Improvements in the fermentation of biomass to ethanol.

Abstract

A method for obtaining ethanol from lignocellulosic biomass, comprising the following steps: step 1: delignification of lignocellulosic biomass using modified Caro's acid; step 2: recovering a solid portion of the delignification reaction mixture, the solid portion comprising a cellulose component that is substantially free of hemicellulose and that contains up to 15% w / w hemicellulose; step 3: exposing the recovered solid portion of the resulting reaction mixture to an enzyme mixture comprising cellulase enzymes to create a saccharification system that degrades cellulose into saccharified compositions, e.g., oligosaccharides; and step 4: feeding the saccharified composition to an organism, e.g., yeast, capable of fermenting sugars into ethanol.

Description

[Technical Field]

[0001] The present invention relates to the use of cellulose that is free or substantially free of hemicellulose in the production of bioethanol. [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 petroleum and coal, the primary feedstock for bioethanol 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 of concentrating only bioethanol during the extraction of 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 bioethanol 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 starch-rich corn and sugarcane as its primary source of fuel remains an obstacle. It is estimated that approximately 45% 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 bioethanol becomes 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, and large plants have had 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 contain a large amount 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 it prohibitively expensive to enter the market. 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 straw, 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 repeating glucose 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 the glucose of each monomer are different between 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. The purpose of starch is as 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. Glucose has two isomers: α-glucose (present in starch as a branched polymer) and β-glucose (present in cellulose, where one β-glucose monomer is linked via a β-1,4-glycosidic bond 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 The hydrolysis of cellulose to glucose is the rate-limiting step in the conversion of cellulose to biofuels. Current methods for using cellulose as a starting material for bioethanol production require the conversion of cellulose to cellobiose and then glucose before ultimately producing ethanol. Fermentation of glucose using yeast leads to the production of ethanol. This rate-limiting step is the most critical and hinders the widespread adoption of biofuels. The difficulty in overcoming this conversion of cellulose to glucose lies in the fact that the crystalline structure of cellulose makes the conversion of cellulose to glucose extremely difficult, as multiple cellulose polymers are tightly packed together. 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, etc. 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 the fermenting yeast.

[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 and hemicellulases). 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) and hemicellulases (e.g., β-xylosidases), require careful control to maximize the reaction rates expected from enzymatic methods. Temperature, pH, salt, 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 to an acid concentration of about 20 to 30% by weight. % by weight of 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 comprising 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 comprising a total of at least about 15% by weight of sugars and no more than 3% by weight of acid.

[0019] 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.

[0020] In a paper titled "Dilute-acid Hydrolysis of Cellulose to Glucose from Sugarcane Bagasse" by Dussan et al. (CHEMICAL ENGINEERING TRANSACTIONS, Vol. 38, 2014), a method for producing ethanol by cellulose hydrolysis was described. 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.

[0021] 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 to glucose. A stable, low-cost process for the production of cellulose has not yet been achieved.

[0022] The benefits of bioethanol are estimated to have the potential to reduce gas emissions by up to 85% over reformulated gasoline. However, the manufacturing challenges of producing bioethanol from lignocellulosic biomass rather than starch have led those skilled in the art to conclude that cellulosic ethanol will not be produced in sufficient quantities to provide at least a partial gasoline replacement or substitute in the near future. It is important that second-generation bioethanol production be based on the use of lignocellulosic biomass as a starting material in order to make this biomass environmentally desirable and economically feasible.

[0023] However, microbial fermentation of xylose, the major pentose sugar present in hemicellulose, is a limiting factor in developing such methods. Some current methods for removing lignin from lignocellulosic biomass leave a large portion of the hemicellulose present along with the cellulose, resulting in high concentrations of xylose present after enzymatic or chemical hydrolysis of cellulose. The presence of xylose poses significant challenges in fermenting cellulose to produce bioethanol, because native species of commonly used fermenters, such as Saccharomyces cerevisiae (S. cerevisiae), are unable to ferment both glucose and xylose. When hemicellulose is converted to xylose, this can lead to the formation of organic acids, such as acetic acid and furaldehyde. The accumulation of these compounds inhibits yeast metabolism, thus reducing bioethanol production. Some approaches used to overcome the limitations caused by the presence of hemicellulose have involved genetically modifying yeast to ferment both glucose and xylose. However, there are multiple genetic barriers to overcome, as fermentation of xylose leads to xylitol, which also inhibits yeast metabolism.

[0024] One approach to addressing the presence of xylose in pulp to be converted to ethanol is to use a mixture of different microorganisms (cocultures), some of which can convert glucose to ethanol, while others can ferment xylose to xylulose and ultimately to ethanol. Examples of organisms capable of accomplishing the latter conversion pathway are Pichia stipites (reclassified as Scheffersomyces stipitis) and Kluyveromyces marxianus. The main drawbacks of using these strains are their low ethanol yields, their inability to grow without oxygen, and their sensitivity to low concentrations of inhibitors in the hydrolysate (i.e., acetate).

[0025] Another technique has recently been developed that involves the use of a strain of the white-rot basidiomycete fungus Trametes versicolor, which was found to be capable of fermenting xylose. This strain was also shown to be able to convert untreated starch, cellulose, xylan, wheat bran, and rice straw to ethanol and to resist the effects of certain common inhibitors. These studies and findings are recent, and therefore further research is needed to assess whether and how they can be implemented on a large scale.

[0026] Another approach to converting xylose to ethanol involves the use of yeast. Some yeasts, such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, have been engineered to be able to convert xylose directly to ethanol, but several challenges exist that make these strains impractical for large-scale applications.

[0027] The pulp produced by many current pulping methods must be bleached to remove residual lignin (approximately 3-4% of the processed biomass) and still contains a non-negligible content of hemicellulose (approximately 20% of the processed residual biomass). Hemicellulose is a source of xylose, and the presence of xylose slows the fermentation of cellulose to ethanol.

[0028] In light of the prior art regarding the use of lignocellulosic biomass in bioethanol production, there remains a need for methods that can be efficiently scaled up and that allow for the use of lignocellulosic biomass in bioethanol production. Preferably, it would also be desirable to overcome the drawbacks associated with the presence of hemicellulose in the pulp that is subjected to fermentation to cellulose. This is further supported by the fact that there have been significant efforts to convert waste biomass into biofuels using various approaches, but nearly all of them have failed to achieve this goal for subsequent conversion to glucose and ultimately ethanol.

[0029] The present inventors have surprisingly and unexpectedly found that the properties of the cellulose resulting from a particular type of delignification procedure have a significant impact on the downstream hydrolysis of cellulose to glucose and subsequent conversion to ethanol. Summary of the Invention

[0030] Lignocellulosic biomass is a widely available resource and is the feedstock used in second-generation bioethanol production. However, incomplete removal of hemicellulose during cellulose pretreatment hinders the efficiency of the fermentation process. This is due, for example, to slow xylose transport, inefficient co-utilization of glucose and xylose, inefficient downstream pathway metabolism, functional expression of xylA, and an overall reduction in ethanol production. Therefore, when using pulp for bioethanol production, minimizing the amount of hemicellulose remaining in the pulp is preferable to maximize bioethanol yield.

[0031] According to one aspect of the present invention, a method for increasing the efficiency of cellulose fermentation to bioethanol is provided by removing or substantially reducing the amount of hemicellulose present in the biomass. Removing the majority of hemicellulose from lignocellulosic biomass material maximizes the amount of ethanol produced from pure cellulose (rather than from a cellulose + hemicellulose mixture). This translates into lower costs in bioethanol production, as fermentation is simpler and does not require genetically modified organisms. When such a cellulose / hemicellulose blend is present, genetically modified yeast must be present to ferment the pentose sugar (xylose), as standard fermenting yeast is less efficient. The presence of hemicellulose / xylose does not provide an advantage to bioethanol production using standard yeast, compromising the overall yield of biomass conversion to bioethanol.

[0032] According to one aspect of the present invention, there is provided a method for increasing the fermentation yield of cellulose by first treating biomass with a delignification reaction, then recovering the remaining solids, which are mostly composed of cellulose, and fermenting the cellulose in a suitable biodigester, whereby the cellulose is fermented to ethanol, preferably in the absence of hemicellulose, resulting in a better ethanol yield compared to hemicellulose-containing cellulose.

[0033] It should be understood that the presence of small amounts of hemicellulose (0.5-15 wt%) generally results in much improved yields compared to conventional pulps containing higher percentages of hemicellulose (15-25 wt%). For example, hemicellulose is generally the second most common component of lignocellulosic biomass and is therefore expected to be present in the range of 15-25% in conventional pulps after delignification using the Kraft process.

[0034] Preferably, when a substantially hemicellulose-free biomass additive is used as part of the organic waste to be fermented or as the total organic load in a saccharification or fermentation unit, the addition of this biomass additive allows for increased ethanol production in the fermentation unit. The substantially hemicellulose-free cellulose is subjected to a hydrolysis reaction using chemicals, organisms, or enzymes to produce glucose and other oligosaccharides, known as saccharification reactants. This saccharified component is then subjected to fermentation to produce ethanol.

[0035] According to one aspect of the present invention, there is provided the following use: use of a cellulosic component comprising cellulose and hemicellulose for saccharification of cellulose to glucose and subsequent fermentation to ethanol, wherein the cellulosic component is obtained from a delignification process using a modified Caro's acid 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 alkylsulfonic 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, compounds containing an amine moiety, Compounds containing sulfonic acid moieties a two-component modifier component comprising: Peroxide Including, The cellulose component comprises at least 85% by weight of the cellulose and at most 15% by weight of the hemicellulose as a result of the delignification process.

[0036] According to one aspect of the present invention, there is provided the following use: use of a cellulosic component comprising cellulose, hemicellulose, and lignin obtained from the treatment of lignocellulosic biomass with modified Caro's acid in the fermentation of cellulose to cellobiose and ethanol, wherein the cellulosic component is characterized by a lignin content of less than 1 wt / w% and a hemicellulose content of less than 15 wt / w%, and the modified Caro's acid is 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 alkylsulfonic 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, compounds containing an amine moiety, Compounds containing sulfonic acid moieties a two-component modifier component comprising: Peroxide Including, use.

[0037] According to a preferred embodiment of the present invention, the weight ratio of cellulose to hemicellulose in the cellulose component is 6:1 or more. Preferably, the weight ratio of cellulose to hemicellulose in the cellulose component is 7:1 or more. Preferably, the weight ratio of cellulose to hemicellulose in the cellulose component is 10:1 or more. More preferably, the weight ratio of cellulose to hemicellulose in the cellulose component is 12:1 or more.

[0038] According to one aspect of the present invention, there is provided the use of a substantially hemicellulose-free cellulosic component as an additive to organic material intended for bioethanol production, to increase the amount of ethanol produced from a fermentation unit.

[0039] According to a preferred embodiment of the present invention, the cellulose component comprises at least 85% cellulose. Preferably, the substantially hemicellulose-free cellulose component comprises at least 90% cellulose. More preferably, the substantially hemicellulose-free cellulose component comprises at least 92.5% cellulose.

[0040] According to another aspect of the present invention, there is provided a method for obtaining ethanol from lignocellulosic biomass, the method comprising the steps of: Step 1: preparing a delignification mixture and delignifying lignocellulosic biomass with a modified Caro's acid 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 alkylsulfonic 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, compounds containing an amine moiety, Compounds containing sulfonic acid moieties a two-component modifier component comprising: Peroxide Including, the delignified mixture comprising the lignocellulosic biomass and the modified Caro's acid; Step 2: Recovering a solid portion of the delignification reaction mixture, wherein the solid portion comprises a substantially hemicellulose-free cellulosic component containing up to 15% w / w hemicellulose; Step 3: exposing the recovered solid portion of the resulting reaction mixture to an enzyme mixture comprising at least one cellulase enzyme to create a saccharification or fermentation system that breaks down cellulose into a saccharified composition, e.g., oligosaccharides, or ferments the saccharified composition into ethanol; Step 4: Optionally, feeding the saccharified composition to an organism, such as yeast, capable of fermenting sugars to ethanol.

[0041] Preferably, the method further comprises step 5, after fermentation, distilling the liquid portion from the fermentation system to recover said ethanol.

[0042] According to another aspect of the present invention, there is provided a method for increasing the amount of ethanol produced from a fermentation system by using a substantially hemicellulose-free cellulosic component containing up to 15% w / w hemicellulose as an additive to organic material intended for bioethanol production, the increase being compared to systems using a cellulosic component with a much higher hemicellulose content, e.g., 20% or more.

[0043] According to yet another aspect of the present invention, there is provided a method for increasing the amount of ethanol produced from a fermentation system by using a cellulosic component that is substantially free of hemicellulose and that contains up to 15% w / w hemicellulose as a partial substitute for organic material intended for bioethanol production.

[0044] Preferably, the substantially hemicellulose-free cellulosic component comprises less than 30% of the amount of hemicellulose present before the lignocellulosic biomass was delignified. More preferably, the substantially hemicellulose-free cellulosic component comprises less than 20% of the amount of hemicellulose present before the lignocellulosic biomass was delignified. Even more preferably, the substantially hemicellulose-free cellulosic component comprises less than 8% of the amount of hemicellulose present before the lignocellulosic biomass was delignified. Even more preferably, the substantially hemicellulose-free cellulosic component comprises less than 4% of the amount of hemicellulose present before the lignocellulosic biomass was delignified.

[0045] According to yet another aspect of the present invention, there is provided a method for hydrolyzing cellulose to glucose, the method comprising the steps of: providing a reaction vessel; providing a cellulose source in the reaction vessel, wherein the cellulose source has a hemicellulose content that is less than 30% of the original hemicellulose content of the lignocellulosic biomass from which the cellulose source is derived; providing a microbial inoculum in said reaction vessel; exposing the inoculum to the cellulose source in an aqueous medium having a pH of about 8, at a temperature ranging from 30° C. to 40° C., for a period ranging from 1 to 42 days; optionally recovering the supernatant containing glucose; and Exposing the glucose to bacteria, fungi, or yeast, or a combination thereof, that ferment the glucose to ethanol.

[0046] According to yet another aspect of the present invention, there is provided a method for hydrolyzing cellulose to glucose, the method comprising the steps of: providing a reaction vessel; providing a cellulose source in the reaction vessel, wherein the cellulose source has a hemicellulose content that is less than 30% of the original hemicellulose content of the lignocellulosic biomass from which the cellulose source is derived; providing an enzyme blend in the reaction vessel; exposing the enzyme blend to the cellulose source in an aqueous medium having a pH of about 4 to 6, at a temperature ranging from 40° C. to 55° C., for a period ranging from 1 to 7 days; optionally recovering the supernatant containing glucose; and Exposing the glucose to bacteria, fungi, yeast, or a combination thereof that ferment the glucose to ethanol.

[0047] The term "saccharification system" refers to a vessel in which biomass or cellulosic components are added along with chemicals, organisms, or enzyme blends under conditions that convert complex sugars (i.e., polysaccharides including cellulose and hemicellulose) into simple sugars, such as oligosaccharides, disaccharides, and monosaccharides (i.e., glucose, xylose, etc.).

[0048] The term "fermentation system" refers to a vessel to which a mixture of oligo-, di-, and monosaccharides (i.e., glucose, xylose, etc.) is added along with an ethanologenic organism and maintained under conditions to convert the sugars to ethanol. In some embodiments of the invention, the term "fermentation system" also refers to a vessel to which a biomass or cellulosic component is added along with a combination of chemicals, organisms, and / or enzyme blends under conditions to convert complex sugars (i.e., polysaccharides including cellulose and hemicellulose) directly to ethanol in a one-pot process.

[0049] The terms "substantially free of hemicellulose" or "substantially free of hemicellulose" refer to a biomass additive or cellulosic component that contains less than 15% hemicellulose, preferably less than 10% hemicellulose, and more preferably less than 7.5% hemicellulose.

[0050] According to a preferred embodiment of the present invention, the biomass additive is cellulose that has been processed to be substantially free of hemicellulose. Preferably, the biomass additive comprises up to 30% of the original hemicellulose content from the harvested lignocellulosic biomass. Preferably, the biomass additive comprises up to 20% of the original hemicellulose content from the harvested lignocellulosic biomass. Preferably, the biomass additive comprises up to 10% of the original hemicellulose content from the harvested lignocellulosic biomass. Preferably, the biomass additive comprises up to 8% of the original hemicellulose content from the harvested lignocellulosic biomass. Preferably, the biomass additive comprises up to 6% of the original hemicellulose content from the harvested lignocellulosic biomass. Preferably, the biomass additive comprises up to 5% of the original hemicellulose content from the harvested lignocellulosic biomass. Preferably, the biomass additive comprises up to 4% of the original hemicellulose content from the harvested lignocellulosic biomass. Preferably, the biomass additive comprises up to 2% of the original hemicellulose content from the harvested lignocellulosic biomass. Preferably, the biomass additive comprises up to 1% of the original hemicellulose content from the harvested lignocellulosic biomass. According to preferred embodiments of the present invention, biomass additives classified as substantially free of hemicellulose also comprise up to 1 w / w% lignin.

[0051] When delignified using modified Caro's acid and reclassified into cellulose according to the methods described herein, residual hemicellulose can remain at low levels of 5% or less of the total weight of the pulp. Similarly, hemicellulose is simultaneously hydrolyzed, and the sugars formed are solubilized and remain in the liquid phase. After delignification is deemed sufficiently complete by the operator, the solids (cellulose and up to 0.5-15 w / w% residual hemicellulose) are separated from the liquid containing the modified Caro's acid and lignin and hemicellulose fragments (the constituents of which are xylose). This approach maximizes the removal of hemicellulose from the cellulose and allows for efficient conversion of the extracted cellulose to ethanol using conventional enzymes, etc. This also eliminates the need to find a mixture of various enzymes capable of converting cellulose and hemicellulose to ethanol, thus streamlining the process and ensuring more efficient conversion of lignocellulosic biomass to ethanol.

[0052] 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]

[0053] [Figure 1] FIG. 1 is a graphical representation of glucose production for samples from Experiment #1. [Figure 2] FIG. 1 is a graphical representation of xylose production for samples from Experiment #1. [Figure 3] FIG. 1 is a graphical representation of ethanol production for samples from Experiment #1. DETAILED DESCRIPTION OF THE INVENTION

[0054] Delignification of biomass by conventional methods, such as kraft pulping, results in a pulp that is still rich in lignin and hemicellulose. The addition of a cellulose-rich additive that is essentially free of hemicellulose (which hydrolyzes to xylose) has made it possible to increase the production of ethanol from the fermentation of glucose.

[0055] According to a preferred embodiment of the present invention, the cellulose is unbleached cellulose with a very low hemicellulose content (preferably in the range of 0.5-15 w / w%). Preferably, the cellulose is obtained by delignification of a lignocellulosic biomass feedstock by exposing the lignocellulosic biomass feedstock to modified Caro's acid according to the following method. A preferred embodiment of the method for delignifying biomass comprises the following steps: providing a container; providing a biomass in the vessel, the biomass comprising lignin, hemicellulose, and cellulose fibers; providing a sulfuric acid component; providing a peroxide component; exposing the biomass to the sulfate source and the peroxide component; Contacting the sulfuric acid source and peroxide component with the biomass for a time sufficient for a delignification reaction to occur, removing greater than 90% by weight of the lignin and hemicellulose from the biomass.

[0056] Preferably, biomass comprising lignin, hemicellulose, and cellulose fiber is exposed 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, compounds containing an amine moiety, Compounds containing sulfonic acid moieties a two-component modifier component comprising: Peroxide Includes:

[0057] According to a preferred embodiment of the present invention, substantially hemicellulose-free cellulose means that the cellulose component comprises at least 85% cellulose. Preferably, the cellulose component comprises at least 90% cellulose. More preferably, the cellulose component comprises at least 92.5% cellulose.

[0058] Preferably, the delignification reaction is carried out at a temperature below 55°C by a method selected from the group consisting of: adding water to the vessel; adding biomass to said vessel; and A method using a heat exchanger.

[0059] Preferably, the sulfuric acid, the modifier component comprising an amine moiety and a sulfonic acid moiety, and the peroxide are present in a molar ratio of 1:1:1 or greater, and 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.

[0060] According to a preferred embodiment of the invention, 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.

[0061] According to a preferred embodiment of the present invention, the compound comprising an amine moiety and a sulfonic acid moiety is selected from the group consisting of taurine, taurine derivatives, and taurine-related compounds.

[0062] According to a preferred embodiment of the present invention, 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.

[0063] According to a preferred embodiment of the present invention, the compound comprising an amine moiety and a sulfonic acid moiety is taurine.

[0064] According to a preferred embodiment of the invention, 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.

[0065] According to a preferred embodiment of the present invention, the compound comprising an amine moiety is an alkanolamine selected from the group consisting of monoethanolamine, diethanolamine, triethanolamine, and combinations thereof.

[0066] According to a preferred embodiment of the present invention, said compound comprising a sulfonic acid moiety is selected from the group consisting of alkylsulfonic acids and combinations thereof.

[0067] According to a preferred embodiment of the present invention, the alkyl sulfonic acid is selected from the group consisting of alkyl sulfonic acids in which the alkyl group is in the range of C1 to C6 and is linear or branched, and combinations thereof.

[0068] According to a preferred embodiment of the present invention, 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.

[0069] According to a preferred embodiment of the present invention, the alkyl sulfonic acid and the peroxide are present in a molar ratio of 1:1 or greater.

[0070] According to a preferred embodiment of the present invention, said compound comprising a sulfonic acid moiety is methanesulfonic acid.

[0071] According to a preferred embodiment of the present invention, in composition C, said sulfuric acid, said compound comprising an amine moiety, and said compound comprising a sulfonic acid moiety are present in a molar ratio of 1:1:1 or greater.

[0072] According to a preferred embodiment of the present invention, 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.

[0073] Preferably, for a modified Caro's acid containing sulfuric acid, peroxide, and taurine (as the modifier component), the molar composition is as follows: the molar ratio of H2O:H2O2:H2SO4:taurine is 56:10:10:1.

[0074] Preferably, for the modified Caro's acid containing TEOA / MSA, the molar composition is as follows: H2O:H2O2:H2SO4:TEOA:MSA molar ratio is 56:10:10:1:1.

[0075] Kraft pulping dissolves and removes approximately 90% of the lignin present in processed biomass. Kraft pulp also contains hemicellulose fragments (including xylose) that are harmful to the proper operation of fermentation units. In fact, kraft pulping dissolves only 0-60% of the hemicellulose originally present in the lignocellulosic feedstock. Therefore, it is clear that the present invention overcomes the shortcomings of the prior art for large-scale bioethanol production using lignocellulosic biomass (or feedstock). Furthermore, large-scale implementation of preferred embodiments of the methods taught herein enables large-scale bioethanol production from lignocellulosic biomass rather than from starch (e.g., corn).

[0076] 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.

[0077] Experiment #1 - Demonstration of increased xylose production with increasing hemicellulose concentration For this experiment, a delignification process disclosed herein using a modified Caro's Acid delignification process was used to produce a substantially hemicellulose-free cellulose component from hardwood forestry waste. As chemicals from the delignification process were recycled, various batches of substantially hemicellulose-free cellulose were produced, categorized by the number of times the chemicals were reused. As the number of reuses increased, the hemicellulose content increased in parallel. The composition of the modified Caro's Acid blend used in Experiment #1 is shown in Table #1 below. [Table 1]

[0078] A sample of hardwood biomass was delignified according to the method described herein, including modified Caro's acid. A mixture containing 38.53% w / w water, 14.46% w / w hydrogen peroxide, 42.69% H2SO4, and 5.32% w / w taurine was prepared. To this, the hardwood biomass was added at a 5.0% loading, and the reaction was allowed to stir in the reaction vessel at 37°C for 20 hours. After the reaction was deemed complete, the substantially hemicellulose-free cellulose was filtered from the liquid stream and neutralized to a pH of 5-7.

[0079] The cellulose composition of the various batches prepared, including hemicellulose content, is shown in Table 2. Substantially hemicellulose-free cellulose was also produced by the same delignification process, but with an additional caustic treatment (CT) to further remove hemicellulose. The hemicellulose content of this cellulose batch is shown in Table 2. By comparison, the hemicellulose content of raw hardwood biomass is between 15 and 25%.

[0080] For the caustic-treated sample, 3.0 g of the substantially hemicellulose-free cellulose (R2B014) produced by fresh delignification using modified Caro's acid as described herein was placed in 100 mL of 8.5% NaOH solution. The reaction mixture was left stirring at room temperature for 30 minutes, after which the solution was filtered and the solids were collected. [Table 2]

[0081] In this experiment, the conversion of various recycled batches of substantially hemicellulose-free cellulose fraction and samples that had been subjected to a caustic treatment after delignification was tested. The cellulose fraction was loaded into a buffer solution at 5% w / w using a commercial blend of cellulase enzymes, and the samples were saccharified at 50°C for one week using the commercial enzyme blend. Subsamples were taken throughout the incubation period to quantify the monosaccharides produced from the conversion of cellulose and hemicellulose.

[0082] Glucose production remains consistent throughout the saccharification reaction for all cellulose recycles, which are essentially hemicellulose-free (Figure 1). However, xylose production increases as the hemicellulose content of the cellulose batch increases (Figure 2). Accumulation of xylose in a bioethanol production system impacts the final ethanol yield. This is because if the yeast used cannot metabolize xylose, an inhibitor forms and accumulates over time. This experiment also demonstrates that commercially available cellulase enzyme blends contain not only cellulase enzymes but also hemicellulase enzymes. Therefore, removing hemicellulose is preferable and more efficient for obtaining optimal bioethanol yields from pure delignified cellulose without the need for genetic modification.

[0083] When using non-genetically modified ethanol-producing organisms (e.g., Saccharomyces cerevisiae), it is understood that a lower hemicellulose and lignin content in the cellulosic component used to produce ethanol results in higher ethanol yields. The presence of lignin is detrimental to the conversion of cellulose to glucose because enzymes are known to adsorb to the surface of lignin, thereby inhibiting the efficiency of enzymatic hydrolysis of cellulose. The presence of hemicellulose is also detrimental to the conversion of glucose to ethanol. Hemicellulose acts as a physical barrier, covering cellulose and preventing cellulase from enzymatically hydrolyzing cellulose. It is known that removing hemicellulose from the cellulosic component is preferable, allowing cellulase to better reach and hydrolyze the cellulose.

[0084] Experiment #2: Conversion to Ethanol As can be seen in Figure 3, conversion of the cellulosic component to ethanol for each batch obtained after a single delignification treatment step using modified Caro's acid resulted in very good ethanol yields. For the caustic-treated samples, ethanol yields increased even further when hemicellulose was almost completely removed (see Figure 3). Subsequent caustic treatment of the delignified cellulosic component produced more ethanol than the single-treatment sample with less than 12.5% residual hemicellulose. This demonstrates the detrimental effect of hemicellulose on the final bioethanol yield.

[0085] Given this information, it is believed that idled ethanol plants around the world could be restarted to convert cellulose to glucose (and subsequently ethanol) using biomass feedstocks, and would likely use the above-described process rather than 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 using lignocellulosic biomass. Those skilled in the art will recognize that using the cellulosic component obtained from the modified Caro's acid delignification approach described herein can avoid the need for additional or subsequent processing steps before using the cellulosic component in bioethanol production. Those skilled in the art will also understand that such additional processing steps (to remove hemicellulose) are likely to be economically infeasible in many, if not all, countries when the ultimate goal of the resulting cellulosic component is further conversion for ethanol production. Those skilled in the art will also appreciate that such pulp with high cellulose purity and low hemicellulose content is beneficial in cellulosic ethanol processes because it minimizes the problems caused by the presence of hemicellulose in processes such as kraft pulp.

[0086] 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. Use of a cellulosic component comprising cellulose and hemicellulose for saccharification of cellulose to glucose and subsequent fermentation to ethanol, wherein the cellulosic component is obtained from a delignification process using a modified Caro's acid 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 Peroxides Including, The composition B is alkyl sulfonic acids, and Peroxides wherein said acid is present in an amount ranging from 40 to 80% by weight of the total weight of the composition, and said 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, compounds containing an amine moiety, Compounds containing sulfonic acid moieties a two-component modifier component comprising: Peroxides Including, The cellulose component comprises at least 85% by weight of the cellulose and at most 15% by weight of the hemicellulose as a result of the delignification process.

2. 1. Use of a cellulosic component comprising cellulose, hemicellulose, and lignin obtained from the treatment of lignocellulosic biomass with modified Caro's acid in the fermentation of cellulose to cellobiose and ethanol, wherein the cellulosic component is characterized in that its lignin content is less than 1 wt. / wt.% and its hemicellulose content is less than 15 wt. / wt.%, and the modified Caro's acid is 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 Peroxides Including, The composition B is alkyl sulfonic acids, and Peroxides wherein said acid is present in an amount ranging from 40 to 80% by weight of the total weight of the composition, and said 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, compounds containing an amine moiety, Compounds containing sulfonic acid moieties a two-component modifier component comprising: Peroxides Including, use.

3. 3. The use according to claim 1 or 2, wherein the weight ratio of cellulose to hemicellulose in the cellulose component is 6:1 or more.

4. 3. The use according to claim 1 or 2, wherein the weight ratio of cellulose to hemicellulose in the cellulose component is 7:1 or more.

5. 3. The use according to claim 1 or 2, wherein the weight ratio of cellulose to hemicellulose in the cellulose component is 10:1 or more.

6. 3. The use according to claim 1 or 2, wherein the weight ratio of cellulose to hemicellulose in the cellulose component is 12:1 or more.

7. 1. Use of a substantially hemicellulose-free cellulosic component as an additive to organic material intended for bioethanol production, which increases the amount of ethanol produced from a fermentation unit.

8. The use according to any one of claims 1 to 7, wherein the cellulose component comprises at least 85% cellulose.

9. The use according to any one of claims 1 to 7, wherein the substantially hemicellulose-free cellulose component comprises at least 90% cellulose.

10. The use according to any one of claims 1 to 7, wherein the substantially hemicellulose-free cellulose component comprises at least 92.5% cellulose.

11. 1. A method for obtaining ethanol from lignocellulosic biomass, comprising the steps of: Step 1: preparing a delignification mixture and delignifying lignocellulosic biomass with a modified Caro's acid 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 Peroxides Including, The composition B is alkyl sulfonic acids, and Peroxides wherein said acid is present in an amount ranging from 40 to 80% by weight of the total weight of the composition, and said 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, compounds containing an amine moiety, Compounds containing sulfonic acid moieties a two-component modifier component comprising: Peroxides Including, the delignified mixture comprising the lignocellulosic biomass and the modified Caro's acid; Step 2: Recovering a solid portion of the delignification reaction mixture, wherein the solid portion comprises a substantially hemicellulose-free cellulosic component comprising up to 15% w / w hemicellulose; Step 3: exposing the recovered solid portion of the resulting reaction mixture to an enzyme mixture comprising at least one cellulase enzyme to create a saccharification or fermentation system that degrades cellulose into a saccharified composition, e.g., oligosaccharides, or ferments the saccharified composition into ethanol; Step 4: Optionally, feeding the saccharified composition to an organism, such as yeast, capable of fermenting sugars to ethanol.

12. 12. The method of claim 11, further comprising step 5, after fermentation, distilling a liquid portion from the fermentation system to recover the ethanol.

13. 1. A method for increasing the amount of ethanol produced from a fermentation system by using a cellulosic component that is substantially free of hemicellulose and contains up to 15% w / w hemicellulose as an additive to organic material intended for bioethanol production.

14. A method for increasing the amount of ethanol produced from a fermentation system by using a cellulosic component that is substantially free of hemicellulose and contains up to 15% w / w hemicellulose as a partial substitute for organic materials intended for bioethanol production.

15. 15. The method of any one of claims 11-14, wherein the substantially hemicellulose-free cellulosic component comprises less than 30% of the amount of hemicellulose present before the lignocellulosic biomass is delignified.

16. 16. The method of any one of claims 11-15, wherein the substantially hemicellulose-free cellulosic component comprises less than 20% of the amount of hemicellulose present before the lignocellulosic biomass is delignified.

17. 17. The method of any one of claims 11 to 16, wherein the substantially hemicellulose-free cellulosic component comprises less than 8% of the amount of hemicellulose present before the lignocellulosic biomass is delignified.

18. 18. The method of any one of claims 11 to 17, wherein the substantially hemicellulose-free cellulosic component comprises less than 4% of the amount of hemicellulose present before the lignocellulosic biomass is delignified.

19. 1. A method for hydrolyzing cellulose to glucose, comprising the steps of: providing a reaction vessel; providing a cellulose source in the reaction vessel, wherein the cellulose source has a hemicellulose content that is less than 30% of the original hemicellulose content of the lignocellulosic biomass from which the cellulose source is derived; providing a microbial inoculum in said reaction vessel; exposing the inoculum to the cellulose source in an aqueous medium at a pH of about 8, at a temperature ranging from 30° C. to 40° C., for a period ranging from 1 to 42 days; optionally recovering the supernatant containing glucose; and Exposing the glucose to bacteria, fungi, or yeast, or a combination thereof, which ferment the glucose to ethanol.

20. 1. A method for hydrolyzing cellulose to glucose, comprising the steps of: providing a reaction vessel; providing a cellulose source in the reaction vessel, wherein the cellulose source has a hemicellulose content that is less than 30% of the original hemicellulose content of the lignocellulosic biomass from which the cellulose source is derived; providing an enzyme blend in the reaction vessel; exposing the enzyme blend to the cellulose source in an aqueous medium having a pH of about 4 to 6, at a temperature ranging from 40° C. to 55° C., for a period ranging from 1 to 7 days; optionally recovering the supernatant containing glucose; and Exposing the glucose to bacteria, fungi, yeast, or a combination thereof that ferment the glucose to ethanol.

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