method of treating a lignocellulose biomass to dissolve lignin in it and process of preparing glucos

专利摘要:
METHOD OF TREATMENT OF A BIOMASS TO DISSOLVE LIGNIN IN IT, PURIFIED LIGNIN, PROCESS OF PREPARING GLUCOSE FROM A BIOMASS OF LIGNOCELLULOSE AND GLUCOSE The present invention relates to a method of treating a lignocellulose biomass in order to dissolve lignin in it, whereas cellulose does not dissolve. The cellulose pulp obtained can be used to produce glucose. Furthermore, lignin can be isolated for subsequent use in the renewable chemicals industry as a source for aromatic platform chemicals.
公开号:BR112013015190B1
申请号:R112013015190-0
申请日:2011-12-15
公开日:2021-05-11
发明作者:Agnieszka Brandt；Richard J. Murphy；David J. Leak；Tom Welton；Jason Hallett
申请人:Imperial Innovations Limited；
IPC主号:

专利说明:

The present invention relates to a method of treating a lignocellulose biomass in order to dissolve the lignin therein while the cellulose does not dissolve. The cellulose pulp obtained can be used to produce glucose. Furthermore, lignin can be isolated for subsequent use in the renewable chemicals industry as a source for aromatic platform chemicals.
Biofuels can be generated by fermenting sugars to produce bioethanol. At present, biofuels are generally derived from food resources. This leads to several problems as there is competition with the food supply for raw materials; yield is low per unit of land area a high energy input is needed to grow crops. It is possible to produce the necessary sugar by hydrolysis of starch, or sucrose produced by plants such as sugar cane or sugar beet can be used. The problems could be alleviated if the woody part of plants from agricultural residues, forest residues and energy crops could be utilized.
The structural or woody parts of plants have evolved to resist degradation. These are mainly made from cellulose, hemicellulose and lignin. Pre-treatment of the material is necessary in order to break the structure. Generally speaking, pretreatment involves one or more of the following: removing the hemicellulose; modify and solubilize lignin; hydrolyzing the hemicellulose-lignin bonds; and reduce the crystallinity of cellulose fibers. This makes cellulose more accessible to enzymes and also removes potential inhibitors from the fermentation stage.
Several pretreatment strategies have been previously described. These include explosion, catalysis with dilute acid or a base, ammonia fiber expansion, the Organosolv pulping technique and biological pretreatment. All of these processes have their downsides. Pretreatment with ionic liquids has also been described. Ionic liquids (ILs) are salts that turn into a liquid at the temperature of interest. The combination of anions and cations can be chosen to match the particular application required.
WO10/0056790 describes the use of substantially water-free ILs to dissolve biomass which can then be separated using various solvents. Documents W008/090155 and W008/090156 describe the use of ILs to dissolve all components of biomass, for example lignin, hemicellulose and cellulose. In these methods, the cellulose is separated from the other components generally by adding a suitable solvent so that the cellulose precipitates out and can be separated. Two recent reports applying [MeSO4]~ containing ionic liquids for biomass pretreatment concluded that the ionic liquid is not able to improve the digestibility of maple wood or corn cob. Document W02008/112291 describes the use of ionic liquids for a pre-treatment of a lignin containing biomass to increase the yield in a subsequent saccharification reaction. IL is used to swell the biomass structure and not achieve any lignocellulose dissolution. Lignin can be recovered as a post-saccharification solid. Document US2010-0081798 describes the use of ILs that contain a polyatomic anion to solubilize lignocellulose. Cellulose dissolves in IL.
The document WO2005/017252 discloses the use of ILs with an aromatic anion to dissolve the lignin from the biomass, allowing the obtained cellulosic fibers to be further processed.
Most prior art processes require the ionic liquid to be substantially free of water so that the biomass dissolves. Therefore IL and biomass must be dried before use, which increases processing costs. A tolerance of up to 15% water by weight in ILs was recorded, but higher levels produced unwanted results, such as reduced cellulose precipitation and reduced saccharification yields.
The pretreatment process can be improved by reducing the processing required to obtain the desired cellulose product. Furthermore, methods that allow lignin to be isolated and used would be desirable as well.
Lignin is produced by current technologies (eg, paper pulping) and is burned as a source of heat and electricity for the process (in paper pulping, it also creates excess electricity that is fed into the grid) . However, if this was available in a purer form, it could be used as the source of aromatic platform chemicals (which contain a benzene ring) for a biorefinery (chemical value chain based on renewable resources). It could also be used with less modification as a polymer additive (eg UV stabilizer) or wood adhesive.
The present inventors have identified a process in which lignin but not cellulose is dissolved by an IL so that the produced cellulose pulp can be mechanically separated before going through saccharification. Lignin can be precipitated out of IL by the simple addition of an anti-solvent, such as water. This means that IL can be recycled. The present invention relates to a method of treating a lignocellulosic biomass to dissolve the lignin in it, but not the cellulose, which comprises: (a) contacting the lignocellulose biomass with a composition comprising an ionic liquid to produce a cellulose pulp, in which the ionic liquid comprises a cation and an anion selected from C-20 alkylsulfate [Alkyl SO4]', C-20 alkylsulfonate [Alkyl SO3]', hydrogen sulfate [HSO4]" , hydrogen sulfite [HSO3]' , dihydrogen phosphate [H2PO4]', hydrogen phosphate [HPO4]2' and acetate, with the proviso that if the anion is acetate then the composition additionally comprises 10 to 40% v/v of water.
The IL is preferably heated with the biomass to 100 to 160 °C, preferably 120 to 140 °C. The reaction is carried out for 1 to 22 hours, preferably 1 to 13 hours, more preferably 1 to 8 hours. Preferably the mixture is stirred.
As used herein, the term "lignocellulosic biomass" refers to living or dead biological material that can be used in one or more of the disclosed processes. It may comprise any cellulosic or lignocellulosic material and includes materials which comprise cellulose, and which additionally and optionally comprise hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides, biopolymers, natural derivatives of biopolymers, mixtures thereof and decomposition products. It may comprise additional components such as protein and/or lipid. Biomass can be derived from a single source, or it can comprise a mixture derived from more than one source. Some specific examples of biomass include, but are not limited to, bioenergy crops, agricultural waste, urban solid waste, industrial solid waste, papermaking sludge, cultural waste, forestry waste and wood. Additional examples of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn waste, grasses including Miscanthus X giganteus, wheat, wheat straw, hay, rice straw, yellow millet, paper waste, sugarcane bagasse, sorghum, soybean, components obtained from grain milling, trees (eg pine), branches, roots, leaves, wood chips, wood pulp, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, multi-component feed and crustacean biomass (ie, chitinous biomass). It may be preferable to treat the biomass of use in the method of the invention. For example, biomass can be mechanically treated, for example by grinding or shredding.
In a preferred modality, the biomass is placed in contact with the ionic liquid composition before mechanical treatment. It was observed that the treatment of biomass, supplied as wood chips, can reduce the energy needed to crush the biomass. The IL composition appears to work as a lubricant during the grinding phase. Lignocellulosic biomass, supplied as wood chips, can be briefly impregnated with an IL composition at a slightly elevated temperature (70 to 100 °C, preferably 90 °C) to take advantage of its lubricating properties before a size reduction step mechanics is applied. The IL composition can come into contact with the biomass for any period of time, from several minutes to 18 hours or even longer, preferably 5 minutes to 1 hour. This can be followed by further treatment with an ionic liquid composition as described herein to solubilize the lignin content of the biomass.
As used herein, "ionic liquid" refers to an ionized species (ie, cations and anions). Typically, they have a melting point below about 100°C. The anion is selected from C-20 alkyl sulfate [Alkyl SO4]', C-2 O alkylsulfonate [Alkyl S03]', hydrogen sulfate [HSO4]", hydrogen sulfite [HSO3]', dihydrogen phosphate [ H2PO4]', hydrogen phosphate [HPO4]2“ and acetate [MeCO2], with the proviso that if the anion is acetate, then the composition comprises 10 to 40% v/v water. Preferably, the anion is selected from methyl sulfate [MeSO4]~, hydrogen sulfate [HSO4]~, methanesulfonate [MeSO3]' and acetate [MeCO2].
The lignin in lignocellulosic biomass is soluble in the ionic liquid at the treatment temperature, but the cellulose is not, so a pulp comprising the cellulose is produced. Other components such as hemicellulose may preferentially dissolve in the ionic liquid as well.
The cation is preferably a protic cation ion, that is, it is capable of donating an H+ (proton).
The cation ion can be an ammonium or a phosphonium derivative. These cations have the general formula
where X is N or P; and
A1 through A4 are each independently selected from H, an aliphatic, C3_5 carbocycle, C6_10 aryl, alkylaryl, and heteroaryl.
The term "aliphatic" as used herein refers to a branched or straight hydrocarbon chain that is completely saturated or contains one or more non-saturation units. Thus, aliphatic can be alkyl, alkenyl or alkynyl, which preferably has 1 to 12 carbon atoms, preferably up to 6 carbon atoms or more preferably up to 4 carbon atoms. The aliphatic can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms.
The term "alkyl" as used herein is typically a branched or linear alkyl group or chemical moiety containing from 1 to 20 carbon atoms, such as 11, 12, 13, 14, 15, 16, 17, 18 or 19 carbon atoms. Preferably, the alkyl group or chemical moiety contains 1 to 10 carbon atoms, i.e. 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms such as a C1-4 alkyl or a C1-4 alkyl group. -6 or chemical moiety, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl and t-butyl, n-pentyl, methylbutyl, dimethylpropyl, n-hexyl, 2-methylpentyl, 3 - methylpentyl, 2,3-dimethylbutyl and 2,2-dimethylbutyl.
The term "carbocycle" as used herein refers to a saturated or partially unsaturated cyclic group having 3 to 6 ring carbon atoms, i.e., 3, 4, 5 or 6 carbon atoms. A carbocycle is preferably a "cycloalkyl", which as used herein refers to a fully saturated cyclic hydrocarbon group. Preferably, a cycloalkyl group is a C3-C' cycloalkyl group.
The term "C6-C10 cycloaryl" used herein means an aryl group which is made up of 6, 7, 8, 9 or 10 carbon atoms and includes condensed ring groups such as monocyclic ring groups or bicyclic ring groups and similar. Specifically, examples of "C6-C10 cycloaryl" include phenyl group, indenyl group, naphthyl group or azulenyl group and the like. It should be noted that condensed rings such as indane and tetrahydro naphthalene are also included in the aryl group.
The term "alkylaryl" as used herein refers to an alkyl group as defined above substituted by an aryl as defined above. The alkyl component of an "alkylaryl" group can be substituted with any one or more of the substituents listed above for an aliphatic group and the heteroaryl component of an "alkylaryl" or "alkylheteroaryl" group can be substituted with any one or more of the substituents listed above for aryl and carbocycle groups. Preferably, alkylaryl is a benzyl.
The term "heteroaryl" as used herein refers to a monocyclic or bicyclic aromatic ring system having from 5 to 10 ring atoms, i.e., 5, 6, 7, 8, 9 or 10 ring atoms, in that at least one ring atom is a heteroatom selected from 0, N or S.
A heteroaryl or carbocycle group as referred to herein may be substituted by one or more substituents independently selected from the group consisting of halo, lower alkyl, -NH 2 , -NO 2 , -OH -COOH or -CN.
The term "halogen atom" or "halo" used herein means a fluorine atom, a chlorine atom, a bromine atom, an iodine atom and the like, preferably a fluorine atom or a chlorine atom and more preferably a fluorine atom.
The cation may also contain a nitrogen-containing heterocyclic chemical moiety which, as used herein, refers to mono- or bicyclic ring systems that include a nitrogen atom and optionally one or more heteroatoms selected from N, S and 0. Ring systems contain 5 to 9 members, preferably 5 or 6 members for monocyclic groups, and 9 or 10 members for bicyclic groups. The rings can be aromatic, partially saturated, and thus include both a "heteroalicyclic" group, which means a non-aromatic heterocyclic, and a "heteroaryl" group, which means an aromatic heterocyclic. The cation is preferably selected from

wherein R and R are independently a C1-6 alkyl or a C1-6 alkoxyalkyl group, and R3, R4, R5, R6, R7, R8 and R9, when present, are independently H, a C1-6 alkyl, C1-6 alkoxyalkyl group. 6, or C2-6 alkoxy group. Preferably R1 and R2 are C1-4 alkyl, one is methyl and R3 to R9 (R3, R4, R5, R6, R7, R8 and R9), when present, are H. Preferably , the cation ring is imidazolium or pyridinium.
"C2-6 Alkoxy" refers to the C1-5 alkyl group above bonded to an oxygen that is also bonded to the cation ring. A "C2-6 alkoxyalkyl group" refers to an alkyl containing an ether group, with the general formula XOY where X and Y are each independently a C1-5 alkyl and the total number of carbon atoms is between 2 and 6 for example 2, 3, 4, 5 or 6.
As used herein, the term "alkenyl" refers to a branched or linear alkenyl group or chemical moiety that contains from 2 to 20 carbon atoms, such as 11, 12, 13, 14, 15, 16, 17, 18 or 19 carbon atoms. Preferably, the alkyl group or chemical moiety contains 2 to 10 carbon atoms, i.e. 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms such as C2-4 alkenyl or a C2-6 alkenyl group or chemical moiety. , for example, ethenyl, 2-propenyl, 1-propenyl.
Preferably, the cation ion is selected from 1-butyl-3-methylimidazolium [CiC1imp, 1-ethyl-3-methylimidazolium [C2C1im]+, 1-methylimidazolium [CiHim]+ and 1-butylimidazolium [C4Him]+.
Preferred ionic liquids for use in the invention are 1-butyl-3-methylimidazolium methyl sulfate [C4C1im] [MeSO4], 1-butyl-3-methylimidazolium hydrogen sulfate [C4C1im] [HSO4], 1-butyl methanesulfonate -3-methylimidazolium [C4C1im] [MeSCq], 1-butylimidazolium hydrogen sulfate [C4Him] [HSO4], and 1-ethyl-3-methylimidazolium acetate [C2C1im] [MeCO2].
Ionic liquids can be prepared by methods known to those skilled in the art or obtained commercially.
It has surprisingly been found that the yield in the saccharification step can be improved if the pretreatment composition comprises water. Therefore, in a preferred embodiment the composition comprises IL and 10 to 40% v/v water. Preferably, the composition comprises 20 to 30% v/v water.
It was also observed that the presence of an excess of acid improves the yield of glucose and hemicellulose. Therefore, in a preferred embodiment the composition additionally comprises 0.01 to 20% v/v acid, preferably 1 to 5% v/v acid. The addition of a small amount of acid significantly speeds up the pretreatment process, while other variables such as water content and temperature are kept constant. The acid can be selected from any known strong acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, perchloric acid and hydrobromic acid. Preferably, the acid is sulfuric or phosphoric acid.
The ionic liquids of the present invention dissolve lignin in biomass, but unlike the ILs described previously, these do not dissolve cellulose. Most of the cellulose remains solid, preferably at least 90%, more preferably 95%. It can easily be removed from the liquid phase mechanically, for example by filtration. The separated pulp can then be washed and used in the saccharification process. This removes the need for a separate precipitation step to obtain the cellulose once the biomass has been treated. Thus, in a preferred embodiment, the method of the invention further comprises the step of separating the ionic liquid from the pulp produced.
In a preferred embodiment, the pulp is washed with an organic solvent miscible with the ionic liquid. The separation efficiency and recovery of the ionic liquid can be improved by washing the pulp with an organic solvent that is miscible with the ionic liquid. The organic solvent is removed before or potentially after the lignin is precipitated. Examples of suitable organic solvents include aliphatic alcohols such as methanol and ethanol.
It is possible to precipitate the dissolved lignin in IL compositions. Therefore, in another preferred embodiment, the method further comprises (c) adding an anti-solvent to the ionic liquid that has been separated from the pulp to precipitate the dissolved lignin; and (d) separating the precipitated solid from the antisolvent/ionic liquid.
As used herein, an "antisolvent" is a liquid that causes lignin to precipitate out of the ionic liquid that contains the solubilized lignin produced in step (a). The anti-solvent is preferably water. The ionic liquid can be recovered by removing the anti-solvent, for example, by evaporation. The resulting ionic liquid can be recycled to be used again in the method. Thus, in another preferred embodiment the method further comprises (e) removing the antisolvent from the ionic liquid obtained in (d). As the presence of some water improves yield, less energy is needed to dry the IL.
The cellulose pulp obtained from the method of the invention can be used to undergo saccharification to obtain glucose. This can be used in the fermentation process to obtain biofuel. Thus, in a second aspect, the invention provides a process for preparing glucose from a lignocellulose biomass which comprises subjecting a cellulose pulp obtainable by suitable methods of the invention to enzymatic hydrolysis. In a further aspect, the invention provides glucose obtained by such hydrolysis.
Suitable enzymes for use in the process include commercially available preparations of cellulases such as T. reseei cellulase and Novozyme 188 cellobiase which also contain hemicellulolytic activity. Other useful enzymes include esterases, or acetyl esterases or feruloyl esterases, which adhere substituents that are esterified to hemicellulose. The process is preferably carried out in an aqueous medium at a pH suitable for the enzymes. Conditions can be optimized in relation to pH, temperature and medium used depending on the enzyme mixture required.
Such methods are well known to those skilled in the art. The process is preferably carried out in accordance with "Enzymatic saccharification of lignocellulosic biomass" (NREL/TP-510-42629), grant date 03/21/2008. In a further aspect, the invention relates to lignin obtained by suitable methods as described herein.
The invention will now be described in the following without limitation examples with reference to the following Figures:
Figure 1 shows the absorption of ionic liquids in Miscanthus chips at 80 °C.
Figure 2 shows images of Miscanthus wood dissolved in wet [C4CimJ [MeSO4] using light transmission microscopy. Left: Outer part with fiber cells; right: parenchyma cells. The magnification was 10 times.
Figure 3 shows crushed Miscanthus after pretreatment with [C4C1im] [MeSO4]. Left: pretreated at 120 °C for 6 hours with pure ionic liquid. Right: pretreated at 120°C for 22 hours with 80/20% v/v ionic liquid/water after washing.
Figure 4 shows the experimental setup for pretreatment of crushed Miscanthus with [C4C1im] [MeSO4]/water mixtures.
Figure 5 shows saccharification yields after 22 hour/120°C pretreatment of Miscanthus ground with [C4Clim][MeSO4] water mixtures. The saccharification proceeded for 48 hours. Yields are based on oven dried sample weight prior to pretreatment.
Figure 6 shows the composition of untreated Miscanthus flour used in this study.
Figure 7 shows saccharification yields of ground Miscanthus after pretreatment with water mixtures with [C4C1im] [HSO4] and [C4C1im] [MeSO4]. The conditions were 120 °C, 13 hours and 22 hours of pretreatment time, respectively. Yields are based on oven dried sample weight prior to pretreatment.
Figure 8 shows the percentage of Miscanthus pulp recovered after pretreatment with various water mixtures with [C4C1im] [HSO4] at 120 °C for 13 hours as well as the glucose yield and total sugar recovery after saccharification.
Figure 9 shows the recovery of lignin after pretreatment with water mixtures with [C4C1im] [HSO4] at 120 °C for 13 hours. Yields are based on oven dried sample weight prior to pretreatment.
Figure 10 shows the mass loss time course for 80% v/v of pre-treated water and ionic liquid mixtures at 120 °C. The ionic liquids were [C4C1im] [MeSOJ and [CziCilm] [HSO4] .
Figure 11 shows the time course study of crushed saccharification yields and Miscanthus lignin yields. Pretreatment with mixtures of [C4Clim][MeS04]80% and [C4C1im][HSO4]80% was carried out at 120°C for up to 26 or 22 hours. Yields are based on oven dried sample weight prior to pretreatment.
Figure 12 shows the effect of pretreatment with [C4C1im][MeSO4]80% and [C4C1im][HSO4]80% at 120°C on the composition of Miscanthus flour for various periods of time.
Figure 13 shows lignin and hemicellulose content and glucose yield after hydrolysis of untreated Miscanthus enzyme and ionic liquid treated Miscanthus pulp. The saccharification yield is based on the oven-dried sample weight prior to pre-treatment.
Figure 14 shows the impact of pretreatment with 80/20% v/v ionic liquid and water mixtures on the Miscanthus composition. Anions are ordered according to their hydrogen bond acceptor strength.
Figure 15 shows qualitative correlation between lignin and hemicellulose content and cellulose digestibility.
Figure 16 shows the saccharification yield after pretreatment with mixtures of ionic liquid and water of 80/20% v/v at 120 °C for 22 hours. Yields were determined after 96 hours. Yields are based on oven dried sample weight prior to pretreatment.
Figure 17 shows concentrations of solubilized sugars and sugar dehydration products pre-treatment liquors. Yields are based on oven dried sample weight prior to pretreatment.
Figure 18 shows the influence of anion on delignification and lignin recovery after precipitation. The crushed Miscanthus was pretreated with mixtures of ionic liquid and water at 120 °C for 22 hours. The ionic liquid cation was [C4C1im]+, except for [CsC1im] [MeCCM . Yields are based on oven dried sample weight prior to pretreatment. The original lignin content was 26.5%.
Figure 19 shows the composition of crushed Miscanthus, willow and pine before and after pretreatment with 80/20% v/v [C4C1im] [HSO4] water mixture.
Figure 20 shows the effect of pre-treatment of different types of lignocellulosic biomass with water mixture with 80/20% v/v [^C1im] [MeCO2] and 80/20% v/v [C4C1im] [HSO4] in composition.
Figure 21 shows the glucose yield after pretreatment with 80/20% v/v ionic liquid and water mixtures and 96 hours of saccharification of the resulting pulp. Yields are based on the weight of the oven dried sample prior to pretreatment.
Figure 22 shows delignification and lignin recovery after pretreatment with 80/20% v/v ionic liquid and water mixtures. The ionic liquids were [C4C1im][HSO4] and [C2C1im] [MeCO2] . No replica was obtained. Yield is displayed as percentage of lignin in untreated biomass.
Figure 23 shows the time course of the saccharification of Miscanthus and willow chips. Yields are based on oven dried sample weight prior to pretreatment.
Figure 24 shows the sugar yields obtained from Miscanthus pulp after pretreatment with water mixtures with [C4C1im] [MeSO4] or [C4C1im] [HSO4] at 120 °C. The pretreatment with [C4C1im] [MeSO4] was carried out for 22 hours, while the treatment with [C4C1im] [HSO4] lasted 13 hours, and the saccharification 96 hours. Yields are based on the glucan and hemicellulose content of the untreated biomass.
Figure 25 shows the ratio of [MeSO4]' anions to the ionic liquid cations in the recycled ionic liquid after Miscanthus pretreatment (detected by 1H-NMR), with the remaining anions being HSO4]~.
Figure 26 shows the glucose and hemicellulose yields after enzymatic hydrolysis of Miscanthus pretreated with [C4C1im] [MeS04]so% and [C4C1im][HS04]80% at 120°C. Yields are based on the glucan and hemicellulose content of the untreated biomass.
Figure 27 shows the composition of Miscanthus before and after pretreatment with [C4C1imMHSO4] 8o% and [C4C1im] [MeS04]8o% at 120°C for 2 hours or 22 hours.
Figure 28 shows the amount of glucose and hemicellulose monomers found in 80% [C4C1im] [HSO4] and 80% [C4C1im] [MeSO4] 80% liquors during pretreatment at 120 °C. Yields are based on the glucan and hemicellulose content of the untreated biomass.
Figure 29 shows the solubilized carbohydrates (monomers only) and the fraction converted to furfural after pretreatment with [C^im][HSO4]80% and [C4C1im][MeSO4]80% liquors. Yields are based on the glucan and hemicellulose content of the untreated biomass.
Figure 30 shows the precipitate yield (in relation to the Klason-lignin content of the untreated biomass) after Miscanthus pretreatment with water mixtures with [C4C1im] [HSO4] at 120 °C for 13 hours.
Figure 31 shows IR spectra of lignin isolated from Miscanthus treated with [C4C1im] [HSO4] 80% for 22 hours (black) and alkaline lignin (from Aldrich, red).
Figure 32 shows IR spectra of lignin isolated from Miscanthus treated with [C4Him][HSO4 ] 80% for 20 hours (blue) and alkaline lignin (from Aldrich, red).
Figure 33 shows IR spectra of lignin isolated from pine treated with [C4C1im] [HSO4]80% for 22 hours (blue) and alkaline lignin (from Aldrich, red).
Figure 34 shows the time course of lignin recovery after Miscanthus pretreatment with [C4C1im][MeS04]so% and [C4C].im][HS04]80% at 120°C. Lignin was isolated from the liquor by precipitation with water.
Figure 35 shows the enzymatic saccharification yield obtained from Miscanthus after pretreatment with [C4Him][HSO4]95% and [C4Him][HSO4]80%. The saccharification was performed for 96 hours. Yields are based on the glucan and hemicellulose content of the untreated biomass.
Figure 36 shows the composition of Miscanthus after pretreatment with [C4Him][HSO4] water mixtures at 120°C.
Figure 37 shows lignin removal and Miscanthus precipitate yield with water mixtures with [C4Him][HSO4] at 120°C. Yields are based on the lignin content of the untreated biomass.
Figure 38 shows the effect of the anion of the ionic liquid on the loss of mass and composition of the pulp recovered after the pretreatment of Miscanthus with 80% ionic liquid and water mixtures at 120 °C for 22 hours. The data is sorted (left to right) according to the basicity of the hydrogen bonding of the ionic liquid, which is, in the case of 1,3-dialkylimidazolium ionic liquids, a property of the anion.
Figure 39 shows the impact of ionic liquid anion on glucose and hemicellulose yields after enzymatic saccharification of Miscanthus pulp pretreated with mixtures of ionic liquid and water of 80/20% by volume at 120 °C for 22 hours. Yields are based on the glucan and hemicellulose content of the untreated biomass.
Figure 40 shows the effect of anion on lignin removal and precipitate yield after Miscanthus pretreatment with 80/20% by volume mixtures of ionic liquid and water. The higher yield of liquors containing [HSO4]’ (compared to Figure 30 and Figure 34) is attributed to the greater amount of ionic liquid and biomass used in this experiment. Values are relative to the lignin content of untreated biomass.
Figure 41 shows furfurals and sugar monomers solubilized in liquors containing 80% by volume of 1,3-dialkylimidazolium ionic liquids with various anions and 20% by volume of water after Miscanthus treatment at 120 °C for 22 hours. Yields are based on the glucan and hemicellulose content of the untreated biomass.
Figure 42 shows the composition of willow (3 bar graphs on the left) and pine (on the right) before and after pretreatment with [C4C1im] [HSO4]80% and [CzjC1im] [MeC02]8o« for 22 hours at 120°C.
Figure 43 shows the lignocellulosic enzymatic saccharification raw materials after pretreatment with [C4C1im] [HSO4] 8Q% or [C2C1im][MeCO2 ] so% for 22 hours at 120 °C. Yields are based on the glucan and hemicellulose content of the untreated biomass.
Figure 44 shows glucose yields after 96 hours of enzymatic saccharification following treatment with [C4Him] [HSO4] where the relative concentrations of acid and base were varied.
Figure 45 shows the yields of xylose, mannose and galactose after 96 hours of enzymatic saccharification following treatment with [C4Him] [HSO4] where the relative concentrations of acid and base were varied.
Figure 46 shows the changes in glucose yields with time during enzymatic saccharification following treatment with [C4Him] [HSO4] where the relative concentrations of acid and base were varied.
Figure 47 shows the changes in xylose yields with time during enzymatic saccharification following treatment with [C4Him] [HSO4] where the relative concentrations of acid and base were varied.
Figure 48 shows precipitate yield versus lignin content in untreated biomass following treatment with [C4Him] [HSO4] where the relative concentrations of acid and base were varied.
Figure 49 shows the energy savings of chipping wood for various pretreatment methods over dry wood.
Figure 50 shows the yields of enzyme-treated wood powder sugar crushed from wood chips pre-treated in different ways as a percentage of the sample weight when dry. Example 1 biomass
The lignocellulosic biomass was pine sapwood (Pinus sylvestris, SCOES variety) from West Sussex, mixed dehulled willow stalks (Salix sp. TORA variety) and Miscanthus X giganteus. All biomass was stored air-dried at room temperature, crushed and sieved (0.18 to 0.85 mm mesh) before use. Giant Miscanthus internodes (0 = 11 mm) were cut into 5 mm high disks to obtain Miscanthus wood chips. The moisture content of untreated lignocellulose was 8.0% (Miscanthus), 8.9% (Pine) and 7.6% (Willow) based on oven drying weight. Biomass was stored in plastic bags at room temperature.
Synthesis of 1-butyl-3-methylimidazolium hydrogen sulfate [C4C1im] [HSO4]170.67 g (682 mmoles) of [C4C1im] [MeSO4] (BASF grade) were mixed with 25 ml of distilled water in a bottom flask round with a Graham condenser followed by a horizontal Liebig condenser. The mixture was heated to reflux. The Graham condenser was cooled to 65°C using a temperature-controlled circulator. The Liebig condenser was cooled with room temperature water and condensed methanol.
The water was refluxed for 24 hours. Most of the water was removed with the rotary evaporator and the ionic liquid vacuum dried at 45 °C. The yield was 98.1% by weight.
Synthesis of 1-butyl-3-methylimidazolium methanesulfonate [C4C1im] [MeSCh] 50.0 ml (0.380 mol) of 1-ethylimidazole and 42 ml (0.495 mol) dimethyl carbonate and 100 ml of methanol were placed in a 300 stainless steel pressure reactor with Teflon coating and stir bar. The mixture was heated at 140 °C for 24 hours, then a yellowish solution containing the ionic product liquid was obtained (conversion: 98%). 33.73 g (351 mmoles) of pure methanesulfonic acid was added to a stirred crude product mixture containing 351 mmoles of 1-butyl-3-methylimidazolium methyl carbonate. Vigorous gas formation was observed. The ionic liquid was vacuum dried until crystallization was observed. The product was recrystallized twice from acetonitrile, washed with ethyl acetate and dried under reduced pressure. The product was a white solid. Yield: 70%
Liquid absorption in Miscanthus flakes
Miscanthus wood chips were coated with ionic liquid when vacuumed to encourage even soaking. Ionic liquids were prepared as described above, but are also commercially available, for example, from Sigma-Aldrich, BASF. The ionic liquids were dried to a water content of <0.3 wt%, with the exception of [C4Him] [HSO4] which had a water content of 1 wt%.
The samples were incubated in snap-cap glass vials with a plastic lid for 20 days and then heated to 80 °C for a few hours. The absorption was calculated according to Eq. 1, where m8o "c is the mass after incubation, mps the mass after pre-soaking at room temperature, and pa density at 25 °C.
Determination of moisture content
To determine the moisture content, 100 to 200 mg of air-dried biomass was wrapped in aluminum foil of known weight and dried in an oven at 105 °C overnight. The samples were transferred to a desiccator with activated silica and the weight determined after 5 minutes. The moisture content was calculated according to Equation 2. The moisture content of air-dried biomass (both treated and untreated) was in the range of 5 to 12%.

Softening of Miscanthus chips in [C4C1im] [MeSO4]
During test measurements that were designed to find ionic liquids that had swelling effects on biomass, an unusual effect of ionic liquid [C4C1im] [MeSO4] on Miscanthus chips was observed when the samples were heated to 80 °C. Rather than swelling, the chips shrank, in addition, they absorbed significantly more liquid than chips immersed in other ionic liquids or water (Figure 1).
The chips immersed in this ionic liquid became soft and even visibly dissolved in the ionic liquid upon agitation. The apparent solution was examined under a microscope and revealed the presence of fiber cells and separate parenchyma (Figure 2).
It looked like the middle lamella, the glue between the cell walls, was affected by the ionic liquid. The middle coverslip in grams consists of hemicelluloses, including pectins, and, in mature tissues, a large proportion of lignin (>50% in fully lignified wood). Therefore, it is possible that one of the major components or both are solubilized by the ionic liquid.
These results suggest that the surface area of Miscanthus chips could be vastly improved with the use of a moderate treatment and also that this ionic liquid could have the ability to improve lignocellulose digestibility by solubilizing lignin and hemicelluloses.
Pre-treatment of lignocellulosic biomass and pulp isolation
In order to ensure homogeneous samples, the Miscanthus stems were ground and particles from 0.18 to 0.85 mm in width were used. Biomass was harvested in winter and air dried. Pre-treatment was carried out in wide-mouth culture flasks with a screw cap and Teflon coating. The bottles were chosen because they guarantee to withstand temperatures up to 120 °C and the Teflon coating ensures chemical resistance as well as a watertight closure. Stirring was not used because the oven could not support stirring. In order to minimize the use of ionic liquid, small batches of 0.5 g of kiln-dried biomass were used, unless otherwise stated. At that, 5 ml of pretreatment solvent was added. This was enough to cover the crushed Miscanthus biomass without compressing it.
After the pretreatment was ended, the samples were cooled to room temperature and mixed with 10 ml of methanol. The suspension was filtered through filter papers (Whatman 541 or equivalent, hardened) after a few hours. The supernatant was set aside for determination of lignin yield and analysis of furfural content. Solids were washed with methanol from a wash bottle and incubated with 10 ml of fresh methanol overnight. The suspension was filtered again, rinsed with methanol from a wash bottle and the solids were dried on filter paper on a laboratory bench overnight. The weight after air drying was recorded and the samples transferred to airtight bags that could be sealed more than once. Moisture content was determined as described above. In order to obtain enough material for compositional analysis, the pretreatment experiments were scaled up 2 to 3 times. Lignin isolation
The supernatant obtained after pretreatment was dried in a moderate vacuum at 40°C to remove the organic wash solvent using a 12-position carousel with glass tubes (Radleys) equipped with a hot plate and stirring bars. rare earth metal. 10 ml of water was added to precipitate the lignin as a fine suspension. The precipitate was washed three times with distilled water, air-dried and subsequently dried in a high vacuum at room temperature. Yield was determined by weighing. Precipitates were stored in glass vials with plastic caps.
The precipitate yield was calculated based on the Klason lignin content of untreated biomass using the equation below. Part of the precipitate may be pseudo-lignin.

The precipitate was characterized by IR spectroscopy using a Spectrum 100 IR machine (Perkin-Elmer) equipped with a universal diamond crystal ATR sampling accessory.
It was surprising to find that treatment of Miscanthus flour with pure [C4C1im][MeSO4] at 120°C resulted in a solid ionic liquid wood slurry (left of Figure 3). The saccharification yield from this paste was low. The addition of water, however, allowed the separation of the ionic liquid and a Miscanthus pulp (right in Figure 3) even after long periods of heating (24 hours) . The liquid turned almost black during pretreatment, but after separating the liquid and solid fraction, a beige pulp was obtained. In preliminary experiments, a very high digestibility was observed. Example 2 saccharification
Enzymatic saccharification was performed according to LAP's "Enzymatic saccharification of lignocellulosic biomass" (NREL/TP-510-42629), grant date 03/21/2008. 150 mg of pre-treated and untreated air-dried sample was used by saccharification. When a pretreatment condition was replicated twice or three times, saccharification was performed only once per sample. If the pretreatment condition was not replicated, saccharification was performed twice. The enzymes were cellulase T.reseei and cellobiase Novozyme 188 which also contain hemicellulolytic activity and can therefore hydrolyze xylan (both from Sigma-Aldrich). Glucose and hemicellulose yields were calculated based on the glucose and hemicellulose content of the untreated biomass, respectively.
compositional analysis
The compositional analysis (lignin, carbohydrates, ash) was performed according to laboratory analytical procedure (LAP) "Determination of structural carbohydrates and lignin in biomass" (NREL/TP-510-42618), grant date 25/04/2008 . No replicas were performed.
Untreated willow and pine biomass extracts were removed by a one-step automated solvent extraction with 95% ethanol using an ASE 300 type accelerated solvent extractor (Dionex) according to "Determination of extractives" of LAP (NREL/TP-510-42619), grant date 07/17/2005. Untreated Miscanthus extracts were removed by a two-step solvent extraction using deionized water and subsequently 95% ethanol according to the same LAP.
HPLC analysis of glucose sugars and hemicellulose was performed on an Agilent 1200 system equipped with an Aminex HPX-87P (Biorad) type column, an ash removal column and a Carbo-P type pre-column. The mobile phase was deionized water. The column temperature was adjusted to 80°C and the flow rate was 0.6 ml/min. Carbohydrate, Klason lignin, ash and extracts (where applicable) content was expressed as a fraction of the sum (normalized to 100%).
Qualification of furfurals and solubilized sugars 200 μl of pretreatment liquor was mixed with 600 μl of deionized water in a 1.5 ml plastic cup, vortexed and centrifuged with a tabletop centrifuge (Biofuge 13, Heraeus) at full speed for 10 minutes. The supernatant was transferred to a clean beaker and centrifuged for 10 minutes. The supernatant was transferred to HPLC sample vials and analyzed on a Jasco HPLC system equipped with an Aminex HPX-87H type column (Biorad) using a 10 mM sulfuric acid mobile phase. Column oven temperature was 55 °C, flow rate 0.6 ml/min and acquisition time 55 minutes. Standard concentrations of 2-furaldehyde (furfural) and concentrations (HMF) of 5-(hydroxymethyl)-2-furaldehyde were prepared in deionized water at the concentration of 0.01, 0.02, 0.1, 0.2 and 0.4 mg/ml. The standards for carbohydrates were 0.1, 1, 2 and 4 mg/ml. The FHPLC(S) factor was obtained from the respective calibration curve. The relative yield of furfurals and solubilized sugar monomers, % by weight(S), was calculated using the Equation. 3. Molecular mass transform factor FT was 1.37 for furfural, 1.28 for HMF, 0.91 for glucose and 0.88 for hemicellulose sugars. The Fc mass fraction factor was 0.243 for hemicellulose and furfural sugars and 0.436 for glucose and HMF.

AHPLC •' HPLC peak area, FHPLC(S): HPLC calibration factor for substance S, FD: dilution factor, VPL: pretreatment liquor volume in ml, mbiomass: biomass (weight after oven drying ) in mg, Fc: fraction of glucan or hemicellulose sugars in untreated biomass as determined by compositional analysis, FT (S): transformation factor count for molecular mass differences between starting material and product.
Sacharification yields after pretreatment with water mixtures with 1-butyl-3-methylimidazolium methyl sulfate
The influence of water content on digestibility was investigated. This confirmed that water was an important factor in the pretreatment with this ionic liquid. The water content ranged between 2% and 80 v/v%. Composition refers to the amount of pure components added, thus mixing effects on volume are neglected. The pretreatment time was set at 22 hours to allow the pretreatment to be completed despite the lack of agitation. A set of control samples were treated with pure deionized water. The samples were processed according to Figure 4. The pulp that could be isolated after the pre-treatment was subjected to saccharification using cellulase and a mixture of hemicellulase (Novozyme 188) in a buffer.
Yields after enzymatic digestion are shown in Figure 5. Sugar yields vary widely. The highest glucose yield was observed in mixtures containing 10 to 40% water. The higher or lower water content reduced the glucose yield. The hemicellulose sugar xylose could be quantified as well. Yield was much lower but followed a similar trend. Other hemicellulose sugars, such as arabinose, mannose and galactose, were below the detection level.
The saccharification yields can be compared with the composition of the original biomass. The amount of glucose in air-dried biomass was 43.6%. A glucose yield between 37.7 and 40.2% by weight after saccharification means that 86 to 92% of the original glucose was recovered in the solid fraction and could be hydrolyzed by cellulases within 48 hours. These yields are very high and in stark contrast to the yields of water-treated controls, for which the glucose yield was marginal.
The hemicellulose yields for the pretreated samples are generally low compared to the hemicellulose content of the untreated biomass. A yield of 5.3 to 6.4% means that only 29 to 35% of the hemicellulose was recovered during saccharification. It's still higher than the recovery of controls. The quasi-qualitative glucose and good hemicellulose recovery is also reflected by the pulp liquefaction during saccharification. Although the untreated Miscanthus hardly changed its appearance during saccharification, the treated material was almost completely solubilized. The leftover was a fine bulky brown powder. It was probably comprised of lignin that precipitated during washing, as will be explained below.
Observations that water was needed for pre-treatment are good news. Water is ubiquitous; it is present not only in biomass, but also the ionic liquid can contain a lot of absorbed moisture. In terms of a process, this means that less energy needs to be expended on drying the biomass and ionic liquid. The results also suggest that a relatively wide range of water concentrations are acceptable, so a change in water content will not necessarily mean a sharp drop in yield. The presence of water improves the process as yield decreases if water content is too low. Example 3
Influence of washing solvent on biomass fractionation and recovery
The previous examples indicate that the cellulose was significantly enriched in the pulp, implying that some of the other components were removed. The low yield of hemicellulose suggests that a significant fraction of the hemicellulose has been solubilized. However, the dark color of the pretreatment liquor also suggests that some of the lignin was solubilized in the ionic liquid. Natural lignin is not colored, however, when lignin is chemically modified it usually takes on a dark color, as seen with commercially available lignin preparations.
During the wash phase, the previously clear liquor became hazy upon dilution with distilled water and a fine precipitate began to settle. Likely it was lignin. The fractionation of lignin from the carbohydrate fraction was part of the Organosolv pre-treatment, from which Organosolv lignin can be obtained. Example 4
Composition of recycled [C4C1im][MeSO4]
The ionic liquid liquor obtained after lignin precipitation was vacuum dried at 40 °C. A sample of the dry ionic liquid was subjected to mass spectrometry. Part of the recovered ionic liquid was dissolved in DMSO-d6 and a 1H NMR spectrum was recorded. The peaks of the methyl group at 3.40 ppm and the C-2 ring hydrogen were used to determine the anion to cation ratio. The pre-treatment was carried out in capped containers, so it is reasonable to assume that the water content did not change substantially during the pre-treatment. The water introduced by the ionic liquid and the air-dry biomass was taken into account, but not the water consumed in hydrolytic reactions.
The spectrum suggests that the recovered ionic liquid appears to be free of degradation products. However, the integral peak for the methyl group on the anion was significantly decreased. A spectrum of the mixture revealed that hydrogen sulphate anions, [HSO4]“, were present along with the methyl sulphate anions. The bond between the methyl group and the rest of the anions is an ester bond, a methoxy sulfate ester and thus, like all esters, subjected to hydrolysis in the presence of water.
Therefore, the loss of the methyl group signal is attributed to a chemical balance between the methyl sulfate ester and the hydrolyzed form (Scheme 1).

Scheme 1: The balance between methyl sulfate and hydrogen sulfate at elevated temperatures in the presence of water
The pre-treatment experiments were carried out in capped containers, it is reasonable to assume that this is a closed system. Example 5
Pretreatment with water mixtures with 1-butyl-3-methylimidazolium hydrogen sulfate [C4C1im] [HSO4]
Pretreatment experiments were conducted with mixtures of water with 1-butyl-3-methylimidazolium hydrogen sulfate. If methyl sulfate anion and methanol were important, yields would be reduced. If hydrogen sulfate was important, yields should be as good as before. The main difference between hydrogen sulfate anion and anions is their acidity. The pKa of hydrogen sulfate is 1.99. This is more acidic than acetic acid (pKa=4.72) but less than hydrochloride acid (pKa=-7).
The saccharification yields for both glucose and xylose after pretreatment with [C4C1im] [HSO4] water mixtures were very similar to the yields observed for [C4C1im] [MeSO4] water mixtures (Figure 7). The glucose yield was high for the ionic liquid which contains 10 to 40% water. This was achieved in less time (13 hours) than previously used (22 hours).
This proves that the hydrogen sulfate anion paired with the 1-butyl-3-methylimidazolium cation is effective in the preparation of Miscanthus lignocellulose.
The recovery of Miscanthus pulp is shown in Figure 8. Although pure water only removed 8% of the matter, the removal by ionic liquid was much more pronounced. The mass loss after pretreatment with 60 to 90% ionic liquid was about 50%. The glucose yield and total sugar yield after saccharification of the recovered solid are also shown in Figure 8. It can be seen that the saccharification yield is higher the more lignin and hemicellulose have been solubilized. The Figure also suggests that cellulose which is the major source of glucose is enriched in the pulp.
The precipitation of lignin from [C^Cdim] [HSO4] was also investigated. Figure 9 shows the lignin yield after washing the biomass with methanol and precipitation with water. The lignin yield correlates well with the ionic liquid concentration. The higher the ionic liquid content, the more lignin can be isolated. Yield only moderately drops from 13.4% and 13.7% at 5% and 10% water content, to 9.5% at 60% water content. A gross drop in lignin yield was observed when the water content increased from 60% to 80%. This is in line with a biomass solubilization reduced by 80% water content (Figure 8). Example 6
Time course of ionic liquid pretreatment The effect of pretreatment of mixtures of ionic liquid and water containing [C4C1im] [HSO4] or [C4C1im] [MeSO4] which contains a mixture of anions [MeSO4]~ and [HSO4]~ under applied conditions, over time was investigated. The 80% ionic liquid pretreatment liquor was used as an example pretreatment liquor.
The gradually resolved recovery of biomass to methyl sulphate and hydrogen sulphate is shown in Figure 10. It can be seen that most of the biomass solubilization occurred within four hours after the start of the pretreatment, accounting for 90% of mass loss. The remaining 10% was solubilized within the next 18 to 20 hours. This was the case for both pretreatment liquors. Small differences were observed before the 4-hour point, where hydrogen sulphate is better at removing the soluble biomass fraction at 45 and 90 minutes.
The saccharification and corresponding lignin yields are shown in Figure 11. The glucose yield rose steeply in the first 8 hours and then leveled off the remaining time. The hemicellulose yield, on the other hand, reached a maximum yield of close to 7% in 8 hours and then declined to 5% at the end of the pretreatment.
Although most mass loss occurred within 4 hours, glucose yield was significantly improved by extending the pretreatment to 8 hours.
Lignin yield followed a similar trend, increasing rapidly in the first 8 hours and changing only slightly afterwards.
In conclusion, the data suggest that pretreatment does not need to be conducted for 22 hours or even 13 hours. 8 to 10 hours seems to be enough to obtain the maximum possible glucose yield. Prolonging the pretreatment does not appear to have a significant impact on the glucose yield, whereas the pulp hemicellulose yield decreases somewhat with more pretreatment time.
The significant mass loss coupled with a high glucose yield suggests that a strong enrichment of the cellulose fraction occurs. In order to confirm this, analysis of the Miscanthus composition before and during pre-treatment was performed. Crushed Miscanthus was pretreated for 2 hours with both [C4C1im] [HSO4] go% and [C4C1im] [MeSO4] 80%. A 2-hour incubation falls into the 'active' phase, when mass loss and yield of saccharification increases rapidly. Therefore, analysis of these samples provides a glimpse of compositional changes during this pretreatment phase.
Figure 12 shows that for 80% [C4C1im] [MeSCg], the 2-hour pretreatment resulted in 10% mass loss. The hemicellulose content was barely changed, while the lignin content was reduced to 70%. Mass loss was significantly greater after pretreatment with [C4C1im] [HSO4] 80%. As the Figure shows, this is due to a reduction in lignin content (by 44%) as well as the removal of sugars from hemicellulose. The hemicellulose content was halved in 2 hours. This can be explained by the acid-labile nature of hemicellulose. It is the fraction that is most susceptible to hydrolysis under acidic conditions. Hemicellulose solubilization is the main mode of action for dilute acid pretreatment and also occurs under acidic conditions of the Organosolv process.
The composition after 22 hours of pretreatment is also shown in Figure 12. The solid fraction after the long pretreatment consisted of only 46% of the original biomass. The largest fraction was glucose, which is made up of 85.2% of the solid residue. This is almost twice as concentrated as in the original biomass, which had a glucose content of 43.6%. Looks like some glucose was lost during pretreatment. It is possible that this is probably the glucose contained in hemicellulose, but also exposed parts of the amorphous regions in cellulose fibrils may have been hydrolyzed. However, 39.5% of the original biomass was recovered as glucose, which is 91% of the glucose fraction. The hemicellulose content of the 22-hour pulp was low, accounting for less than 9.4% of the solid fraction. This translates to an 80% removal of hemicellulose sugars (not glucose). A similar trend is seen in lignin. The lignin content after 22 hours was only 4.1%. This means that 93% of the original lignin has been solubilized.
The highly enriched cellulose contained in this pulp is very susceptible to enzymatic hydrolysis. The relationship between lignin and hemicellulose removal and glucose saccharification yield is depicted in Figure 13. A correlation between lignin removal by ionic liquid pretreatment and saccharification yields has been previously suggested. Example 7
The influence of anion on biomass composition and saccharification yields
The impact of water mixtures with [C4C1im] [MeSO4] and [G^C^im] [HSO4] was investigated and revealed that efficient lignocellulose pretreatment can be performed using these pretreatment solvents. To further assess which features make the ionic liquid effective, the influence of the anion was investigated. The anion effect of the ionic liquid has been a focus before and has proven to be an important factor in promoting cell wall swelling.
The condition that has been most thoroughly investigated so far, namely pretreatment with [C4C1im] [HS04] 80% at 120°C for 22 hours, was also selected for investigation of the anion effect. The anions used for comparison were acetate, chloride, methanesulfonate and trifluoromethanesulfonate. The effectiveness of the pretreatment could not be correlated with the basicity of the hydrogen bonding of the anion.
The impact on composition and mass loss is represented in Figure 14. The results were ordered according to the basicity of the hydrogen bonding of the respective anions, with high values on the left. It is clear that the anion [HSO4]~ caused the greatest mass loss, concomitant with a complete removal of lignin and hemicellulose.
The second most efficient pulping was observed in the anion [MeSO3]“. The hemicellulose content was reduced to similar levels, but the lignin content was significantly higher. A significant reduction in hemicellulose and lignin content was observed in the pretreatment with the ionic acetate liquid. However, pulping was significantly less efficient under these conditions. The effect of the chloride containing ionic liquid was surprisingly small. The least impact on composition was exerted by [C4C1im][OTf] (trifluoromethanesulfonate).
A correlation between hemicellulose and lignin removal and saccharification yields was found (Figure 15). The highest yields of glucose were obtained with mixtures of water with [C4C1im] [MeSO3] and [C4C1im] [HSO4] . Pretreatment with [C2C1im] [MeCO2] resulted in good glucose yields, but did not reach the same level of digestibility as methanesulfonate and hydrogensulfate based pretreatment solvents. Glucose yields from mixtures containing chloride and triflate ions were very low.
The hemicellulose yields behaved slightly differently (Figure 16). The yield was highest after treatment with [CsC1im] [MeCO2] (9.6%), intermediate after pretreatment with [C^im] [MeSO3] (6.0%) and lowest after treatment with [C4C1im] [HSO4] (3.3%). This appears to correlate with the acidity of the ionic liquid mixtures, which has a profound effect on the stability of the hemicellulose fraction.
The acetate anion can exert a buffering effect in an aqueous solution that limits the hydrolysis of glycosidic bonds. Methanesulphonate, as the base of a strong acid, cannot perform this function and further hydrolysis is observed. Hydrogen sulfate is even more acidic and will lower the pH to 2 or lower. This will not only assist in the hydrolysis of hemicellulose but encourage further reactions of sugar monomers with furfurals and possibly other degradation products.
The amounts of sugar monomers and furfurals found in the pretreatment liquor are shown in Figure 17. Pretreatment with [C2C1im] [MeCO2] resulted in a very small amount of monomers and virtually no furfural. A large fraction of hemicellulose is recovered in pulp (63% of all hemicellulose). The remaining cellulose was likely solubilized but oligomeric and could not be detected with the available HPLC configuration.
Substantial amounts of monomers and furfurals were found in the liquors containing [MeSCq]" and [HSO4]". 13.3% by weight and 12.1% by weight of the original biomass were detected as monomers or monomer dehydration products in both The distribution among the various products varied considerably, with monomeric hemicellulose being the prevalent fraction in the methanesulfonate liquor, while furfural was the largest fraction in the hydrogensulfate liquor.
The chloride and liquors containing [OTf]‘ both had an abundance of monomers and dehydration products, which is not surprising given the negligible fractionation and little mass loss they both achieved.
Lignin recovery was also determined for mixtures of ionic liquid and water of 80/20% by volume (Figure 18). The removal of lignin from the biomass (delignification) is also shown in this diagram.
Lignin recovery was better in [C4C1im [HSO4] 80% with a recovery of 64% of the original lignin, followed by 31% recovery of lignin from 80% [C4C1im[MeSO3] and 18% of all lignin from of [C2C1im [MeCCç] eo% • Example 8
The influence of the type of lignocellulose
Pretreatment with 80% [C4C1im[HSO4] at 120 °C for 22 hours was carried out in crushed willow and crushed pine. The impact on biomass composition is shown in Figure 19.
It shows that pretreatment has a similar effect on other types of biomass. In particular, the impact on willow was very similar compared to the effect on Miscanthus, with advanced solubilization of hemicellulose and lignin. The effect on the pine was less pronounced.
The various types of biomass were pretreated with 80% [C2C1im] [MeCO2] • The impact on biomass composition is shown in Figure 20. Less delignification and less hemicellulose removal was observed when the anion was acetate, regardless of the type of biomass. The general trend was, again, that pine flour was more recalcitrant to pretreatment. The [C2C1im] [MeC02]so% liquor had very little impact on the pine composition, with some lignin solubilization. There was also some loss of the glucose fraction during pretreatment with [C2C1im] [MeCO2]80%. This is attributed to the solubility of cellulose in the ionic acetate liquid. Although most of the cellulose was precipitated during washing the pulp with methanol, it was observed that some biomass remained dispersed in the diluted liquor and would only precipitate after further dilution.
The reduced activity in willow and pine compared to Miscanthus can be explained by their thicker cell walls and smaller pores. This will result in mass transfer limitation. Differences in the composition and natural abundance of chemical bonds in lignin or between lignin and hemicellulose could also be responsible.
The saccharification results reflect the compositional changes. The removal of lignin and hemicellulose coincided with a better digestibility of the cellulose fraction (Figure 21). The highest digestibility was obtained in the Miscanthus biomass pretreated with [C4C1im] [HSO4]80%. The second best yield was obtained in willow with the same pre-treatment liquor. The order of biomass anion combinations tested was Miscanthus [HSO4]~ > Willow [HSO4]~ > Miscanthus [MeCO2]~ > Willow 5 [MeCO2]~ > Pine [HSO4]~ > Pine [MeCO2]'. saccharification for pine were surprisingly low. Significant delignification (66%) and removal of hemicellulose (69%) was observed for pine pretreated with [C4C1im] [HS04]8o%< however, the glucose yield was only 30% of the theoretically possible yield. .
The lignin yields are shown in Figure 22, along with the delignification efficiencies. Delignification was generally higher [C4C1im] [HSO4]80% than with [C2C1im] [MeCO2] 80% • In addition, lignin 15 recovery appears to be better for [C4C1im] [HSO4]80% liquor.
Acidification of pretreatment liquor has been reported to improve lignin recovery. This may be the advantage of using an ionic liquid of hydrogen sulphate, as this ionic liquid is already acidic by itself.
The unsatisfactory delignification with 80% [C2C1im] [MeCO2] is probably due to the high water content. A negative correlation between water content and lignin removal with [C^Cj-im] [MeCO2] has already been demonstrated. Example 9
Pre-treatment of Miscanthus and Willow Chips
Both Miscanthus and willow seem to have very good substrates for pretreatment with [C4C1im] [HSO4]80%. Until now, the substrate was crushed biomass. A truly energy efficient pre-treatment process, however, will use coarsely chopped biomass, as crushing is an energy-intensive operation. Therefore, the pre-treatment efficiency of 80% [C4C1im] [HSO4] was tested on chip-size biomass. For Miscanthus chips, substantial disintegration of the less recalcitrant sap was observed. The structure was softened and was fragile under mechanical impact. There was also a fine dust that settled on the filter paper, which must be parts of the cell wall that have been dissociated from the chips.
Willow chips have also undergone significant changes through pre-treatment. In addition to the discoloration, the chips were significantly easier to break. Untreated pine chips require strong mechanical impact in order to break them, eg sawing or grinding, while pre-treated chips can be broken using a hard spatula. This suggests that pre-treatment of chips with ionic liquids prior to crushing can reduce the energy required for shredding.
Glucose yields obtained from chip-sized biomass are depicted in Figure 23. The enzyme hydrolysis of Miscanthus cellulose proceeded faster but stopped after 48 hours. Only 70% of the glucose was released on saccharification, compared to >85% obtained from crushed material. A visual check of the residue shows that what was left was recalcitrant outer ring material. The saccharification yield of willow chips was surprisingly good, reaching levels comparable to crushed biomass (84% glucose recovery compared to 81% for crushed willow). The thicker hardwood cell walls and reduced surface area were likely responsible for the slower saccharification. Example 10
The influence of water on the effectiveness of ionic liquid pretreatment.
A notation to indicate the amount of ionic liquid contained in the pretreatment solvent/liquor was developed. This involves a subscript that is added to the common ionic liquid notation that indicates the ionic liquid content in percent by volume (% by volume), where the remainder is water. An example is [C^im] [MeSO4] 80%, which is a mixture of 80% by volume of [C4C1im] [MeSO4] and 20% by volume of water. The conversions of % by volume to percent by weight (% by weight) and percent by mol (% by mol) were calculated and are listed in Table 1. When [C4C1im] [MeSO4] was allowed to balance with moisture in the laboratory air, a water content of 70,400 ppm or 7.0 wt% was measured (last entry of Table 1). Although the moisture content of air is variable, the measurement demonstrates the highly hygroscopic nature of this ionic liquid. Table 1: Concentration of ionic liquid in aqueous pretreatment liquors.
*These ionic liquids are solid at room temperature. Therefore the % by volume and % by weight were
The aim of this work is to investigate the effect of ionic liquid liquor composition in the pre-treatment. The recovery of solids, pulp composition, its enzymatic digestibility, the precipitation of a fraction containing lignin and the production of furfurals in the liquor were investigated. The application of an ionic liquid with a monoalkylated imidazolium cation was also examined. The pre-treatment of different raw materials was performed to assess their recalcitrance in relation to pre-treatment with mixtures of ionic liquid and water.
Fabric softening of Miscanthus flakes
In preliminary experiments, substantial disintegration of cross sections of Miscanthus immersed in the ionic liquid 1-butyl-3-methylimidazolium methyl sulfate, [C4C1im][MeSO4] when heated above 80 °C was observed. This encouraged us to investigate the application of this ionic liquid for biomass pretreatment. The use of [C^iim] [MeSO4] , dried to a water content below 0.3% by weight, resulted in the formation of a biomass-degraded ionic liquid composite that was not enzymatically digestible. In contrast, using a mixture of 80% by volume of ionic liquid and 20% by volume of water yielded a solid fraction that was separable from the (intensely colored) ionic liquid fraction and highly digestible. It was concluded that a certain amount of water was required for successful [C4C1im] [MeSO4] pretreatment. In the "dry" sample, 0.3% by weight of water was contaminated in the ionic liquid as residual moisture and 0.7% by weight was introduced with the air-dried biomass which contains 8% by weight of moisture, providing 1.1 % by weight or 15% by mole of water in total. This apparently was not enough to obtain an enzymatically digestible pulp. Example 11
Influence of water content on saccharification yield after pretreatment with ionic liquid with [C4C1im] [MeSOJ
A range of mixtures of ionic liquid and water were used for the Miscanthus pretreatment to explore the effect of water content in more detail. The effect of water on the enzymatic release of glucose and hemicellulose is shown in Figure 24. Yields are calculated based on the content of glucose and hemicellulose found in the untreated Miscanthus feedstock (on an oven-drying basis), which were 43 .6% by weight and 24.3% by weight, respectively. In preliminary experiments, it was shown that the only detectable hemicellulose sugar during saccharification was xylose.
The best saccharification yields were obtained after pretreatment with mixtures containing 60 to 90% by volume of ionic liquid. Pretreatment with [C4C1im] [MeS04]go% resulted in the release of 92% of the glucose originally contained in the biomass. Pretreatment with [C4C1im] [MeS04]8o% and [C4C1im] [MeSO4] 6o% resulted in 89% and 87% release based on the original glucan content. Glucose yields decreased when the ionic liquid content was higher or lower. The hemicellulose yield was significantly lower than the glucose yield, regardless of the mix composition; 24% of the hemicellulose sugars (based on the initial hemicellulose content) were released after pretreatment with [C4C1im] [MeSO4 ] 6o% • Similar yields were obtained with blends containing 40 to 90% by volume [C4C1im] [ MeSO4]. Example 12
Attempt to recycle [C4C1im] [MeSO4]
When trying to recycle the [C4C1imHMeSO4] , we observed that the anion of the ionic liquid was partially hydrolyzed. After recording a mass spectrum of the recovered ionic liquid, a high abundance of a negatively charged species at am/z=97 was detected, which was related to the hydrogen sulfate anion, [HSO4]'. This led to the conclusion that the ester bonds in methyl sulfate anions are hydrolytically unstable under the pretreatment conditions and mixtures of the ester and the hydrolyzed form are produced.

The extent of anion hydrolysis depended on the water content of the liquor (Figure 25). The more water that was present in the mixture, the greater the hydrolysis of the anion, with the exception of mixtures where the water content was greater than 90% in mol. These results suggest that, without extreme precautions to protect ionic liquids that contain [MeSO4]”, [HSO4]~ will be present and other studies using these ionic liquids should be interpreted in this light.20 Example 13
Influence of water content on enzymatic saccharification of Miscanthus treated with [C4C1im] [HSO4]
With the knowledge that binary 1-butyl-3-methylimidazolium methyl sulfate mixtures were transformed into quaternary mixtures of two ionic liquids plus two molecular solvents (water and methanol), we began to identify the active component(s) (s). Miscanthus was pretreated with aqueous mixtures of [C4C1im][HSO4], which allowed us to exclude methyl sulfate and methanol. The saccharification yields obtained from pulps pretreated with various water mixtures with [C4C1im] [HSO4] are shown in the Figure. Glucose yields were almost identical to the glucose yields obtained with the quaternary mixtures. The pattern of hemicellulose release was also similar, however, after blackening with [C4C1im][HSO4]40% to 80%, less hemicellulose was recovered than after treatment with equivalent mixtures 5 containing methyl sulfate.
A 90% glucose recovery after pretreatment with the ionic liquid is a substantial improvement compared to saccharification yields reported after pretreatment with other ionic liquids.
It was reported that 74% of glucose was enzymatically released from the ground maple wood after treatment with [C4C1im] [MeCO2] at 90 °C for 24 hours. 70% of glucose was released from the maple wood after treatment with [C2C1im] [MeCO2] at 90 °C for 24 hours. Li et al. reported 15% glucose release from ground eucalyptus pretreated with 1-allyl-3-methylimidazolium chloride, [C=C2C1im] Cl, at 120 °C for 5 hours, while 55% of glucose was released after treatment with 1-ethyl-3-methylimidazolium diethyl phosphate, [C2C4im] [Et2PO4], from wheat straw ground at 130 °C for 30 minutes. It should be noted that saccharification yields obtained from ball milled lignocellulose samples were not considered for this listing because fine grinding can have a considerable effect on cellulose digestibility.22 The use of crushed material reduces economic viability .31 but the use of fine powders obtained by grinding with a ball mill is of very little relevance for an industrial process. Studies using 3,5-dinitrosalicylic acid (DNS) for the determination of glucose yield were also not considered. Test 30 is not specific for glucose and therefore glucose yields from lignocellulose are generally overestimated. Example 14
Effect of pretreatment time on enzymatic saccharification
It then focused on optimizing the pre-treatment time. Figure 26 shows the saccharification yields for the treatments with both [C4C1im][MeS04]80% 5 and [C4C1im][HSO4]80% after various periods of time. It can be seen that the improvement in the digestibility of cellulose and hemicellulose occurred mainly in the first 4 hours. This was also the period when mass loss increased significantly. The pretreatment was practically complete after 8 hours, 10 reaching around 80% glucose and 30% hemicellulose release. By prolonging the pretreatment, the glucose yield increased slightly to above 85%, but the hemicellulose yield decreased to just above 20%. This experiment shows that the presence or absence of methyl sulphate 15 in the pretreatment mixture does not significantly influence the speed of pretreatment. It is anticipated that the pretreatment time can be reduced with the application of higher temperatures, but it must be balanced with the stability of the ionic liquid and potential side reactions. Example 15
The effect of [C4C1im] [MeSO4]80% and [C4C1im] [HSO4]80% pretreatment on biomass composition
The composition of untreated Miscanthus and pretreated pulp is shown in Table 2 and Figure 27. The untreated biomass contained 43.6% glucose, 24.3% hemicellulose and 26.5% lignin. After pretreatment with [C4C1im] [MeS04]8o% for 2 hours, the main effect was a reduction in lignin content. Treatment with [C4C1im] [HSO4]8Q% 30 for 2 hours resulted in the removal of lignin and hemicellulose. After an extended pretreatment for 22 hours, most of the lignin and hemicellulose was solubilized and the glucan content increased from 44% in the untreated biomass to 85% in the pretreated biomass. 91% of the original glucan was still present in the pulp. Biomass recovery after 22 hours was less than 46%, showing that more than half of the wood was solubilized in the ionic liquid. Tan et al. reported a mass recovery between 46% and 55% after pretreatment with I^C1im] [ABS] at 170 to 190 °C, which indicates that this ionic liquid mixture may be capable of fractionating similar biomass. Simultaneous removal of lignin and hemicellulose was also reported for [CzC1im] [MeCCt] , although less completely than seen in this study with [C^C1im] [HSO4]80%.
Table 2: Composition of Untreated Miscanthus and Miscanthus pretreated with [C4C1im] [MeSO4] and [C4C1im] [HSO4] .
Example 16
Production of sugars and furfurals As seen above, hemicellulose was removed from the biomass during treatment with water mixtures of [C4C1im] [HSO4] and [C^im] [MeSO4] . It is likely that, under the pretreatment conditions, (partial) hydrolysis of solubilized hemicellulose has occurred.
Therefore, the concentration of monomeric carbohydrates in the pretreatment liquor was investigated. Figure 28 shows the relative amount of hemicellulose sugars and glucose in [C4C1im] [HSO4 ] 80% θ [C4C1im] [MeSO4]80% liquors at different time points. The amount of hemicellulose monomers in the liquor increased in the first 4 hours. The increase was most pronounced in the [C4C1im] [HSO4] 80% liquor • The maximum amount of hemicellulose monomers was detected around 4 to 8 hours later.
This coincided with a greater increase in cellulose digestibility after 4 to 8 hours of treatment. Subsequently, the concentration of hemicellulose in the pretreatment liquor decreased, suggesting that the conversion of carbohydrate monomers to furfurals was taking place.
Furfural was detected in ionic liquid liquors and quantified for selected mixtures (Figure 29). The glucose content was significantly less than the sugar content of hemicellulose and hardly changed over time. The smaller amount of solubilized glucose is related to the slow hydrolysis of cellulose under pretreatment conditions and the decomposition of glucose to HMF. The small amount of HMF may be due to its decomposition to other decomposition products in the presence of water. Example 17
Lignin Recovery An attempt was made to recover lignin from the liquor (Figure 30), as this has been successfully demonstrated for other ionic liquids. It was observed that diluting the ionic liquid liquor with water precipitated a fine powder. The powder was characterized by IR spectroscopy and comparison with a spectrum of a reference lignin (alkaline lignin) showed that the precipitate is probably mostly lignin (Figures 31 to 34). When methanol was used to wash the pulp instead of water, most of the precipitate remained in solution and a 15-20% improvement in precipitate recovery was observed. Therefore, washing the pulp with methanol was preferred. The final protocol consisted of washing the pulp with methanol, drying the combined ionic liquid fractions by methanol evaporation and lignin precipitation by diluting the ionic liquid liquor with dry water. The precipitate was washed with copious amounts of water and dried before the yield was determined. The data (Figure 30) show that the precipitate yield was up to 50% of the Klason lignin content of the untreated biomass. More precipitate was obtained when the ionic liquid content in the pretreatment liquor was high.
The time dependence of the precipitate yield was also examined and we observed that the precipitate yield stabilized within 8 hours (Figure 34). The yield of [C4C1im][HSO4]80% was slightly higher compared to that of [C^im][MeSO4]80%. Example 18
The cation effect of the ionic liquid
The use of ionic liquids with monoalkylated imidazolium cations (1-alkylimidazolium, [CnHim]+) is advantageous from an industrial point of view, as ionic liquids are easier to synthesize and thus cheaper to produce.
Therefore, an exemplary Miscanthus pretreatment with 1-butylimidazolium hydrogen sulfate, [C4Him] [HSO4] was performed. Sugar yields after treatment with [C4Him] [HSO4] at % θ a subsequent enzymatic saccharification are shown in Figure 35. After 4 hours of pretreatment, 25 69% of the original glucose and 10% of the original hemicellulose were released from enzymatic mode. Yield was somewhat improved by extending the treatment to 20 hours, when 75% of the glucose was recovered. However, the xylose yield was reduced to only 3%. Pretreatment with [HC4im][HSO4]95% resulted in significantly reduced glucose yields (44%).
The compositional analysis and mass loss results of Miscanthus treated with [C4Him] [HSO4] are shown in Table 3 and Figure 36. 80 to 93% of the lignin and more than 95% of the hemicellulose were removed. Complete removal of hemicellulose is reflected by the low yields of xylose obtained during saccharification. Treatment with [C4Him][HSO4]g5% not only resulted in the solubilization of lignin and hemicellulose, but also a substantial removal of the cellulose fraction (51% of the glucan), explaining the reduced glucose yield shown in Figure 35. results indicate that pretreatment with [C4Him] [HSO4] was harder than with [C4C1im] [HSO4] under comparable conditions, potentially due to the increased acidity of [C4Him][HSO4] compared to [C4C1im][HSO4] . Table 3: Composition of Miscanthus pretreated with [C4Him] [HS04]80% θ [C4Him] [HSO4]95% at 120 °C. Values are given in %; Glu=glucan, Xyl=Xylane, Man=mannan, Gal=galactan, Ara=arabinan.

It was also possible to obtain a precipitate by diluting the ionic liquid liquor (Figure 37). For the [C4Him][HSO4]80% liquor, the yield was almost 100% of the lignin content. For 95% of the liquor, the amount of precipitate was almost double the amount of lignin content. The high yield of the unusual precipitate with the formation of pseudo-lignin was explained. The formation of water-insoluble carbohydrate degradation products was observed during biomass pretreatment under strong acidic conditions and it was observed that it obscured the Klason lignin yield.
Therefore, it was called pseudo-lignin. The formation of such degradation products is undesirable and optimization of pretreatment conditions is necessary to minimize this. Example 19
The effect of ionic liquid anion on the Miscanthus composition treated with ionic liquid
The effect of [C4C1im] [HSO4]8Q% treatment on the Miscanthus composition was compared with the effect that other mixtures of water and ionic liquid with 20/80% by volume dialkylimidazolium have on the composition. The anions examined were trifluoromethanesulfonate, [OTf]”, methanesulfonate, [MeSCq]”, chloride, Cl”, and acetate, [MeCO2]’. It should be noted that the acetate-containing ionic liquid, [C2C1im] [MeCO2] , was commercial grade.
Table 4: Pretreated Miscanthus composition after treatment with 80/20% ionic liquid and water mixtures at 120 °C for 22 hours. Values are given in %; Glu=glucan, Xyl=Xylane, Man=mannan, Gal=galactan, Ara=arabinan.

Figure 38 and Table 4 show that the nature of the anion has a profound effect on mass loss and pulp composition. [C4C1im] [HSO4] 80% removed lignin and hemicellulose almost completely, followed by [C4C1im] [MeSO3] 80% and then [C2C1im] [MeCO2] 80%. Almost no compositional change was observed when the biomass was treated with [C4C1im] Cl80% and [C4C1im] [OTf ] 80%, despite the fact that the high solubility of Kraft lignin was reported for both ionic liquids (in an anhydrous form). The contradiction could be resolved if lignin solubilization and lignin extraction (which usually involve chemical modifications) were considered different properties. Example 20
The effect of anion on saccharification yield
Enzymatic saccharification of Miscanthus treated with ionic liquid liqueurs was also performed (Figure 39). In general, enzymatic glucose release appears to reflect the extent of compositional change/mass loss achieved during pretreatment with ionic liquid. The highest glucose yield was observed after pretreatment with [C4C1im][MeSO3]80% and [C4C1im][HSO4]80%. The hemicellulose yield behaved slightly differently. The xylose yield was the highest after pretreatment with [C2C1im][MeCO2]80%. The yield was significantly lower after pretreatment with [C4C1im][MeSO3]80% and [C4C1im][HSO4]80%. Comparatively high hemicellulose yields after treatment with [C4C1im][MeCO2] can also be found in the literature. The increased hemicellulose recovery after treatment with [C2C1im][MeCO2]80% may be due to a buffering effect exerted by the basic acetate anion. Its ability to combine with protons to form acetic acid may limit the acid-catalyzed hydrolysis of hemicellulose polymers. Inhibition of cellobiose hydrolysis by [C4C1im][MeCO2] was observed in mixtures of ionic liquid, water and strong acid catalyst amounts. Binder et al. also observed inhibition of cellulose depolymerization to [C4C1im][MeCO2] despite the addition of catalyst amounts of HCl. The methanesulfonate anion appears to have a less protective effect and the acid catalysts that are released from the biomass (acetic acid and hydroxycinnamic acids) can help in the hydrolysis of xylan. The hydrogen sulfate increases the amount of available protons, which may explain the particularly low content of xylan in the pulp. The glucose and xylose yields obtained after treatment with [C4C1im] Cl80% and [C4C1im] [OTf ] 80% were low, despite their ability to dissolve cellulose and lignin preparations (in the case of triflate, only the solubility of lignin ). Example 21
The effect of anion on delignification and precipitate recovery
The precipitate yield to be related to the liquor's ability to extract lignin (Figure 40). The best delignification and the highest yield of the precipitate was obtained with [C4C1im] [HSO4]8Q%, followed by [C4C4im] [MeSO3]80% and then [C2C1im] [MeCO2]80%. This supports the notion that the precipitate comprises lignin, although it was shown in Figure 37 that pseudo-lignin also precipitates upon dilution of the ionic liquid liquor. Example 22
The effect of anion on the formation of soluble degradation products
The amounts of carbohydrate monomers and 25 dehydration products solubilized in the pretreatment liquors are shown in Figure 41. The [C4C1im] [HSO4] 80% and [C4C1imHMeSO3] 80% liquors contained approximately 45% of the total hemicellulose as the sugar monomers or furfural. In [C4C1im] [HSO4] 80%, most of the larger fraction 30 was furfural. The conversion of pentoses to furfurals was also observed in [C^Chim] [MeSO3) 8%, but to a lesser extent. This is related to the non-acidic nature of this ionic liquid. Only small amounts of monomers were detected in the acetate-containing liquor, which is probably due to the fact that the solubilized carbohydrates are mostly in oligomeric form. No furfural was formed in [C2C1im] [MeCO2]80% in our experiment. It is likely that the acidity of the liqueur is responsible for the concentrations of varying sugar and furfural monomers found in the liqueur. Similar to the hydrolysis of glycosidic bonds, the rate of furfural formation depends on the acid concentration. Since the acidity/basicity of 1,3-dialkylimidazolium ionic liquids is determined by the anion, their nature should have a profound impact on the fate of the solubilized hemicellulose. The amount of solubilized glucose and HMF was small in all cases. This is related to the improved stability of the cellulose fraction towards hydrolysis under pretreatment conditions and the propensity of HMF to react with levulinic and formic acids in the presence of water. Example 23 The effect of biomass type: willow and pine pretreatment
Pretreatment with 80% [C^im][HSO4] was also carried out on crushed willow (a hardwood species) and pine (a softwood species). For comparison, willow and pine were also pretreated with [C2C!ím] [MeC02]8o%. The effect of pretreatment on biomass composition is shown in Table 5 and Figure 42.Table 5: Composition of untreated willow and pine and pulps after treatment with [C4C1im] [HSO4]80% and [C4C1im] [MeC02]so%-


For both substrates, the removal of lignin and hemicellulose was more extensive after pretreatment with [C4C1im] [HS04] 80% than treatment with [C2C1im] [MeCO2]80«. The degree of cellulose enrichment after pretreatment with [C4C1im] [HSO4] 80% of the willow was almost as good as the enrichment observed in the Miscanthus pulp. A precipitate can be recovered from all samples. Significantly higher yields were obtained from [C4C1im] [HSO4 ] liqueurs so% • Glucose yields obtained by enzymatic saccharification are shown in Figure 43. More than 80% of the original glucose was released from the treated willow pulp [C4C1im] [HSO4] 80«, approaching the saccharification yields obtained from Miscanthus pretreated with this liquor. However, enzymatic saccharification of pine pulp only released up to 30% of the glucose; the type of ionic liquid that plays a small role. The generally higher yields obtained after pretreatment with [C4C1im] [HSO4] 8o% could be due to the enhanced lignin and hemicellulose removal by the liquor containing hydrogen sulfate, as noted in Miscanthus. Example 24 Solvent properties of ionic liquid and biomass digestibility
The Kamlet-Taft property (as described in A. Brandt, JP Hallett, DJ Leak, RJ Murphy and T. Welton, Green Chemistry, 2010, 12, 672 to 679) of [C4C1im] [HSO4] and [C4C1im] was measured ] [MeSO3] (Table 6), as it has not been reported in the literature. Three parameters are used to determine the strength of solvent solute interactions. The parameter α describes the acidity of the solvent hydrogen bond, β the basicity of the hydrogen bond and π* the polarizability. Our measurements showed that the β parameter of [C4C1im] [HSOJ is the same as the value for [C4C1im] [MeSO4] . The acidity of the hydrogen bond is very different, in fact the α value cannot be determined for [C4C1im] [HSO4] , because it protonates one of the dye probes.
It is highlighted that high glucose yields were achieved without complete biomass solubilization. This is due to the relatively low β values of [C4C1im] [MeSO4], [C4C1im] [HSO4] and [C4C4im] [MeSOβ] that do not allow solubilization of cellulose. The β parameters are smaller than the values of [C4C1im] [MeCO2] ( [β]=1.20), 1-butyl-3-methylimidazolium dimethyl phosphate, [C4C1im] [Me2PO4] , (β=1.12 ) and [C4C1im]Cl (β=0.83) .19 Although [C2C1im] [MeCO2] can dissolve cellulose when it is anhydrous, the presence of 20% by volume of water impedes the solubility of cellulose. Table 6: Kamlet-Taft parameters of selected ionic liquids used in this work.

An attempt was also made to correlate glucose yields to the basicity of hydrogen bonding of ionic liquids. Although it is clear that the nature of the anion affects the saccharification yield, it cannot be correlated to the β value of the ionic liquid.
It was first demonstrated that the ionic liquids [ChC1im] [HSO4] , [C4C1im] [MeSO3] and the ionic liquid mixture [C4C1im] [MeSO4] / [HSO4] can be used to pre-treat lignocellulosic biomass. These ionic liquids worked effectively in the presence of significant amounts of water, eliminating the need for anhydrous conditions during pretreatment. Commercial [C2C1im] [MeCh] was also effective in the presence of 20% by volume of water, but the saccharification yield was lower. The lignin and hemicellulose were solubilized during the pretreatment, leaving behind a solid residue that was highly enriched in cellulose. Enzymatic saccharification of Miscanthus pulp pretreated at 120 °C with liquors containing 80% by volume of ionic liquid resulted in glucose yields of ca. 90%. Hemicellulose was partially recovered as solid and readily hydrolysable during enzymatic saccharification. However, a significant portion of the hemicellulose remained in the pretreatment liquor as sugar monomers and was partially converted dehydration products. The amount of furfurals generated during pretreatment with ionic liquid originates from the acidity of ionic liquid liquors. In the presence of 20% by volume of water, treatment with [C-jC1imJCl and [C4C1im] [OTf] had little effect on biomass, showing that the anion of 1,3-dialkylimidazolium ionic liquids plays an important role in determining efficacy pretreatment with ionic liquid and water tolerance. It was not possible to find a correlation between pretreatment efficacy and anion basicity, as found previously for cellulose solubility or wood chip swelling. While enzymatic sugar release from grass and hardwood pulps was very good, yields from softwood pulp were only moderate. Upon dilution with water, a precipitate was recovered which likely contains lignin as well as pseudo-lignin. This study also suggests that monoalkylated imidazolium ionic liquids, such as [C4Him] [HSO4] , appear to be industrially relevant and promising alternatives to dialkylimidazolium ionic liquids. Example 25
Effect of acid:base ratio on yield
In order to investigate the effect of acid/base properties of monoalkylated hydrogen sulphate/imidazolium sulphate ILs on biomass pretreatment, a number of different ILs with cation [C4Him] and different anion ratios [HSO4]/[ SO4] and/or an excess of H2SO4 were prepared. ILs were synthesized by adding different ratios of sulfuric acid to 1-butylimidazole in water (Table 7).
A solution of H2SO4 (95%) in water (3 ml water/ml H2SO4) was added dropwise to a solution of 1-butylimidazole (98.4%) in water (1 ml water/ml 1-butylimidazole ). The mixture was stirred at room temperature for several hours.
Once the reaction was complete, the ILs were decolorized by the addition of charcoal and filtered through neutral alumina. The water was then removed by heating at 50 °C for 48 hours and the ILs were obtained as colorless liquids at room temperature with high yields and high purity. The structure and composition of ILs were confirmed by -1 ! NMR, 13 C NMR, mass spectroscopy and elemental analysis. The final amounts of [C4Him], [HSO4] , [SO4] and H2SO4 present in the prepared ILs are shown in Table 8.

Table 7. Rates of H2SO4 and 1-butylimidazole used in the synthesis of ILs.
Table 8. Amounts of [C4Him], [HSO4], [SO4. and H2SO4 present in the prepared ILs.
The relative concentrations of acid and base varied in the C4Him HSO4 system as described above. The saccharification yields relative to glucose or hemicellulose content in the untreated Miscanthus were measured and are shown in Figures 44 and 45. The best results were obtained with a small excess (1%) of the acid (group 5 column from left. ), in which a maximum glucose yield and a decent hemicellulose yield were achieved after 4h. It appears that the addition of a little acid significantly speeds up the pretreatment process, when other variables such as water content and temperature are kept constant. Too much acid appears to carbonize the biomass and excess base increases the hemicellulose yield slightly but also the time taken until the maximum glucose yield is reached. Example 26
Wood chip crushing energy Pine wood chips (8 chips, sized 8x7x7 mm, approximately 1.3 g) were placed in 5 ml of the pretreatment liquid in a glass tube and heated to 90°C in an oven for 1 or 18 hours. The chips were then cooled and excess liquid removed from their surfaces with a paper towel, then crushed in an analytical crusher for 30 seconds. The power consumption of the crusher was determined using a power analyzer. Energy savings (Table 9, Figure 49) is calculated per gram of wood by subtracting the energy used by the shredder when empty and compared to the energy used to grind dry wood.
Table 9: Energy savings in wood chip crushing for various pretreatment methods compared to dry wood
After grinding, the samples were soaked overnight in 20 ml of an appropriate volatile solvent (in all cases methanol except petroleum ether for silicone oil and perfluorohexane for Fomblin). They were then filtered, rinsed twice with 5 ml of solvent and left to air dry for at least 24 hours. The resulting dry powder was checked using a gravimeter for significant amounts of residual treatment liquid. The powder was then passed through a nested column with sieves of decreasing pore sizes (2 mm to 53 µm) by oscillating on a vibrating sieve oscillator for 8 minutes. The weight percentage of material retained by each sieve was measured, and from these data the log normal distribution mass mean diameter (D5O) was calculated, that is, the particle size that 50% of the sample is smaller than in mass. . (Table 10) . It is used in this document as a measure of the average particle size of wood dust and was calculated by linear interpolation using the Equation below.
where Xi and x2 are the pore sizes of the sieves that allowed slightly below and slightly above 50% of the sample to pass through by weight, respectively, and Yi and 72 are the percentages of material passing through these sieves.
Table 10: Average particle size (D50) of wood dust obtained by grinding pretreated wood chips in various ways
Sacharification 150 mg of wood dust was taken from a certain particle size fraction of each sample. For comparison, wood chips that were pretreated but not crushed were also prepared. This was added to a buffering solution containing cellulose hydrolysis enzymes and incubated for 96 hours at 50 °C. The preparations used were Celluclast, a mixture of cellulase from Trichoderma reesei, and β-glucosidase from Novozyme 188 which can also hydrolyze xylan due to its hemicellulolytic activity. 60 μl of each preparation were used.
The amount of glucose and hemicellulose present thereafter was determined using HPLC (Table 11, Figure 50). Sugar yields are given as a percentage of the weight of each sample when dry.

Table 11: Sugar Yields of Enzymatically Crushed Wood Powder from Wood Chips Pretreated in Different Ways as a Percentage of Sample Weight when Dry.

权利要求:
Claims (17)
[0001]
1. METHOD OF TREATMENT OF A LIGNOCELLULOSE BIOMASS TO DISSOLVE LIGNIN IN IT, but not cellulose, characterized by comprising: (a) placing the lignocellulose biomass in contact with a composition comprising an ionic liquid and 10-40% v /v of water to produce a cellulose pulp, wherein the ionic liquid comprises an anion selected from C1-C20 alkyl sulfate [AlkylSO4]-; C1-C20 alkylsulfonate [AlkylSO3]-; hydrogen sulphate [HSO4]-, hydrogen sulphite [HSO3]- dihydrogen phosphate [H2PO4]- and hydrogen phosphate [HPO4]2- and a cation selected from an imidazolium derivative, pyridinium derivative and ammonium derivative.
[0002]
2. METHOD according to claim 1, characterized in that the anion is selected from [MeSO4]-, [HSO4]- and [MeSO3]-.
[0003]
3. METHOD, according to any one of claims 1 or 2, characterized in that said cation is a protic cation.
[0004]
4. METHOD according to any one of claims 1 or 2, characterized in that the cation is selected from 1-butyl-3-methylimidazolium [C4C1im]+, 1-ethyl-3-methylimidazolium [C2C1im]+, 1-methylimidazolium [C1Him]+ and 1-butylimidazolium [C4Him]+.
[0005]
5. METHOD according to any one of claims 1, 2 or 4, characterized in that said ionic liquid is selected from 1-butyl-3-methylimidazolium methyl sulfate [C4C1im][MeSO4], hydrogen sulfate of 1 -butyl-3-methylimidazolium [C4C1im][HSO4], 1-butyl-3-methylimidazolium methanesulfonate [C4C1im][MeSO3] and 1-butylimidazolium hydrogen sulfate [C4Him][HSO4].
[0006]
6. METHOD, according to any one of claims 1 to 5, characterized in that the composition comprises 20 to 40% volume/volume (v/v) of water.
[0007]
7. METHOD according to any one of claims 1 to 6, characterized in that the ionic liquid additionally comprises 0.01 to 20% v/v of acid.
[0008]
8. METHOD, according to any one of claims 1 to 7, characterized in that lignocellulose biomass is placed in contact with the composition at 100 to 160°C.
[0009]
9. METHOD, according to any one of claims 1 to 8, characterized in that lignocellulose biomass is placed in contact with the composition for 1 to 22 hours.
[0010]
10. METHOD, according to any one of claims 1 to 9, characterized in that it additionally comprises the step of: (b) separating the ionic liquid from the pulp produced in (a).
[0011]
11. METHOD, according to any one of claims 1 to 10, characterized in that the biomass is placed in contact with the composition before mechanical processing, such as grinding or crushing.
[0012]
12. METHOD, according to any one of claims 1 to 11, characterized in that the biomass is placed in contact with the composition after mechanical processing, such as grinding or crushing.
[0013]
13. METHOD, according to any one of claims 1 to 12, characterized in that it additionally comprises the step of washing the pulp with an organic solvent that is miscible with the mixture of ionic liquid and water.
[0014]
14. METHOD, according to claim 13, characterized in that it further comprises the step of: (c) adding an anti-solvent to the ionic liquid obtained in (b) to extract the dissolved lignin by precipitation; and (d) separating the precipitated solid from the antisolvent/ionic liquid.
[0015]
15. METHOD according to claim 14, characterized in that it further comprises the step of: (e) removing the antisolvent from the ionic liquid obtained in (d).
[0016]
16. METHOD according to any one of claims 14 or 15, characterized in that the antisolvent is water.
[0017]
17. GLUCOSE PREPARATION PROCESS FROM A LIGNOCELLULOSE BIOMASS, characterized in that it comprises subjecting a cellulose pulp obtainable by the method, as defined in any one of claims 1 to 13, to enzymatic hydrolysis.

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同族专利:

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公开号 | 公开日

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EP2652193A2|2013-10-23|

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US20140073016A1|2014-03-13|

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CN103370469B|2015-12-16|

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PL2652193T3|2021-01-25|

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WO2012080702A3|2012-08-23|

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ES2828748T3|2021-05-27|

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US9765478B2|2017-09-19|

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EP2652193B1|2020-09-16|

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BR112013015190A2|2020-07-21|

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CN103370469A|2013-10-23|

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CA2821403C|2020-08-04|

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WO2012080702A2|2012-06-21|

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CA2821403A1|2012-06-21|

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法律状态:
2020-07-28| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-10-20| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-11-24| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-02-23| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-05-11| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 15/12/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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GBGB1021319.7A|GB201021319D0|2010-12-15|2010-12-15|Treatment|

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GBGB1021319.7|2010-12-15|

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GBGB1109119.6|2011-05-27|

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GBGB1109119.6A|GB201109119D0|2011-05-27|2011-05-27|Treatment|

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PCT/GB2011/001723|WO2012080702A2|2010-12-15|2011-12-15|Treatment|

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