enough liquid) at a concentration of 50 grams per liter. S samples dissolved easily and could later be added to the flasks in a higher concentration. Samples A and G could be added at 100 grams per liter later.

Testing was performed using the two microorganisms as described below.

Saccharomyces cerevisiae ATCC 24858 (American Type Culture Collection)

An S. cerevisiae ATCC 24858 working cell bank was prepared from a rehydrated lyophilized culture obtained from the American Type Culture Collection. Cryoprasks containing S. cerevisiae culture in 15% v / v glycerol are stored at -75 ° C. A part of the material from the frozen working cell bank will be placed on a Yeast Mold Broth (Yeast Mold - YM) + 20 g / L agar (pH 5.0) and incubated at 30 ° C for 2 days. 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 of 5.0) was inoculated with a colony of YM slide and incubated for 24 hours at 25 ° C and 200 rpm. After 23 hours of growth, the sample was taken and analyzed for optical density (600 nm in a UV spectrophotometer) and purity (Gram staining). Based on these results, a flask (called the Culture Flask) with an OD of 9-15 and pure Gram staining was used to inoculate the growth flasks. After 23 hours of growth, the culture flask had a low OD (5.14) and cell count (1.35 x 10 ⁸ cells / ml). Of note, the colony taken from the culture slide was smaller than usual. Therefore, 0.5 ml of culture material (as opposed to planned 0.1 ml) was added to each test vessel.

The test containers were 500 ml Erlenmeyer flasks containing 100 ml of the sterile medium described above. All vials were autoclaved at 121 ° C and 103.42 kPa (15psi) before adding the

102/117 test materials. The test materials were not sterilized, since being subjected to the autoclave would alter the content of the samples. Test samples were added at the time of inoculation (rather than before) to reduce the possibility of contamination. In addition to the test samples, 0.5 -1.0 mL (0.5-1.0% v / v) of material from the culture flask was added to each flask. The flasks were incubated as described above for 72 hours.

Pichia stipitis (ARS Culture Collection)

A working cell bank of P. stipitis NRRL Y-7124 was prepared from a rehydrated lyophilized culture obtained from the ARS Culture Collection. Cryoprasks containing P. stipitis culture in 15% v / v glycerol are stored at -75 ° C. A part of the material from the frozen working cell bank was placed on a Yeast Mold Broth (Yeast Mold - YM) + 20 g / L agar (pH 5.0) and incubated at 30 ° C for 2 days. The slides were kept for up to 5 days at 4 ° C before use. One 250 mL Erlenmeyer flask containing 100 mL of medium (40 g / L glucose, 1.7 g / L nitrogen base for yeast, 2.27 g / L urea, 6.56 g / L peptone , 40 g / L xylose, pH 5.0) was inoculated with a colony and incubated for 24 hours at 25 ° C and 125 rpm. After 23 hours of growth, the sample was taken and analyzed for optical density (600 nm in a UV spectrophotometer) and purity (Gram staining). Based on these results, a flask (called the Culture Flask) at an optical density of 5-9 and with a pure Gram stain was used to inoculate all test flasks.

The test containers were 250 ml Erlenmeyer flasks containing 100 ml of the sterile medium described above. All vials were autoclaved empty at 121 ° C and 103.42 kPa (15psi) and sterile filter medium (0.22 pm filter) added to the vials before adding the test materials. The test materials were not sterilized, since subjection to the autoclave would alter the content of the samples and filter sterilization is not suitable for sterilization of solids. Test samples were added at the time of inoculation (rather than before) to

103/117 reduce the possibility of contamination. In addition to the test samples, 1 mL (1% v / v) of material from the culture flask was added to each flask. The flasks were incubated as described above for 72 hours.

Analyze

Samples were taken from the culture flasks just before inoculation and each test flask at 24 and 72 hours and analyzed for cell concentration using direct counts. Appropriately diluted samples of S. cerevisiae and P. stipitis were mixed with 0.05% Trypan blue, loaded on a Neubauer hemocytometer. The cells were counted at 40 X magnification.

Samples were taken from each bottle at 0, 6, 12, 24, 36, 48 and 72 hours and analyzed for ethanol concentration using the YSI Biochem Analyzer based on the alcohol dehydrogenase assay (YSI, Interscience). Samples were centrifuged at 14,000 rpm for 20 minutes and the supernatant stored at -20 ° C. The samples should be diluted to 0-3.2 g / L of ethanol before analysis. A standard of 2.0 g / L of ethanol was analyzed approximately every 30 samples to ensure that the integrity of the membrane was maintained during analysis.

Calculations

The following calculations were used to compare cell counts and ethanol concentration with the control flasks:

performance% = (concentration of ethanol in the test bottle / ethanol in the control) * 100% cells = (number of cells in the test bottle / number of cells in the control bottle) * 100

Results

The flask of culture with S. cerevisiae had an optical density (600 nm) of 5.14 and a cell concentration of 1.35 χ 10 ⁸ cells / mL. 0.5 ml of material from the culture flask was added to each of the test flasks. Therefore, the initial cell concentration in each flask was 6.75 x 10 ⁵ / ml. During the second week of testing, the S. cerevisiae culture flask had an optical density (600 nm) of 4.87 and a cell concentration of 3.15 χ 10 ⁷ cells / mL. One mL of material

104/117 of the culture flask was added to each of the test flasks. Therefore, the initial cell concentration in each flask was 6.30 x 10 ⁵ / ml. The pH of the flasks with S. cerevisiae at a sampling time of 0 hour is shown in Table 71. The pH of the flasks with contents of the flask was within the optimal pH for growth of S. cerevisiae (pH 4-6). No pH adjustment was required.

Table 71. pH of flasks with S. cerevisiae at 0 hour sample time

Sample number pH Sample number PH P 5.04 Ç 5.46 P1E 4.99 C1E 5.54 P5E 5.04 C5E 5.50 P10E 4.98 C10E 5.33 P50E 4.67 C30E 5.12 P100E 4.43 C50E 4.90 G 5.45 C100E 4.66 G1E 5.47 ST 5.11 G5E 5.46 ST1E 5.06 G10E 5.39 ST5E 4.96 G50E 5.07 ST10E 4.94 THE 5.72 ST30E 5.68 A1E 5.69 ST50E 4.48 A5E 5.62 ST100E 4.23 A10E 5.61 control A 5.02 A50E 5.74 control B 5.04 S* 5.10 S1E 5.08 S5E 5.07 S10E 5.04 S30E 4.84 S50E 4.57 S100E 4.33

* S refers to sucrose * C refers to corn * ST refers to starch

Ethanol concentration and performance in flasks with S. cerevisiae are shown in Tables 72 and 73. The highest concentrations of ethanol were produced by the S. series.

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Table 72. Ethanol concentration in flasks with S. cerevisiae

sample 0 6 12 24 36 48 72 P 0.02 0.04 0.38 5.87 7.86 5.41 1.04 P1E 0.03 0.03 0.28 5.10 8.03 5.46 0.58 P5E 0.03 0.04 0.57 8.84 6.38 3.40 0.04 P10E 0.06 0.05 0.65 6.63 7.66 5.57 1.40 P50E 0.04 0.03 0.26 2.80 5.85 8.59 5.68 P100E 0.04 0.02 0.12 3.64 8.26 7.51 3.03 G 0.04 0.04 0.57 10.20 8.24 6.66 2.84 G1E 0.04 0.05 0.46 10.20 9.24 6.94 2.84 G5E 0.11 0.11 0.44 10.00 8.7 6.36 0.88 G10E 0.05 0.04 0.40 9.97 8.41 5.79 0.11 G50E 0.05 0.05 0.48 9.72 8.33 6.13 2.38 THE 0.29 0.38 0.48 8.43 8.76 7.09 4.66 A1E 0.34 0.44 0.79 9.66 8.9 7.18 2.64 A5E 0.55 0.45 0.99 9.44 8.96 7.56 3.80 A10E 0.55 0.55 0.93 9.58 8.33 6.28 1.40 A50E 0.22 0.08 0.38 9.38 8.01 5.99 0.98 s 0.03 0.03 0.39 5.73 7.06 10.10 15.90 S1E 0.05 0.06 0.31 7.24 9.52 12.10 14.90 S5E 0.02 0.05 0.34 5.87 7.68 11.90 19.00 S10E 0.03 0.04 0.35 5.88 TJ1 11.50 19.30 S30E 0.03 0.05 0.09 5.94 7.97 11.20 20.40 S50E * 0.13 0.19 0.47 5.46 7.96 13.00 18.30 S100E 0.11 0.10 0.21 7.00 10.6 13.80 12.70 Ç 0.01 0.04 0.32 8.47 7.57 5.48 6.40 C1E 0.00 0.06 0.37 8.93 7.86 5.99 1.37 C5E 0.03 0.05 0.48 9.32 7.92 5.69 1.41 C10E 0.02 0.04 0.52 9.14 7.67 5.34 0.35 C30E 0.02 0.05 0.28 9.15 8.15 5.84 2.47 C50E 0.03 0.06 0.44 9.31 7.79 5.78 1.79 C100E 0.03 0.06 0.58 9.06 6.85 5.95 1.09 ST 0.02 0.05 0.99 8.54 6.69 5.09 0.42 ST1E 0.03 0.04 0.70 8.87 7.29 4.81 1.04 ST5E 0.02 0.04 0.52 8.61 7.16 4.97 0.85 ST10E 0.02 0.05 0.33 8.97 7.05 5.26 0.68 ST30E 0.03 0.04 0.71 8.47 6.96 4.89 0.21 ST50E 0.04 0.07 0.34 8.46 8.19 7.04 3.20 ST100E 0.03 0.10 0.30 9.30 8.62 7.29 4.23 control A 0.01 0.07 0.85 5.92 8.18 7.81 6.26 control B 0.01 0.04 0.27 4.86 6.43 8.01 6.75 control THE* 0.04 0.21 1.36 5.19 7.31 7.55 5.16 control B* 0.03 0.20 1.18 5.16 5.96 7.62 5.32

* analyzed in week 2

See Table 72 for sample number key

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Table 73. Performance in flasks with S. cerevisiae

Sample number Performance (%) in the following times (hours) 24 36 48 72 P 108.9 107.6 68.4 16.0 P1E 94.6 109.9 69.0 8.9 P5E 164.0 87.3 43.0 0.6 P10E 123.0 104.9 70.4 21.5 P50E 51.9 80.1 108.6 87.3 P100E 67.5 113.1 94.9 46.5 G 189.2 112.8 84.2 43.6 G1E 189.2 126.5 87.7 43.6 G5E 185.5 119.1 80.4 13.5 G10E 185.0 115.1 73.2 1.7 G50E 180.3 114.0 77.5 36.6 THE 156.4 119.9 89.6 71.6 A1E 179.2 121.8 90.8 40.6 A5E 175.1 122.7 95.6 58.4 A10E 177.7 114.0 79.4 21.5 A50E 174.0 109.7 75.7 15.1 s 106.3 96.6 127.7 244.2 S1E 134.3 130.3 153.0 228.9 S5E 108.9 105.1 150.4 291.9 S10E 109.1 105.7 145.4 296.5 S30E 110.2 109.1 141.6 313.4 S50E * 105.5 119.9 171.3 349.2 S100E 129.9 145.1 174.5 195.1 Ç 157.1 103.6 69.3 98.3 C1E 165.7 107.6 75.7 21.0 C5E 172.9 108.4 71.9 21.7 C10E 169.6 105.0 67.5 5.4 C30E 169.8 111.6 73.8 37.9 C50E 172.7 106.6 73.1 27.5 C100E 168.1 93.8 75.2 16.7 ST 158.4 91.6 64.3 6.5 ST1E 164.6 99.8 60.8 16.0 ST5E 159.7 98.0 62.8 13.1 ST10E 166.4 96.5 66.5 10.4 ST30E 157.1 95.3 61.8 3.2 ST50E 157.0 112.1 89.0 49.2 ST100E 172.5 118.0 92.2 65.0 Control A 109.8 112.0 98.7 96.2 Control B 90.2 88.0 101.3 103.7 Control A * 100.3 110.1 99.5 98.5 Control B * 99.7 89.8 100.4 101.5

* analyzed in week 2

The concentration of cells and% of cells in the flasks with S. cerevisiae are shown in Table 74. High cell counts were observed in all flasks; however, not all cells appear

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Table 74. S cerevisiae cell counts and% cells

Sample number Cell count (cells x 10 ⁸ / mL) Cell% (count / count control) * 100 24 hours 72 hours 24 hours 72 hours P 0.62 0.96 97.7 139.0 P1E 0.35 1.18 54.1 170.9 P5E 1.13 1.93 177.3 279.5 P10E 0.59 1.42 91.8 205.6 P50E 0.32 1.40 49.4 202.8 P100E 0.45 1.94 70.6 281.0 G 0.74 3.48 116.5 504.0 G1E 0.68 3.65 107.1 528.6 G5E 0.62 3.87 96.5 560.5 G10E 0.70 2.73 109.5 395.4 G50E 0.46 2.10 71.8 304.1 THE 0.55 3.53 86.0 511.2 A1E 0.83 3.45 130.7 499.6 A5E 0.67 3.53 104.8 511.2 A10E 0.53 1.95 83.6 282.4 A50E 0.66 1.62 103.5 234.6 s 0.44 1.11 69.5 160.8 S1E 0.44 1.10 68.2 159.3 S5E 0.23 0.99 36.5 143.4 S10E 0.39 0.73 61.2 105.4 S30E 0.31 0.71 48.3 102.1 S50E * 0.44 0.90 86.5 196.5 S100E 0.53 0.84 82.4 121.7 Ç 0.45 1.81 70.6 262.1 C1E 0.71 2.40 110.6 347.6 C5E 0.53 2.33 83.6 337.4 C10E 0.77 1.55 120.0 224.5 C30E 0.75 1.80 117.6 260.7 C50E 0.64 1.70 100.1 246.2 C100E 0.81 1.51 127.1 218.7 ST 0.75 1.75 117.6 253.4 ST1E 0.57 1.36 89.4 197.0 ST5E 0.58 1.49 90.7 215.8 ST10E 0.61 1.32 95.4 191.2 ST30E 0.59 0.60 91.8 86.9 ST50E 0.59 1.30 91.8 188.3 ST100E 0.41 1.24 63.5 179.6 control A 0.81 0.79 127.1 114.1 control B 0.47 0.59 72.9 85.9 control A * 0.66 0.42 131.2 91.7 Control B * 0.35 0.50 69.0 108.1

The P. stipitis culture flask had an optical density (600 nm) of 5.01 and a cell concentration of 3.30 x 10 ⁸ cells / ml.

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One ml of material from the culture flask was added to each of the test flasks. Therefore, the initial cell concentration in each flask was 3.30 x 10 ⁶ / ml. During the second week of testing, the P. stípitis culture flask had an optical density (600 nm) of 5.45 and a cell concentration of 3.83 x 10 ⁸ cells / ml. One ml of material from the culture flask was added to each of the test flasks. Therefore, the initial cell concentration in each flask was 3.83 x 10 ⁶ / ml. The pH of the flasks with P. stípitis at a sampling time of 0 hour is shown in Table 75. The pH of the flasks with contents of the flask was within the optimum pH for growth of P. stípitis (pH 4-7). No pH adjustment was required.

ábela 75. pH of flasks with P. stípitis at 0 hour sample time

Sample number pH Sample number pH P 4.91 Ç 5.36 P1E 4.87 C1E 5.30 P5E 4.90 C5E 5.29 P10E 4.78 C10E 5.06 P50E 4.46 C30E 4.89 P100E 4.24 C50E 4.70 G 5.45 C100E 4.59 G1E 5.43 ST 4.93 G5E 5.48 ST1E 4.90 G10E 5.32 ST5E 4.81 G50E 4.99 ST10E 4.83 THE 5.69 ST30E 4.91 A1E 5.66 ST50E 4.24 A5E 5.60 ST100E 4.07 A10E 5.58 control A 4.93 A50E 5.69 control B 4.91 s 5.00 S1E 4.94 S5E 4.86 S10E 4.78 S30E 4.51 S50E 4.27 S100E 4.08

The ethanol concentration and performance in the flasks with P. stipitis are shown in Tables 76 and 77. The highest concentrations of 15 ethanol were the G and A series. Flasks C-30e, C-50e and C-100e also had 109 high concentrations of ethanol. The concentration of cells and% of cells in the flasks with P. stipitis are shown in Table 78. Low concentrations of cells were observed in flasks with the designations S. Low cell counts were also observed in flasks containing samples C, C1E, C5E and C10E at 24 hour sampling time.

Table 76. Ethanol concentration in bottles with P. stipitis

Number in sample Ethanol concentration (g / L) in the following times (hours) 0 6 12 24 36 48 72 P 0.01 0.05 0.26 4.98 8.57 14.10 17.00 P1E 0.02 0.03 0.04 4.24 9.03 12.40 17.30 P5E 0.02 0.03 0.42 6.72 12.40 15.60 18.60 P10E 0.02 0.02 0.01 1.38 8.69 13.00 17.00 P50E 0.01 0.02 0.02 0.03 3.77 10.50 16.90 P100E 0.02 0.03 0.02 3.75 10.50 15.60 18.80 G 0.02 0.08 0.20 10.80 17.70 19.40 25.40 G1E 0.04 0.12 0.50 12.20 19.60 23.80 28.60 G5E 0.07 0.14 0.73 12.50 19.10 24.50 27.50 G10E 0.04 0.19 0.42 10.20 19.10 22.90 28.20 G50E 0.05 0.22 0.25 8.73 18.40 22.20 28.00 THE 0.13 0.28 0.82 1.08 16.10 19.40 19.30 18.60 A1E 0.22 0.59 16.10 22.40 27.60 27.70 A5E 0.32 0.43 0.43 10.60 22.10 27.10 28.10 A10E 0.33 0.61 1.15 14.90 22.00 27.10 27.90 A50E 0.30 0.10 0.47 13.40 20.20 24.80 27.10 s 0.01 0.01 0.26 3.68 7.50 10.20 13.30 S1E 0.02 0.02 0.22 4.98 9.22 11.60 14.20 S5E 0.02 0.02 0.19 4.25 8.50 11.70 14.70 S10E 0.03 0.02 0.17 2.98 8.87 11.90 14.70 S30E 0.08 0.05 0.03 2.96 8.73 12.60 16.50 S50E 0.08 0.05 0.04 2.24 6.13 7.95 12.50 S100E 0.11 0.10 0.08 3.36 7.82 10.50 13.90 Ç* 0.02 0.03 0.05 0.23 1.66 2.68 6.57 C1E * 0.03 0.03 0.03 0.07 0.95 1.85 10.20 C5E * 0.03 0.02 0.04 0.05 0.37 1.59 4.80 C10E * 0.03 0.04 0.04 0.05 3.91 15.20 28.30 C30E 0.01 0.03 0.60 12.30 21.20 26.00 27.20 C50E 0.02 0.02 0.45 12.30 19.50 23.80 29.20 C100E 0.05 0.04 0.38 11.40 18.70 22.90 27.70 ST 0.03 0.03 0.37 6.69 10.70 13.50 10.90 ST1E 0.01 0.00 0.48 5.24 9.37 12.50 15.70 ST5E 0.02 0.03 0.29 5.45 10.10 11.90 14.70 ST10E 0.02 0.02 0.42 5.60 9.44 12.20 14.90 ST30E 0.05 0.04 0.73 5.70 9.50 12.10 15.20 ST50E 0.02 0.05 0.19 5.16 9.47 12.70 15.20 ST100E * 0.07 0.15 0.11 4.98 10.70 15.40 18.80

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Number in sample Ethanol concentration (g / L) in the following times (hours) 0 6 12 24 36 48 72 control THE 0.02 0.03 0.37 4.05 7.50 9.24 11.50 control B 0.02 0.02 0.30 4.22 7.44 9.44 11.50 control THE* 0.02 0.05 0.69 4.86 8.69 11.10 16.40 control B* 0.02 0.05 0.74 5.96 10.80 13.00 14.00

* analyzed in week 2

Table 77. Performance in bottles with P. stipitis

Sample number Performance (%) in the following times (hours) 24 36 48 72 P 120.3 114.7 151.0 147.8 P1E 102.4 120.9 132.8 150.4 P5E 162.3 166.0 167.0 161.7 P10E 33.3 116.3 139.2 147.8 P50E 0.7 50.5 112.4 147.0 P100E 90.6 140.6 167.0 163.5 G 260.9 236.9 207.7 220.9 G1E 294.7 262.4 254.8 248.7 G5E 301.9 255.7 262.3 239.1 G10E 246.4 255.7 245.2 245.2 G50E 210.9 246.3 237.7 243.5 THE 388.9 259.7 206.6 161.7 A1E 388.9 299.9 295.5 240.9 A5E 256.0 295.9 290.1 244.3 A10E 359.9 294.5 290.1 242.6 A50E 323.7 270.4 265.5 235.7 s 88.9 100.4 109.2 115.7 S1E 120.3 123.4 124.2 123.5 S5E 102.7 113.8 125.3 127.8 S10E 72.0 118.7 127.4 127.8 S30E 71.5 116.9 134.9 143.5 S50E 54.1 82.1 85.1 108.7 S100E 81.2 104.7 112.4 120.9 Ç* 4.2 17.0 22.2 43.2 C1E * 1.4 9.7 15.4 67.1 C5E * 0.9 3.8 13.2 31.6 C10E * 0.9 40.1 126.1 246.1 C30E 297.1 283.8 278.4 236.5 C50E 297.1 261.0 254.8 253.9 C100E 275.4 250.3 245.2 240.9 ST 161.6 143.2 144.5 94.8

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Sample number Performance (%) in the following times (hours) 24 36 48 72 ST1E 126.6 125.4 133.8 136.5 ST5E 131.6 135.2 127.4 127.8 ST10E 135.3 126.4 130.6 129.6 ST30E 137.7 127.2 129.6 132.2 ST50E 124.6 126.8 136.0 132.2 ST100E * 120.3 109.7 127.8 123.7 control A 97.8 100.4 98.9 100.0 control B 101.9 99.6 101.1 100.0 control A * 89.8 89.1 92.1 107.9 control B * 110.2 110.8 107.9 92.1

* analyzed in week 2

Table 78. P. stipitis cell counts and% of cells

Sample number Cell count (cells x 10 ⁸ / mL) Cell% (count / count control) * 100 24 hours 72 hours 24 hours 72 hours P 2.78 11.00 80.6 148.0 P1E 2.10 7.20 60.9 96.9 P5E 2.93 9.68 84.9 130.3 P10E 1.42 7.73 41.2 104.0 P50E 0.33 8.63 9.6 116.2 P100E 1.58 8.25 45.8 111.0 G 1.50 14.20 43.5 191.1 G1E 3.90 8.10 113.0 109.0 G5E 2.93 6.45 84.9 86.8 G10E 4.35 13.30 126.1 179.0 G50E 3.75 11.60 108.7 156.1 THE 7.43 8.55 215.4 115.1 A1E 4.13 9.53 119.7 128.3 A5E 3.68 9.75 106.7 131.2 A10E 4.50 7.50 130.4 100.9 A50E 6.23 5.33 180.6 71.7 s 3.53 5.55 102.3 74.7 S1E 3.00 3.30 87.0 44.4 S5E 3.68 3.00 106.7 40.4 S10E 1.73 5.78 50.1 77.8 S30E 2.55 5.48 73.9 73.8 S50E 2.63 6.15 76.2 82.8 S100E 2.25 4.43 65.2 59.6 Ç* 0.00 0.26 0.00 7.2 C1E * 0.00 0.36 0.00 9.9 C5E * 0.00 0.08 0.00 2.1 C10E * 0.00 5.85 0.00 160.7 C30E 5.78 4.20 167.5 56.5 C50E 3.40 7.35 98.6 98.9

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Sample number Cell count (cells x 10 ⁸ / mL) Cell% (count / count control) * 100 24 hours 72 hours 24 hours 72 hours C100E 1.98 6.60 57.4 88.8 ST 2.55 7.65 73.9 103.0 ST1E 2.00 8.70 58.0 117.1 ST5E 1.85 6.75 53.6 90.8 ST10E 1.83 5.40 53.0 72.7 ST30E 2.78 6.15 80.6 82.8 ST50E 1.33 3.45 38.6 46.4 ST100E * 4.35 3.83 59.8 105.2 control A 3.60 7.13 104.3 96.0 Control B 3.30 7.73 95.7 104.0 Control THE* 7.50 3.23 103.0 88.7 control B * 7.05 4.05 96.8 111.3

* analyzed in week 2

Summary of Zymomonas mobilis cell toxicity results

As shown in Graph 1A, elevated cell numbers (for example, greater than the control) were observed in samples containing P-132-10, G-132-10 and WS-132-10 at the 24 hour time point. The cell numbers in the presence of all other samples were comparable to the control. This observation indicates that the substrates were not toxic to Z. mobilis for up to 24 hours after culture.

At the 36 hour time point, a decrease in cell numbers (for example, due to cell loss or cell death) was observed for all samples, including the control. The greatest decrease in cell numbers was observed for those samples containing P-132-10, G-132-10. The probable cause of this effect is common to all samples, including the control. Thus, the cause of this effect is not the test substances, since they vary in each sample and are not present in the control. Possible explanations for this observation include inappropriate culture conditions (for example, temperature, medium compositions) or concentrations of ethanol in the sample.

As shown in Graph 1B, all cells produced comparable amounts of ethanol (for example, 5-10 g / L) at each time point, regardless of the substrate. Consistent with the number of

113/117 cells shown in Graph 1A, the ethanol concentration in each sample peaked at the 24 hour time point. In contrast to the cell number data, the ethanol concentration does not decrease at subsequent time points. This was expected, since ethanol was not removed from the system. In addition, these data suggest that ethanol production in these samples may have resulted from glucose fermentation in the culture medium. None of the tested substrates appeared to increase ethanol production.

Pichia stipitis

As shown in Graph 2A, the cell numbers were comparable to the control. In addition, although slightly reduced cell numbers were present in samples containing G-132 and WS132, reduced cell numbers were not observed for G-132-10, ΟΙ 32-100, A-132-10 or A-132-100 . Thus, G or A substrates are unlikely to be toxic. Previously, the reduced cell numbers observed for G-132 and WS-132 were probably caused by an experimental anomaly or by the presence of unprocessed substrate, somewhat impeding cell growth. In general, these data suggest that the glucose present in control and experimental samples is likely to be sufficient to promote optimal growth of P. stipitis and that the presence of an additional substrate in the sample does not increase this growth rate. These results also suggest that none of the samples are toxic to P. stipitis.

As shown in Graph 2B, despite the numbers of similar cells reported in Graph 2B, greatly increased ethanol production was observed in all samples containing an experimental substrate. Ethanol concentrations increased over time for each of the three time points tested. The highest concentration of ethanol was observed for A-132-10 at the 48 hour time point (for example, approximately 26.0 g / L). In comparison, the substrate concentrations with the highest levels of ethanol production with the cell number data presented in Graph 2B, it can be seen that P. stipitis does not

114/117 appears to be sensitive to increasing concentrations of ethanol. In addition, ethanol production does not seem to be related to the number of cells, but rather it seems to be related to the type of substrate present in the sample.

Together, the results presented in Graphs 2A and 2B suggest that the experimental substrates do not promote increased growth of P. stipitis, however, they greatly increase the amount of ethanol produced by this type of cell. These data are also presented as a normalized percentage against the control, as shown in Graph 2C.

Saccharomyces cerevisiae

As shown in Graph 3A, G-132-100, A-132, A-132-10, A-132-100 and WS-132 promoted slightly higher cell numbers compared to the control. No significant reduction in the number of cells was observed for any sample. These results suggest that none of the samples is toxic to S. cerevisiae.

As shown in Graph 3B, increased ethanol production was observed in cells treated with each cell type compared to the control. Comparison of those samples containing the largest amount of ethanol with the cell number data shown in Graph 3A suggests that concentrations of ethanol above 5 g / L may have had an adverse effect on cell numbers. However, this observation is not the case for all samples.

These data are also presented as a normalized percentage against the control, as shown in Graph 3C.

In conclusion, none of the samples appeared to be toxic to Z. mobilis, P. stipitis or S. cerevisiae. In addition, P. stipitis appeared to be the most efficient of the three cell types for producing ethanol from the experimental substrates tested.

OTHER MODALITIES

A number of embodiments of the invention have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of the invention.

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For example, the fibers can be in any desired shape and can have a variety of different morphologies. In general, it is desirable that the cellulosic material has a high surface area. In some cases, the fibers may be a part of a HEPA filter or the like. The sheet material can have a surface area, for example, from about 1 to 500 m ² / g. The fibrous material can be deposited, for example, melt blown, folded, in the form of a canvas or mesh or supplied in other geometries. The fibers can be extruded or co-extruded.

The fibers can have any desired particle size, from the nanoscale, for example, less than about 1000 nm, for example, less than 500 nm, 250 nm, 100 nm, 50 nm, 25 nm or even less than 1 nm, up to large particle sizes, for example, more than 100 microns, 200 microns, 500 microns or even 1000 microns or clusters of particles.

Although biomass substrates have been discussed here, such substrates can be used in combination with other substrates, for example, the inorganic and synthetic substrates disclosed in US Provisional Application No. 61 / 252,300, filed on October 16, 2009, the full disclosure of which is incorporated herein by reference.

The fibers or a fibrous material containing the fibers can be pretreated with a microorganism and / or enzyme and / or the fibers or fibrous material can be contacted with a microorganism and / or enzyme during a bioprocess, such as saccharification or fermentation.

As discussed above, enzymes can be immobilized on fibers, rather than or in addition to microorganisms.

Biomass-destroying enzymes and organisms that break down biomass, such as cellulose and / or lignin portions of biomass, contain or manufacture various cellulolytic enzymes (cellulases), ligninases or various metabolites that destroy small molecule biomass. These enzymes can be a complex of enzymes that act synergistically to degrade crystalline cellulose or lignin portions of biomass. Examples of cellulolytic enzymes include: endoglucanases, cellobiohydrola116 / 117 ses and cellobiases (β-glucosidases). During saccharification, a cellulosic substrate is initially hydrolyzed by endoglucanases at random locations, producing oligomeric intermediates. These intermediates are then substrates for exo-dividing glucanases, such as cellobiohydrolase, which produce cellobiose from the ends of the cellulose polymer. Cellobiosis is a water-soluble 1,4-bonded glucose dimer. Finally, celobiase divides cellobiosis to provide glucose.

A cellulase is capable of degrading biomass and can be of fungal or bacterial origin. Suitable enzymes include cellulases of the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, Chrysosporium and Trichoderma and include species of Humicola, Coprinus, Thielavia, Fusarium, Myceliophthora, Acremonium, Cephalosporium, Scytalidium, Penicillium or Asperg, or EP 458162), especially those produced by a selected strain of Humicola insolens (reclassified as Scytalidium thermophilum, see, for example, US Patent No. 4,435,307), Coprinus cinereus, Fusarium oxysporum, Myceliophthora thermophila, Mcripilus giganteus, Thielavia terrestris , Acremonium sp., Acremonium persicinum, Acremonium acremonium, Acremonium brachypenium, Acremonium dichromosporum, Acremonium obclavatum, Acremonium pinkertoniae, Acremonium roseogriseum, Acremonium incoloratum and Acremonium furaturrr, preferably of the species Humicola insolensio DSM672, 6Mystery, 265, Fusarium , Cephalosporium sp . RYM-202, Acremonium sp. CBS 478.94, Acremonium sp. CBS 265.95, Acremonium persicinum CBS 169.65, Acremonium acremonium AHU 9519, Cephalosporium sp. CBS 535.71, Acremonium brachypenium CBS 866.73, Acremonium dichromosporum CBS 683.73, Acremonium obclavatum CBS 311.74, Acremonium pinkertoniae CBS 157.70, Acremonium roseogriseum CBS 134.56, Acremonium incoloratum CBS 146.62 and Acremonium furatum. Cellulolytic enzymes can also be obtained from Chrysosporium, preferably a strain of Chrysosporium lucknowense. Additionally, Trichoderma (particularly Trichoderma viride, Trichoderma reesei and Trichoderma koningii),

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Alkalophilic bacillus (see, for example, U.S. Patent No. 3,844,890 and EP 458162) and Streptomyces (see, for example, EP 458162) can be used.

Suitable cellobiases include an Aspergillus ni5 ger cellobiasis sold under the trademark NOVOZYME 188 ™.

Enzyme complexes can be used, such as those available from Genencor under the trademark ACCELLERASE®, for example, Accellerase® 1500 enzyme complex. The Accellerase® 1500 enzyme complex contains multiple enzyme activities, mainly exoglucanase, endoglucanase (2200-2800 CMC U / g), hemi-cellulase and beta-glucosidase (525-775 pNPG U / g) and has a pH of 4.6 to 5.0. The endoglucanase activity of the enzyme complex is expressed in units of carboxymethyl cellulose (CMC U), whereas beta-glucosidase activity is reported in units of pNP-glucoside (pNPG U). In one embodiment, a mixture of the Accellerase® 1500 enzyme complex and NOVOZYME ™ 188 celobiase is used.

Consequently, other modalities are within the scope of the following claims.

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Claims (11)

1. Method, characterized by the fact that it comprises: contacting a low molecular weight sugar, in a medium, with a layer of oxidized biomass fibers and a fermenting microorganism immobilized on the fibers; and fermenting low molecular weight sugar using an ethanol fermenting microorganism, in which low molecular weight sugar is selected from glucose and xylose; wherein the biomass is a vegetable biomass, more specifically a biomass of a cellulosic or lignocellulosic material; where the microorganism is S. cerevisiae, P. stipitis or Zymomonas mobilis. 2. Method according to claim 1, characterized by the fact that the biomass fibers were oxidized in an oxidizing medium by a radiation method. Method according to claim 2, characterized in that the radiation delivers a radiation dose between 1 Mrad to 100 Mrad of radiation. Method according to claim 1, characterized in that the fibers have been oxidized in an oxidizing environment by irradiation with ionizing radiation, in which preferably the irradiation is carried out using a particle beam. 5. Method according to claim 1, characterized by the fact that the biomass fibers are derived from biomass selected from the group consisting of paper, paper products, waste paper, wood, particulate board, sawdust, agricultural waste, water served, silage, grasses, rice husks, bagasse, jute, hemp, flax, bamboo, sisal, abaca, straw, corn cobs, corn straw, fast-growing grass, alfalfa, hay, coconut hair, cotton, seaweed marine, algae and mixtures thereof; Petition 870180072916, of 8/20/2018, p. 10/20 2/2 and / or the biomass fibers are derived from a biomass raw material that has internal fibers and that has been cut to a point where its internal fibers are exposed. 6. Method according to claim 1, characterized by the fact that the biomass fibers have a porosity greater than 70%. 7. Method according to claim 1, characterized by the fact that it comprises recovery of the biomass fibers after conversion and re-use of the fibers in a second subsequent conversion process. 8. Method according to claim 1, characterized by the fact that the layer is multilayer. Method according to claim 1, characterized in that the biomass fibers are supplied in the form of a fibrous material that is superimposed, folded or in the form of a screen or mesh. 10. Method according to claim 1, characterized by the fact that the biomass fibers are extruded or co-extruded. 11. Method according to claim 1, characterized by the fact that the fibers have a nanoscale average particle size. Petition 870180072916, of 8/20/2018, p. 11/20 1/22 ο ο _. LO “* Ο 2/22