Improved cellulose for use in cellulosic ethanol production applications

Abstract

1. A method for hydrolyzing cellulose to cellobiose, comprising the steps of: providing a reaction vessel; providing in the reaction vessel an inoculum of bacteria or fungi capable of expressing one or more endo-β-glucanases or exo-β-glucanases; exposing the bacteria or fungi to a cellulose source having a kappa number of less than 10 and a hemicellulose content of less than 15% in an aqueous medium having a pH of 5 to 9, at a temperature in the range of 20°C to 40°C, for a period in the range of 1 to 30 days; and exposing the cellobiose to bacteria, fungi, yeast, or a combination thereof that converts the cellobiose to glucose or ethanol.

Description

[Technical Field]

[0001] The present invention relates to the use of cellulose with a low kappa number and low hemicellulose content to provide significantly improved conversion of cellulose to ethanol. [Background technology]

[0002] Biofuels are becoming increasingly necessary to reduce human consumption of fossil fuels in aspects of daily life, namely transportation and home heating, two of the largest industries of note. As an alternative energy source to oil and coal, the primary feedstock for biofuel production is starch, which can produce its sugars much more easily than cellulose. This is due to structural differences: in starch, the glucose molecules are linked by α-1,4 linkages, while in cellulose, the glucose molecules are linked by β-1,4 linkages. These β-1,4 linkages allow cellulose to crystallize, resulting in a more rigid and less fragile structure.

[0003] The limitations imposed by focusing biofuel production solely on extracting sugars from starch prevent the utilization of the majority of biomass in the form of lignocellulosic biomass (containing lignin, cellulose, and hemicellulose), which is present in nearly all plants on Earth. Delignification allows for the recovery of cellulose from these lignocellulosic plants. Once the cellulose is separated from the other two biomass components, lignin and hemicellulose, further degradation of the cellulose produces cellobiose and / or glucose, which can be further processed into bioethanol.

[0004] The biofuel industry is seen as a sustainable alternative to gasoline and has the goal of reducing many countries' dependence on foreign oil, but its reliance on corn and sugarcane, both rich in starch, as its primary source of fuel remains an obstacle. It is estimated that approximately one-third of all corn production in the United States is directed toward the production of ethanol fuel. This is a situation that could have devastating consequences if gasoline prices become so low that corn-based biofuels become unsustainable from a price perspective.

[0005] Many other countries around the world that produce ethanol on a large scale, such as China and Brazil, have struggled somewhat in producing ethanol from biomass because many companies have incurred significant debt from implementing these methods, forcing large plants to stop or reduce production.

[0006] In Asia, palm oil prices have recently risen to their highest levels in many years, hindering the ability of Indonesia and Malaysia to produce local biofuels. Oil palm trunks, a valuable and abundant resource in these countries, contain large amounts of starch, which is more easily soluble in water than cellulose. Therefore, the starch can be heated and hydrolyzed to glucose by amylolytic enzymes without pretreatment. However, conventional oil palm trunk processing requires high capital and operating costs, making market entry prohibitive. Furthermore, this process has a high potential for microbial contamination during starch processing.

[0007] In Europe, the biofuel industry (both biodiesel and bioethanol production) relies heavily on food-based feedstocks such as virgin vegetable oils (i.e., rapeseed, palm oil, and soybeans) for biodiesel, and corn, wheat, and sugar beet for bioethanol. At the same time, concerns have been raised that the production of fuel from crops could displace other crops and increase food prices. These concerns have led to policy changes encouraging a transition away from food-based biofuels.

[0008] Conversion of starch to cellulose for glucose production is desirable because cellulose provides a nearly unlimited amount of feedstock from waste biomass, reducing competition with food-source materials for glucose production. However, the cost of doing so is currently prohibitive. Cellulosic ethanol utilizes the non-food parts of plants used to produce ethanol, potentially replacing the more prevalent current method of using corn or sugarcane to produce bioethanol. The diversity and abundance of these types of cellulose-rich plants makes it possible to maintain nearly intact food sources and utilize waste products from these sources (e.g., corn stalks) to produce ethanol. If commercially viable methods can be developed, other cellulose sources, such as grasses, algae, and even trees, represent cellulose-rich biomass that could be used for ethanol production.

[0009] The reason starch is preferred over cellulose-rich sources for ethanol production is that extracting glucose from cellulose is substantially more difficult and resource intensive. To better understand the differences that create this challenge, it is useful to point out the similarities and differences between starch and cellulose.

[0010] Cellulose and starch are polymers with the same glucose repeating unit. However, the difference between starch and cellulose can be found in the way the repeating glucose monomers are linked to each other. In starch, the glucose monomers are oriented in the same direction. In cellulose, each successive glucose monomer is rotated 180 degrees relative to the previous glucose monomer. This ensures that the bonds between each glucose monomer are different in starch and cellulose. In starch, the bonds (or linkages as they are known) are called α-1,4 linkages, while in cellulose, these bonds are called β-1,4 linkages.

[0011] The differences between these bonds affect the properties of starch and cellulose. Starch is soluble in warm water, while cellulose is not. Starch is digestible by humans, while cellulose is not. Starch is weaker than cellulose, in part due to the fact that its structure is less crystalline than cellulose. Starch is essentially a way for plants to store energy, and therefore, extracting sugars from starch is much easier than extracting sugars from cellulose. This is because the central function of cellulose is to provide structural support.

[0012] Cellulose, the main component of lignocellulosic biomass, is a biopolymer composed of many glucose units linked via β-1,4-glycosidic bonds (see Figure 1). D-glucose is a building block of many polysaccharides, including cellulose. Glucose has two isomers: α-glucose (present in starch as a branched polymer) and β-glucose (present in cellulose as repeating β-glucose subunits, with one β-glucose monomer rotated 180 degrees relative to its neighbor). A single cellulose molecule can contain hundreds to thousands of glucose units. Because cellulose molecules are linear, due in part to intermolecular hydrogen bonding, adjacent cellulose molecules can be very tightly packed, which may provide the structural strength necessary to support plants.

[0013] Cellulose hydrolysis Cellulose hydrolysis is the rate-limiting step in the conversion of cellulose to bioethanol. Current methods for using cellulose as a starting material for bioethanol production require the conversion of cellulose to oligomers and then glucose before ultimately producing ethanol. Fermentation of glucose using yeast leads to the production of ethanol. While the final step in biofuel production has long been established, the rate-limiting step is the most important and has hindered the wider adoption of bioethanol. The difficulty in overcoming this conversion of cellulose to glucose lies in the fact that cellulose's crystalline structure, in which multiple cellulose polymers are tightly packed, makes the conversion of cellulose to glucose extremely difficult. This tight packing gives cellulose its inherent stability under various chemical conditions. Cellulose polymers are generally insoluble in water and many organic solvents. Cellulose is also generally insoluble when exposed to weak acids or bases.

[0014] Generally, there are two main methods for hydrolyzing cellulose: chemical and enzymatic. Chemical methods rely on the use of concentrated, strong acids to hydrolyze cellulose under high temperature and pressure conditions. Many different types of acids, such as HCl and H2SO4, have been used in the past to achieve this. The use of one of these acids usually results in at least one of the following drawbacks: corrosion of the reaction vessel, difficulty in disposing of the discharged reactants, and the cost of a highly energy-intensive process. The biofuel industry is generally reluctant to use chemically hydrolyzed cellulose because of the presence of toxic by-products in the resulting glucose. When introduced into the fermentation process, these by-products adversely affect the delicate balance of fermenting microorganisms.

[0015] Cost of Enzymatic Hydrolysis The cost of extracting biofuels from cellulose is known to be higher than the cost of extracting them from starch. Depending on the location and availability of biomass, the cost of converting cellulose is estimated to be about 50% higher on average than converting starch to glucose. This means that there are currently clear barriers for producers to produce glucose from biomass using cellulose rather than corn or other starch sources.

[0016] It is generally understood that approximately half of the total cost of producing biofuels from cellulose is due to the price of enzymes (cellulases). The production of enzymes for the enzymatic hydrolysis of cellulose is a time-consuming process, and large amounts of enzymes are required to make the process commercially viable. One possible approach is to improve the rate of the hydrolysis reaction, which would reduce the overall cost of the process.

[0017] Enzymatic methods for cellulose hydrolysis use enzymes to carry out the hydrolysis reaction. Enzymes, such as cellulases (including endo-1,4-β-glucanases, exo-1,4-β-glucanases, and β-glucosidases), require careful control to maximize the reaction rates expected from enzymatic methods. Temperature, pH, salt content, and substrate and product concentrations are all factors that can affect enzyme activity. Even slight deviations from the enzyme's optimum can lead to loss of function. The conversion of cellulose to glucose is carried out by several different enzymes, namely endo-1,4-β-glucanases, exo-1,4-β-glucanases, and β-glucosidases, all of which have specific environmental conditions that must be met. These controls can sometimes make the process prohibitively expensive and / or limit its implementation.

[0018] PCT patent application WO9640970(A1) discloses a method for producing sugar from a material containing cellulose and hemicellulose, the method comprising the steps of: mixing the material with a solution of about 25 to 90% by weight of acid, thereby at least partially decrystallizing the material and forming a gel containing a solid material and a liquid portion; diluting the gel to an acid concentration of about 20 to 30% by weight and heating the gel to a temperature of about 80 to 100°C, thereby partially hydrolyzing the cellulose and hemicellulose contained in the material; separating the liquid portion from the solid material, thereby obtaining a first liquid containing sugar and acid; and diluting the gel so that the acid concentration of the gel is about 20 to 30% by weight. The method includes the steps of mixing the separated solid material with a solution of about 25-90% acid until it becomes acidic, heating the mixture to a temperature of about 80°C-100°C, thereby further hydrolyzing the cellulose and hemicellulose remaining in the separated solid material and forming a second solid material and a second liquid portion; separating the second liquid portion from the second solid material, thereby obtaining a second liquid containing sugars and acid; combining the first liquid and the second liquid; and separating the sugars from the acid in the combined first and second liquids to produce a third liquid containing a total of at least about 15% by weight of sugars and no more than 3% by weight of acid.

[0019] A paper by Ichikawa S et al. titled "Glucose production from cellulose through biological simultaneous enzyme production and saccharification using recombinant bacteria expressing the β-glucosidase gene" (J Biosci Bioeng. March 2019, Vol. 127, No. 3, pp. 340-344) describes a saccharification technology for cellulosic biomass. Glucose was produced by hydrolyzing 100 g / L of Avicel cellulose for 10 days using biological simultaneous enzyme production and saccharification (BSES). The product yield was similar to that obtained using BSES supplemented with purified β-glucosidase.

[0020] A paper titled "A novel facile two-step method for producing glucose from cellulose" (Bioresource Technology, Vol. 137, June 2013, pp. 106-110) discloses a two-step acid-catalyzed hydrolysis method that hydrolyzes cellulose to glucose in high yield and selectivity under mild conditions. The method involves a multi-step hydrolysis, the first step of which involves depolymerizing a phosphoric acid solution of microcrystalline cellulose into cellulose oligomers at 50°C. The second step involves precipitation of the oligomers with ethanol and subsequent hydrolysis with dilute sulfuric acid.

[0021] A review paper by Akhtar et al., "BIODIVERSITY OF CELLULASE-PRODUCING BACTERIA AND THEIR APPLICATIONS" (2014), reports that many microorganisms are capable of producing and secreting cellulolytic, hemicellulolytic, and ligninolytic enzymes. These microorganisms are found among a highly diverse range of taxa inhabiting a variety of habitats, from extremely thermophilic conditions to polar regions and from aerobic to anaerobic systems. By 1976, an impressive collection of over 14,000 fungi active against cellulose and other insoluble fibers had been reported. Most cellulolytic bacteria belong to the phyla Actinobacteria, Bacteroidetes, Fibrobacteria, Firmicutes, and Proteobacteria. Actinobacteria and Firmicutes comprise 80% of isolated cellulolytic bacteria.

[0022] In the paper "Cleaning carbohydrate impurities from lignin using Pseudomonas fluorescens," Ghosh et al. (2019) demonstrated that lignin biomass could be selectively biodegraded to cellulose and hemicellulose by exposing it to Pseudomonas fluorescens (P. fluorescens). P. fluorescens is a nonpathogenic bacterium capable of producing cellulolytic enzymes and has adapted to cleave polymeric carbohydrates into free sugars and utilize them as a carbon source. The paper concludes that P. fluorescens is yet another organism that can be used to convert lignocellulosic biomass into free sugars.

[0023] In their paper "Direct ethanol production from cellulose by a consortium of Trichoderma reesei and Candida molischiana" (2019), Bu et al. describe the use of Trichoderma reesei and Candida molischiana to produce ethanol from cellulose. They state that cellulose was hydrolyzed by an enzymatic saccharification method using Trichoderma reesei cellulase. The resulting sugars were then utilized by Candida molischiana to produce ethanol.

[0024] Dussan et al., in their paper entitled "Dilute-acid Hydrolysis of Cellulose to Glucose from Sugarcane Bagasse" (CHEMICAL ENGINEERING TRANSACTIONS, Vol. 38, 2014), describe a method for producing ethanol by hydrolysis of cellulose. Sugarcane bagasse was used as a substrate for ethanol production, and the optimal conditions for acid hydrolysis of the cellulose fraction were evaluated. The glucose thus produced was fermented to ethanol using the yeast Scheffersomyces stipitis.

[0025] As can be seen from the above, the hydrolysis of cellulose is limited not only by the structure of the cellulose itself, but also by the approach taken to break it down into biofuels. A stable, low-cost process for the production of biofuels from cellulose has not yet been realized.

[0026] U.S. Patent No. 9,663,807 discloses the preparation of ethanol using lignocellulosic biomass, such as corn stover. This involves pretreatment to remove C5 compounds (derived from hemicellulose), leaving C6 solids, which are then subjected to a simultaneous saccharification and fermentation (SSF) process. It should be noted that simultaneous saccharification and fermentation could be performed at temperatures suitable for yeast ethanol production (e.g., approximately 37°C), but this was not optimal for cellulase enzymes. As a result, yields from these enzymes were lower because their activity was hindered by the presence of lignin to which the cellulase enzymes could bind. It was discovered that the addition of lignin-binding agents, such as clarified thin stillage and / or anaerobic membrane bioreactor (AnMBR) effluent, could increase glucose yield during enzymatic hydrolysis.

[0027] In light of the above, there is a critical need to develop methods for producing biofuels from waste biomass as an abundant, untapped renewable biofuel source that does not compete with food sources such as corn. In this regard, lignocellulosic biomass, from which cellulose is extracted, is much more attractive, as it leaves the food source available for its primary intended purpose while still producing a significant cellulose yield. This is further supported by the fact that although significant efforts have been made to convert waste biomass into biofuels using various methods, nearly all of them have failed to achieve this goal for subsequent conversion to glucose and ultimately ethanol.

[0028] The inventors have surprisingly and unexpectedly found that the properties of cellulose resulting from a particular type of delignification procedure significantly affect its downstream hydrolysis. Cellulose with a low lignin content provides an advantageous substrate for producing glucose. More preferably, cellulose with a low lignin and low hemicellulose content provides a substrate that can be more easily converted to glucose. The subsequent conversion of glucose to ethanol results in significant savings in the production of the latter and increased use of cellulose to produce ethanol. Summary of the Invention

[0029] According to another aspect of the present invention, there is provided a method for hydrolyzing cellulose to cellobiose, said method comprising the steps of: providing a reaction vessel; providing a cellulose source in the reaction vessel, wherein the cellulose source has a kappa number of less than 10, preferably the kappa number is 5 or less, more preferably the kappa number is 2 or less, and has a hemicellulose content of less than 15%, preferably less than 10%, even more preferably less than 5% w / w, based on the total weight of the cellulose source; providing an inoculum of an organism capable of expressing one or more β-glucanases in said reaction vessel; exposing said organism capable of expressing one or more β-glucanases to said cellulose source in an aqueous medium; and Optionally, recovering the supernatant containing cellobiose.

[0030] According to a preferred embodiment of the present invention, the method further comprises exposing the cell supernatant to an organism to convert cellobiose to glucose, wherein the organism is selected from the group consisting of bacteria, fungi, yeast, and combinations thereof, such as Aspergillus brasiliensis, Trichoderma reesei, and Pseudomonas protegens. Preferably, the organism capable of expressing one or more β-glucanases is selected from the group consisting of bacteria and fungi capable of producing endo-β-glucanase enzymes or exo-β-glucanase enzymes. Also preferably, the organism capable of expressing one or more β-glucanases is a bacterium of the phylum Bacteroidetes. Also preferably, the organism capable of expressing one or more β-glucanases is Cytophaga hutchinsonii.

[0031] According to a preferred embodiment of the present invention, the organism capable of expressing one or more β-glucanases is a bacterium of the phylum Proteobacteria, preferably Pseudomonas protegens.

[0032] According to a preferred embodiment of the invention, the obtained glucose is exposed to one of the bacteria Zymomonas mobilis or the yeast Saccharomyces cerevisiae in a step of converting the glucose to ethanol.

[0033] According to a preferred embodiment of the present invention, the organism used to convert cellobiose into glucose is a bacterium or a fungus capable of producing the β-glucosidase enzyme.

[0034] According to a preferred embodiment of the present invention, the organism used to convert cellobiose into glucose is a fungus of the Ascomycota phylum.

[0035] According to a preferred embodiment of the present invention, the organism used to convert cellobiose to glucose is a bacterium of the phylum Proteobacteria.

[0036] Preferably, the cellulose source is exposed to the organism at a temperature between 20° C. and 40° C. Preferably, the organism is incubated with the cellulose source for a period ranging from 1 to 30 days. Preferably, the aqueous medium has a pH of about 5.0 to 8.0.

[0037] According to another aspect of the present invention, there is provided a method for hydrolyzing cellulose to glucose, said method comprising the steps of: providing a reaction vessel; providing a cellulose source in the reaction vessel, wherein the cellulose source has a kappa number of less than 10, preferably the kappa number is 5 or less, more preferably the kappa number is 2 or less, and has a hemicellulose content of less than 15%, preferably less than 10% w / w, even more preferably less than 5% w / w, based on the total weight of the cellulose source; providing an inoculum of an organism capable of expressing one or more β-glucanases and β-glucosidases in the reaction vessel; exposing said organism capable of expressing one or more β-glucanases and β-glucosidases to said cellulose source in an aqueous medium; and Optionally, recovering the supernatant containing glucose.

[0038] Preferably, the method uses a bacterium or fungus capable of producing the organism capable of expressing endo- or exo-β-glucanase and β-glucosidase. Preferably, the bacterium or fungus capable of producing the organism capable of expressing endo- or exo-β-glucanase and β-glucosidase is selected from the group consisting of Ascomycota, Proteobacteria, and combinations thereof. Preferably, the organism capable of expressing one or more β-glucanases and β-glucosidases is Trichoderma reesei. Also preferably, the organism capable of expressing one or more β-glucanases and β-glucosidases is Pseudomonas protegens.

[0039] According to a preferred embodiment of the present invention, the cellulose source is exposed to the organism at a temperature of 30°C to 40°C.

[0040] According to a preferred embodiment of the present invention, the organism is incubated with the cellulose source for a period ranging from 1 to 30 days. Preferably, the aqueous medium has a pH of about 5.0 to 9.0.

[0041] According to one aspect of the invention, there is provided a method for converting cellulose to cellobiose (and optionally glucose or ethanol), said method comprising: providing a reaction vessel; providing a cellulose source in the reaction vessel, wherein the cellulose source has a hemicellulose content of less than 15%, preferably less than 10%, more preferably less than 5%, and a kappa number of less than 10, more preferably less than 5, and even more preferably less than 2; providing in said reaction vessel an organism capable of expressing one or more β-glucanases; exposing the organism to the cellulose source in an aqueous medium having a pH of 5 to 9 at a temperature in the range of 30°C to 35°C for a period in the range of 1 to 30 days; optionally recovering the supernatant containing cellobiose, optionally exposing the supernatant containing cellobiose to a bacterium or fungus producing β-glucosidase for the conversion of cellobiose to glucose, optionally recovering the supernatant containing glucose, and optionally exposing said supernatant containing glucose to an ethanologenic organism for fermenting glucose to ethanol; A method is provided, comprising:

[0042] Preferably, the temperature within the reaction vessel during said exposure time does not exceed 70°C, more preferably 60°C, even more preferably 50°C.

[0043] According to another aspect of the invention, there is provided a method for converting cellulose to cellobiose (and optionally glucose or ethanol), said method comprising: providing a reaction vessel; providing a cellulose source in the reaction vessel, wherein the cellulose source has a hemicellulose content of less than 15%, preferably less than 10%, more preferably less than 5%, and a kappa number of less than 10, more preferably less than 5, and even more preferably less than 2; providing a Cytophaga hutchinsonii inoculum in the reaction vessel; exposing the Cytophaga hutchinsonii to the cellulose source in an aqueous medium having a pH of 5 to 9 at a temperature ranging from 20°C to 40°C for a period ranging from 0 to 30 days; optionally recovering the supernatant containing cellobiose, optionally exposing the supernatant containing cellobiose to a bacterium or fungus producing β-glucosidase for the conversion of cellobiose to glucose, optionally recovering the supernatant containing glucose, and optionally exposing said supernatant containing glucose to an ethanologenic organism for fermenting glucose to ethanol; A method is provided, comprising:

[0044] According to another aspect of the invention, there is provided a method for converting cellulose to cellobiose (and optionally glucose and / or ethanol), said method comprising: providing a reaction vessel; providing a cellulose source in the reaction vessel, wherein the cellulose source has a hemicellulose content of less than 15%, preferably less than 10%, more preferably less than 5%, and a kappa number of less than 10, more preferably less than 5, and even more preferably less than 2; providing a Pseudomonas protegens inoculum in the reaction vessel; exposing the Pseudomonas protegens to the cellulose source in an aqueous medium having a pH of 5 to 9 for a period ranging from 0 to 30 days; optionally recovering the supernatant containing cellobiose, optionally exposing the supernatant containing cellobiose to a bacterium or fungus producing β-glucosidase for the conversion of cellobiose to glucose, optionally recovering the supernatant containing glucose, and optionally exposing said supernatant containing glucose to an ethanologenic organism for fermenting glucose to ethanol; A method is provided, comprising:

[0045] Preferably, the temperature within the reaction vessel during said exposure time does not exceed 70°C, more preferably 60°C, even more preferably 50°C.

[0046] According to another aspect of the present invention, there is provided a method for converting cellulose to glucose (and optionally ethanol), said method comprising: providing a reaction vessel; providing a cellulose source in the reaction vessel, wherein the cellulose source has a hemicellulose content of less than 15%, preferably less than 10%, more preferably less than 5%, and a kappa number of less than 10, more preferably less than 5, and even more preferably less than 2; providing a fungal or bacterial inoculum, preferably Trichoderma reesei, in said reaction vessel; exposing the Trichoderma reesei to the cellulose source in an aqueous medium having a pH of 5 to 9 at 200 rpm for a period ranging from 0 to 30 days; optionally recovering the supernatant containing glucose, and optionally exposing the supernatant containing glucose to an ethanologenic organism for fermentation of glucose to ethanol; A method is provided, comprising:

[0047] Preferably, the temperature within the reaction vessel during said exposure time does not exceed 70°C, more preferably 60°C, even more preferably 50°C.

[0048] According to a preferred embodiment of the present invention, a method for delignification of biomass materials to produce low lignin and low hemicellulose cellulose (also called modified Caro's acid delignified (MCA delignified) cellulose), also called low kappa number and low hemicellulose content cellulose, used in experiments to convert cellulose to cellobiose (and ultimately glucose and ethanol), comprises: the cellulose source is lignocellulosic biomass that has been delignified by exposure to a modified Caro's acid composition selected from the group consisting of Composition A, Composition B, and Composition C; The composition A is sulfuric acid in an amount ranging from 20 to 70% by weight of the total weight of the composition, a modifier component comprising an amine moiety and a sulfonic acid moiety, selected from the group consisting of taurine, taurine derivatives, and taurine-related compounds; and Peroxide Including, The composition B is alkyl sulfonic acids, and Peroxide wherein the acid is present in an amount ranging from 40 to 80% by weight of the total weight of the composition, and the peroxide is present in an amount ranging from 10 to 40% by weight of the total weight of the composition; The composition C is ·Sulfuric acid, A two-component modifier component, a compound containing an amine moiety, and Compounds containing sulfonic acid moieties a two-component modifier component comprising: Peroxide conducted for a period of time sufficient to remove substantially all of the lignin present on the biomass material; For brevity, the above process will be referred to hereinafter as the modified Caro's acid delignification process, and the resulting cellulose will be referred to as modified Caro's acid delignified cellulose or "MCA cellulose" to indicate the delignification process used to obtain said cellulose.

[0049] Preferably, the sulfuric acid, the compound comprising an amine moiety and a sulfonic acid moiety, and the peroxide are present in a molar ratio of 15:1:1 or less, and preferably, the sulfuric acid, and the compound comprising an amine moiety and a sulfonic acid moiety are present in a molar ratio of 3:1 or greater.

[0050] According to a preferred embodiment of the procedure for obtaining low-lignin cellulose, said delignification lasts from 2 to 20 hours.

[0051] According to a preferred embodiment of the procedure for obtaining low-lignin cellulose, said delignification is carried out at a temperature below 50° C. Preferably, the delignification is carried out at a temperature below 40° C.

[0052] According to a preferred embodiment of the present invention, the method described herein for producing cellobiose (or glucose) from cellulose uses cellulose having a low kappa number and a low hemicellulose content, said cellulose having the following characteristics: a particle size in the range of 0-1000 microns; a hemicellulose content of less than 15%, preferably less than 10%, more preferably less than 5% w / w, based on the total weight of said cellulose; and a kappa number of less than 10, more preferably less than 5, even more preferably less than 2.

[0053] According to another aspect of the present invention, there is provided use of a cellulose source having a kappa number of less than 10, preferably said kappa number equal to or less than 5, more preferably said kappa number equal to or less than 2, and a hemicellulose content of less than 15%, preferably less than 10% w / w, even more preferably less than 5% w / w, relative to the total weight of the cellulose source, in a method for hydrolyzing cellulose to cellobiose, said method comprising the steps of: providing a reaction vessel; providing the cellulose source in the reaction vessel; providing in said reaction vessel an organism capable of expressing one or more β-glucanases; exposing said organism capable of expressing one or more β-glucanases to said cellulose source in an aqueous medium; and Optionally, recovering the supernatant containing cellobiose.

[0054] According to another aspect of the present invention, there is provided the use of a cellulose source having a kappa number of less than 10, preferably said kappa number equal to or less than 5, more preferably said kappa number equal to or less than 2, and a hemicellulose content of less than 15%, preferably less than 10% w / w, even more preferably less than 5% w / w, relative to the total weight of the cellulose source, in a method for hydrolyzing cellulose to cellobiose (and optionally glucose or ethanol), said method comprising the steps of: providing a reaction vessel; providing the cellulose source in the reaction vessel; providing an inoculum of an organism capable of expressing one or more β-glucanases and β-glucosidases in the reaction vessel; exposing said organism capable of expressing one or more β-glucanases and β-glucosidases to said cellulose source in an aqueous medium; optionally recovering the supernatant containing cellobiose, optionally exposing the supernatant containing cellobiose to a bacterium or fungus producing β-glucosidase for the conversion of cellobiose to glucose, optionally recovering the supernatant containing glucose, and Optionally, exposing said supernatant containing glucose to ethanologenic bacteria or fungi for fermenting glucose to ethanol.

[0055] According to another aspect of the present invention, there is provided a method characterized in that the cellulose has not been previously exposed to a bleaching chemical selected from the group consisting of sodium hydrosulfite (NaSO), diethylenetriaminepentaacetic acid pentasodium salt, amine borane (CH)CNH-BH, borane ammonia complex BH-NH, sodium percarbonate, formamidine sulfinic acid, sodium perborate, and chlorine dioxide. Those skilled in the art will appreciate that, in the context of this application, reference to bleaching of pulp should be understood to refer to a separate and distinct step in pulp processing. Accordingly, the pulp used in accordance with the preferred method of the present invention is intended to be pulp that has not undergone a separate bleaching step after delignification. It is understood that such a treatment step would not be economically feasible if the ultimate purpose of the cellulose is to be used to produce ethanol.

[0056] Features and advantages of the embodiments of the present application will become apparent from the following detailed description and accompanying drawings. [Brief explanation of the drawings]

[0057] [Figure 1] FIG. 1 is a diagram of the glucose monomers and their β-1,4-linkages present in a cellulose polymer, showing the binding sites for the β-glucanase and β-glucosidase enzymes. [Figure 2] FIG. 2 shows a graphical representation of β-glucanase enzyme activity of various microorganisms exposed to modified Caro's acid delignified cellulose. [Figure 3] FIG. 3 shows a graphical representation of β-glucosidase enzyme activity of various microorganisms exposed to modified Caro's acid delignified cellulose and cellobiose. DETAILED DESCRIPTION OF THE INVENTION

[0058] The following description and the embodiments described therein are provided as illustrations of example(s) of particular embodiments of the principles of the present invention. These examples are provided for purposes of explanation, not limitation, of those principles and the invention.

[0059] According to a preferred embodiment of the present invention, Cytophaga hutchinsonii, Pseudomonas protegens, and Trichoderma reesei are used to hydrolyze modified Caro's acid delignified (MCA delignified) cellulose with a kappa number ranging from 0 to 2.

[0060] According to a preferred embodiment of the present invention, a method for hydrolyzing cellulose to cellobiose comprises the following steps: providing a reaction vessel; providing in said vessel an organism capable of expressing one or more β-glucanases; exposing said organism in an aqueous medium to a source of cellulose having a kappa number of less than 10, more preferably less than 5, even more preferably less than 2.

[0061] According to a preferred embodiment of the invention, said organism capable of expressing one or more β-glucanases is a bacterium or a fungus.

[0062] Preferably, the bacterium is a member of the phylum Bacteroidetes or Proteobacteria, and more preferably, the bacterium is Cytophaga hutchinsonii or Pseudomonas protegens.

[0063] Preferably, the fungus is a member of the Ascomycota phylum, and more preferably, the fungus is Trichoderma reesei.

[0064] According to a preferred embodiment of the present invention, the method of exposing the cellulose source to the organism is carried out at a temperature below 40° C. Preferably, the method is carried out at a temperature between 25° C. and 37° C.

[0065] According to a preferred embodiment of the present invention, the method of exposing the cellulose source to the organism is carried out for a period ranging from 1 to 60 days, preferably from 3 to 30 days.

[0066] According to a preferred embodiment of the present invention, the aqueous medium has a pH of about 5.0 to 8.0. Preferably, the aqueous medium is maintained at a pH of 6.0 to 7.5.

[0067] Preferably, the method further comprises the step of exposing the cell supernatant to an ethanologenic organism that converts cellobiose to glucose or ethanol.

[0068] According to a preferred embodiment of the present invention, the ethanol-producing organism is a bacterium or a fungus, more preferably Saccharomyces cerevisiae.

[0069] Internally generated data indicate that modified Caro's acid delignified (MCA delignified) cellulose, with a lower kappa number, produced higher enzyme activity upon initial exposure to organisms producing endo- and exo-β-glucanases. This initial exposure hydrolyzes the β-1,4-glycosidic linkages of cellulose to produce oligosaccharides, thus producing the majority of the glucose precursor material (in this case, cellobiose). The second step of the method according to a preferred embodiment of the present invention is understood to be straightforward because it does not deviate from the general approach of cellobiose-to-glucose conversion. It has been established that the first step is more decisive for the extent of biomass-to-glucose conversion, since cellobiose is produced as the major primary product and subsequently used for glucose conversion. It is desirable to maximize the degradation of cellulose to cellobiose during this step; otherwise, the second step of the method would not have a significant impact.

[0070] Method for obtaining modified Caro's acid delignified cellulose According to a preferred embodiment of the present invention, a method for delignification of biomass material to produce modified Caro's acid delignified cellulose (also called MCA cellulose) for use in cellulose to cellobiose (and ultimately glucose) conversion experiments comprises: Providing a biomass material comprising cellulose fibers and lignin; exposing the biomass material requiring delignification to a modified Caro's acid composition selected from the group consisting of Composition A, Composition B, and Composition C; Including, The composition A is sulfuric acid in an amount ranging from 20 to 70% by weight of the total weight of the composition, a modifier component comprising an amine moiety and a sulfonic acid moiety, selected from the group consisting of taurine, taurine derivatives, and taurine-related compounds; and Peroxide Including, The composition B is alkyl sulfonic acids, and Peroxide wherein the acid is present in an amount ranging from 40 to 80% by weight of the total weight of the composition, and the peroxide is present in an amount ranging from 10 to 40% by weight of the total weight of the composition; The composition C is ·Sulfuric acid, A two-component modifier component, a compound containing an amine moiety, and Compounds containing sulfonic acid moieties a two-component modifier component comprising: Peroxide conducted for a period of time sufficient to remove substantially all of the lignin present on the biomass material; This process can be carried out for various periods depending on the particle size of the biomass fed to the process. The process can last from 2 to 20 hours depending on its characteristics. Furthermore, the temperature of the resulting mixture also influences the duration of the process. Because the reaction is highly exothermic, precautions are taken to prevent runaway decomposition of the cellulose, which would result in a worthless carbon black product. The process is preferably carried out at a temperature below 50°C, more preferably below 40°C. The delignification process is preferably carried out using cooling means adapted to control the heat generated by the delignification chemical reaction and maintain the temperature to avoid undesirable "runaway" reactions.

[0071] Preferably, the sulfuric acid, the compound comprising an amine moiety and a sulfonic acid moiety, and the peroxide are present in a molar ratio of 15:1:1 or less.

[0072] According to a preferred embodiment of the procedure for obtaining low-lignin cellulose, said sulfuric acid and said compound comprising an amine moiety and a sulfonic acid moiety are present in a molar ratio of 3:1 or greater.

[0073] Preferably, the modifier component comprising an amine moiety and a sulfonic acid moiety is selected from the group consisting of taurine, taurine derivatives, and taurine-related compounds.

[0074] According to a preferred embodiment of the method for obtaining low-lignin cellulose, the taurine derivative or taurine-related compound is selected from the group consisting of taurolidine, taurocholic acid, taurocercholic acid, tauromustine, 5-taurinomethyluridine and 5-taurinomethyl-2-thiouridine, homotaurine (tramiprosate), acamprosate, and taurate, as well as aminoalkylsulfonic acids, where alkyl is selected from the group consisting of C1-C5 linear alkyls and C1-C5 branched alkyls. Preferably, the linear alkyl aminosulfonic acids are selected from the group consisting of methyl, ethyl (taurine), propyl, and butyl. Preferably, the branched aminoalkyl sulfonic acids are selected from the group consisting of isopropyl, isobutyl, and isopentyl.

[0075] According to a preferred embodiment of the method for obtaining low-lignin cellulose, said compound comprising an amine moiety and a sulfonic acid moiety is taurine.

[0076] According to a preferred embodiment of the procedure for obtaining low-lignin cellulose, the sulfuric acid and the compound comprising an amine moiety and a sulfonic acid moiety are present in a molar ratio of 3:1 or greater.

[0077] According to a preferred embodiment of the approach to obtain low-lignin cellulose, said compound comprising an amine moiety is an alkanolamine selected from the group consisting of monoethanolamine, diethanolamine, triethanolamine, and combinations thereof.

[0078] According to a preferred embodiment of the approach to obtain low-lignin cellulose, said compound comprising a sulfonic acid moiety is selected from the group consisting of alkyl sulfonic acids, aryl sulfonic acids, and combinations thereof.

[0079] Preferably, the alkyl sulfonic acid is selected from the group consisting of alkyl sulfonic acids in which the alkyl group ranges from C1 to C6 and is linear or branched, and combinations thereof. More preferably, the alkyl sulfonic acid is selected from the group consisting of methanesulfonic acid, ethanesulfonic acid, propanesulfonic acid, 2-propanesulfonic acid, isobutylsulfonic acid, t-butylsulfonic acid, butanesulfonic acid, isopentylsulfonic acid, t-pentylsulfonic acid, pentanesulfonic acid, t-butylhexanesulfonic acid, and combinations thereof.

[0080] Preferably, the aryl sulfonic acid is selected from the group consisting of toluene sulfonic acid, benzene sulfonic acid, and combinations thereof.

[0081] According to a preferred embodiment of the method for obtaining low-lignin cellulose, said alkylsulfonic acid and said peroxide are present in a molar ratio of 1:1 or greater.

[0082] Preferably, the compound containing a sulfonic acid moiety is methanesulfonic acid.

[0083] According to a preferred embodiment of the method for obtaining low-lignin cellulose, composition C may further comprise a compound containing an amine moiety. Preferably, the compound containing an amine moiety has a molecular weight of less than 300 g / mol. Preferably, the compound containing an amine moiety is a primary amine. More preferably, the compound containing an amine moiety is an alkanolamine. Preferably, the compound containing an amine moiety is a tertiary amine. According to a preferred embodiment of the method for obtaining low-lignin cellulose, the alkanolamine is selected from the group consisting of monoethanolamine, diethanolamine, triethanolamine, and combinations thereof. Preferably, the alkanolamine is triethanolamine.

[0084] According to a preferred embodiment of the method for obtaining low-lignin cellulose, in composition C, the sulfuric acid, the compound comprising an amine moiety, and the compound comprising a sulfonic acid moiety are present in a molar ratio of 1:1:1 or greater.

[0085] Preferably, in composition C, the sulfuric acid, the compound comprising an amine moiety, and the compound comprising a sulfonic acid moiety are present in a molar ratio ranging from 28:1:1 to 2:1:1.

[0086] Preferably, in composition C, the compound containing an amine moiety is triethanolamine and the compound containing a sulfonic acid moiety is methanesulfonic acid.

[0087] Those skilled in the art know that cellulose biodegradation utilizes two distinct enzymes: endo- or exo-1,4-β-glucanase, and β-glucosidase. The 1,4-β-glucanase enzyme hydrolyzes the glycosidic bonds between glucose monomers within the cellulose chain. The β-glucosidase enzyme catalyzes the hydrolysis of the glycosidic bonds in cellobiose, or the glucose monomers at the ends of the cellulose chain. The different locations on cellulose where these two enzymes interact are shown in Figure 1. Alone, β-glucanase produces cellobiose, but when acting in conjunction with β-glucosidase, glucose is produced.

[0088] Various bacteria and fungi were compared for the biodegradability of MCA delignified cellulose, using a known microorganism capable of converting cellulose to cellobiose as a control. Four additional microorganisms, including bacteria and fungi, were tested to measure cellulase enzyme activity (Table 1). Organisms were cultured at temperatures ranging from 25 to 40°C, pH ranging from 5 to 9, and under shaking at 150 rpm. The media and culture conditions for each microorganism were optimized. MCA cellulose was added at a loading of 1% w / w of the total culture volume. All cultures were supplemented with 0.5% cellobiose to induce β-glucosidase activity.

[0089] Referring to Figure 2, a comparative graph of the maximum β-glucanase enzyme activity of different microorganisms on MCA cellulose obtained from the method described above can be observed. All bacteria and fungi tested showed some enzyme activity, demonstrating that the conversion of cellulose to cellobiose was occurring. Cytophaga hutchinsonii (C. hutchinsonii) and Pseudomonas protegens (P. protegens) achieved less than half the maximum enzyme activity of the control microorganism tested within the same time period. Meanwhile, Trichoderma reesei (T. reesei) achieved the same level of activity as the control but 7 days earlier (see Table 1). [Table 1]

[0090] Figure 3 shows the maximum β-glucosidase activity of different microorganisms incubated with MCA cellulose and cellobiose obtained from the method described above. Although all microorganisms listed in Table 1 were tested, only P. protegens and T. reesei, in addition to the control microorganisms, showed enzymatic activity toward cellobiose. T. reesei showed activity comparable to that of the control microorganisms at the same incubation time (Table 2). [Table 2]

[0091] According to a preferred embodiment of the method of the present invention, exposing microorganisms containing cellulase enzymes to MCA cellulose results in enzymatic activity, thus converting the cellulose to cellobiose and ultimately glucose. Cellulose obtained by delignification of biomass feedstocks using modified Caro's acid (such as MCA cellulose) has been shown to be biodegradable by several different bacteria and fungi. As is known, the higher the kappa number, the greater the amount of lignin, making biodegradation of cellulosic materials much more difficult. According to a preferred embodiment of the method of the present invention, this delignification method increases the bioavailability of cellulose to the microorganisms or enzymes utilized, thereby overcoming the first step in the reaction sequence—the conversion of cellulose to cellobiose—and enabling higher conversion of cellulose to glucose.

[0092] Cellulose obtained from various kraft processes is known to those skilled in the art to have a lignin content of 2.5% to 4.5% and a hemicellulose content of 9% to 25%. The MCA cellulose produced from the delignification process described herein results in cellulose with a lignin content of less than 1% and a hemicellulose content of less than 15%.

[0093] Those skilled in the art will recognize that the method described herein offers significant advantages over existing state-of-the-art biomass delignification processes due to the reduced energy requirements resulting from the ambient conditions employed. Furthermore, because the presence of lignin is known to be detrimental in currently existing processes due to residues in the equipment and the adsorption and deactivation of enzymes, a high delignification yield makes subsequent cellulose hydrolysis and fermentation highly efficient. As a result of the lack of lignin, the resulting solids, which primarily contain cellulose, have significantly greater surface area available for degradation by enzymes and / or organisms. This makes the method highly efficient in terms of yield (both mono- and oligosaccharides, and fermentation products), making it more cost-effective.

[0094] Given this information, idled ethanol plants around the world could resume operations converting cellulose to glucose (and subsequently ethanol) if they used biomass feedstock according to the following specifications rather than using corn, sugarcane, or traditional kraft pulp. Furthermore, implementation of a method according to a preferred embodiment of the present invention would be essentially "compatible" with delignification of lignocellulosic biomass using modified Caro's acid and the production of ethanol using the cellulose obtained from the delignification process. As previously mentioned, those skilled in the art will recognize that using cellulose obtained from a modified Caro's acid process avoids the need for an additional or subsequent bleaching step after delignification. It should be understood that this bleaching refers to a separate and distinct step in pulp processing. Thus, the pulp used using a modified Caro's acid-driven delignification process is intended to be pulp that has not undergone a separate bleaching step after delignification. As those skilled in the art will appreciate, such a treatment step (bleaching) is not economically feasible when the ultimate purpose of the cellulose is to be further converted to produce ethanol. Those skilled in the art will also appreciate that such high-purity, low-kappa cellulose is beneficial for cellulosic ethanol processes because it minimizes the problems caused by the presence of lignin in kraft pulp processes or unbleached cellulose. Those skilled in the art know that lignin causes problems during processing of the cellulose fraction as well as during distillation of the hydrolyzate. Utilizing low-kappa cellulose obtained from processes using modified Caro's acid avoids these problems and results in increased bioethanol yields.

[0095] Although the foregoing invention has been described in some detail for purposes of clarity and understanding, those skilled in the art, once familiar with the present disclosure, will appreciate that various changes in form and detail can be made therein without departing from the true scope of the invention as set forth in the appended claims.

Claims

1. 1. A method for hydrolyzing cellulose to cellobiose, said method comprising the steps of: - providing a reaction vessel; providing a cellulose source in the reaction vessel, wherein the cellulose source has a kappa number of less than 10, preferably said kappa number is 5 or less, more preferably said kappa number is 2 or less, and has a hemicellulose content of less than 15%, preferably less than 10%, even more preferably less than 5% w / w, based on the total weight of the cellulose source; providing an inoculum of an organism capable of expressing one or more β-glucanases in said reaction vessel; exposing said organism capable of expressing one or more β-glucanases to said cellulose source in an aqueous medium; and Optionally, recovering the supernatant containing cellobiose.

2. 2. The method of claim 1, further comprising exposing the cell supernatant to an organism to convert cellobiose to glucose, wherein the organism is selected from the group consisting of bacteria, fungi, yeast, and combinations thereof, such as Aspergillus brasiliensis, Trichoderma reesei, and Pseudomonas protegens.

3. 3. The method of claim 1 or 2, wherein the organism capable of expressing one or more β-glucanases is selected from the group consisting of bacteria and fungi capable of producing endo-β-glucanase enzymes or exo-β-glucanase enzymes.

4. The method according to claim 1 or 2, wherein the organism capable of expressing one or more β-glucanases is a bacterium of the phylum Bacteroidetes.

5. 3. The method of claim 1, wherein the organism capable of expressing one or more β-glucanases is Cytophaga hutchinsonii.

6. The method according to claim 1 or 2, wherein the organism capable of expressing one or more β-glucanases is a bacterium of the phylum Proteobacteria.

7. 3. The method according to claim 1, wherein the organism capable of expressing one or more β-glucanases is Pseudomonas protegens.

8. 3. The method of claim 1 or 2, wherein the obtained glucose is exposed to one of Zymomonas mobilis or the yeast Saccharomyces cerevisiae in a step of converting the glucose to ethanol.

9. 3. The method of claim 1 or 2, wherein the organism used to convert cellobiose to glucose is a bacterium or a fungus capable of producing a β-glucosidase enzyme.

10. 3. The method of claim 1 or 2, wherein the organism used to convert cellobiose to glucose is a fungus of the phylum Ascomycota.

11. 3. The method according to claim 1 or 2, wherein the organism used to convert cellobiose to glucose is a bacterium of the phylum Proteobacteria.

12. 3. The method of claim 1 or 2, wherein the cellulose source is exposed to the organism at a temperature of 20°C to 40°C.

13. 3. The method of claim 1 or 2, wherein the organisms are incubated with the cellulose source for a period ranging from 1 to 30 days.

14. 3. The method of claim 1, wherein the aqueous medium has a pH of about 5.0 to 8.

0.

15. 1. A method for hydrolyzing cellulose to glucose, said method comprising the steps of: - providing a reaction vessel; providing a cellulose source in the reaction vessel, wherein the cellulose source has a kappa number of less than 10, preferably said kappa number is 5 or less, more preferably said kappa number is 2 or less, and has a hemicellulose content of less than 15%, preferably less than 10% w / w, even more preferably less than 5% w / w, based on the total weight of the cellulose source; providing an inoculum of an organism capable of expressing one or more β-glucanases and β-glucosidases in said reaction vessel; exposing said organism capable of expressing one or more β-glucanases and β-glucosidases to said cellulose source in an aqueous medium; and Optionally, recovering the supernatant containing glucose.

16. 11. The method according to claim 10, wherein a bacterium or a fungus capable of producing said organism capable of expressing endo-β-glucanase or exo-β-glucanase and β-glucosidase is used.

17. 12. The method of claim 11, wherein the bacterium or fungus capable of producing the organism capable of expressing endo-β-glucanase or exo-β-glucanase and β-glucosidase is selected from the group consisting of Ascomycota, Proteobacteria, and combinations thereof.

18. 11. The method of claim 10, wherein the organism capable of expressing one or more β-glucanases and β-glucosidases is Trichoderma reesei.

19. 11. The method of claim 10, wherein the organism capable of expressing one or more β-glucanases and β-glucosidases is Pseudomonas protegens.

20. 11. The method of claim 10, wherein the cellulose source is exposed to the organism at a temperature of 30°C to 40°C.

21. 11. The method of claim 10, wherein the organisms are incubated with the cellulose source for a period ranging from 1 to 30 days.

22. The method of claim 10, wherein the aqueous medium has a pH of about 5.0 to 9.

0.

23. 1. Use of a cellulose source having a kappa number of less than 10, preferably said kappa number equal to or less than 5, more preferably said kappa number equal to or less than 2, and a hemicellulose content of less than 15%, preferably less than 10% w / w, even more preferably less than 5% w / w, relative to the total weight of the cellulose source, in a process for hydrolyzing cellulose to cellobiose, said process comprising the steps of: - providing a reaction vessel; providing the cellulose source in the reaction vessel; - providing in said reaction vessel an organism capable of expressing one or more β-glucanases; exposing said organism capable of expressing one or more β-glucanases to said cellulose source in an aqueous medium; and Optionally, recovering the supernatant containing cellobiose.

24. 1. Use of a cellulose source having a kappa number of less than 10, preferably said kappa number equal to or less than 5, more preferably said kappa number equal to or less than 2, and a hemicellulose content of less than 15%, preferably less than 10% w / w, even more preferably less than 5% w / w, relative to the total weight of the cellulose source, in a process for hydrolyzing cellulose to cellobiose (and optionally glucose or ethanol), said process comprising the steps of: - providing a reaction vessel; providing the cellulose source in the reaction vessel; providing an inoculum of an organism capable of expressing one or more β-glucanases and β-glucosidases in said reaction vessel; exposing said organism capable of expressing one or more β-glucanases and β-glucosidases to said cellulose source in an aqueous medium; Optionally, recovering the supernatant containing cellobiose; Optionally, exposing the supernatant containing cellobiose to a bacterium or fungus producing β-glucosidase for the conversion of cellobiose to glucose, optionally recovering the supernatant containing glucose, and Optionally, exposing said supernatant containing glucose to ethanologenic bacteria or fungi for fermenting glucose to ethanol.

Images