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|CLAIMS What is claimed is:
1. A method for producing carbohydrate-enriched biomass comprising:
(a) providing lignocellulosic biomass comprising lignin;
(b) suspending the biomass of (a) in an organic solvent solution comprising water and one or more sulfide salt under alkaline conditions whereby a biomass-solvent suspension is formed; (c) heating the biomass-solvent suspension to a temperature of about 100 0C to about 220 0C for about 5 minutes to about 5 hours whereby lignin is fragmented and is dissolved in the suspension; and
(d) filtering free liquid whereby the dissolved lignin is removed and whereby readily carbohydrate-enriched biomass is produced.
2. The method of claim 1 further comprising:
(e) washing the biomass produced in step (d) with a solvent solution.
3. The method of Claim 2, further comprising:
(f) washing the biomass produced in step (e) with water to produce readily sacchahfiable carbohydrate-enriched biomass.
4. The method of Claim 2 further comprising drying biomass produced in step (e) to produce readily saccharifiable carbohydrate-enriched biomass.
5. The method of Claims 2 or 3, further comprising repeating steps
(e) and (f) one or more times.
6. The method of Claim 1 wherein the heating step of (c ) occurs in a sealed pressure vessel.
7 The method of Claim 1 wherein the filtering step of (d) occurs under pressure.
8. The method of Claim 1 wherein the organic solvent solution further comprises an additional nucleophile selected from the group consisting of NH3, one or more alkylamines, NaOH, polysulfide, hydropolysulfide reagents, and combinations thereof.
9. The method of Claim 8 wherein the additional nucleophile is one or more alkylamine and said one or more alkylamine is at a concentration of about up to 20% by weight of dry biomass
10. The method of Claim 1 wherein the solvent solution to biomass in step (b) has a weight ratio of about 10 to 1 to 0.5 to 1.
11. The method of Claim 1 , wherein the heated suspension of step (c) is cooled to room temperature before filtering in step (d).
12. The method of Claim 2 further comprising evaporating off the solvent under vacuum of the filtered and washed biomass after step (e).
13. The method of Claims 3, 4 or 12, further comprising saccharifying the biomass with an enzyme consortium whereby fermentable sugars are produced.
14. The method of Claim 3, further comprising saccharifying the biomass without drying by contacting said biomass with an enzyme consortium after washing in step (f), whereby fermentable sugars are produced.
15. The method of Claims 13 or 14, further comprising fermenting the sugars to produce a target product.
16. The method of Claim 15 wherein the target product is selected from the group consisting of alcohols, organic acids, amino acids and gases.
17. The method of Claim 1 wherein the biomass is selected from the group consisting of switchgrass, waste paper, sludge from paper manufacture, corn fiber, corn cobs, corn husks, corn stover, grasses, wheat, wheat straw, hay, barley, barley straw, rice straw, sugar cane bagasse, sugar cane straw, yellow poplar, sorghum, soy, components obtained from processing of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure and combinations thereof.
18. A method of simultaneous fragmentation and selective extraction of lignin from lignocellulosic biomass to produced a substantially lignin-free biomass comprising: (a) providing:
1 ) an amount of lignocellulosic biomass;
2) a multi-component organic solvent solution comprising from about 40% to about 70% ethanol in water;
3) one or more sulfide salt(s);and
4) one or more alkylamine(s) under alkaline conditions; (b) contacting said biomass with the multi-component solvent solution of (a) whereby a solvent-biomass mixture is produced; (c) placing the solvent-biomass mixture in a sealed pressure vessel whereby the mixture of (b) is heated from about 100 0C to about 220 0C for about 5 minutes to about 5 hours whereby lignin is fragmented and dissolved in the solvent;
(d) removing the dissolved lignin of (c) by filtration; and
(e) washing the residual biomass with organic solvent, whereby substantially lignin-free biomass is produced.
19 The method of Claim 18 wherein the solvent at step (e) may contain water
20. The method of Claim 18 wherein the substantially lignin-free biomass is from about 60% to about 100% original weight of the biomass.
21. The method of any one of Claims 1 or 18, wherein the organic solvent solution further comprises one or additional component selected from the group consisting of alkali or alkaline earth hydroxides or carbonates, ammonia, thiols, polysulfides, hydropolysulfides and combinations thereof.
22. The method of Claims 1 or 18 wherein the solvent solution, and any unreacted sulfide salts or other unreacted components are recyclable.
23. The method of Claims 1 or 18 wherein said organic solvent solution comprises a solvent selected from the group consisting of alcohols, diols and aprotic solvents
24. The method of Claim 23 wherein the organic solvent solution comprises a solvent selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol and hexanol, isomers thereof, and diols thereof.
25. The method of Claims 1 or 18 wherein the lignocellulosic biomass of step (a) has a carbohydrate content that is highly conserved through steps (a) through (d).
26. The method of Claims 8 or 18 wherein the one or more alkylamines is selected from the group consisting of R-NH2, R2-NH, R3N, (H2N-R-NH2), (H2N-R(NH2)2), (HO-R-NH2), ((HO)2-R-NH2), (HO-R-(NH2)2), (HS-R-NH2), ((HS)2-R-NH2), (HS-R-(NH2J2) and (H2N-R(OH)(SH) and combinations thereof, wherein R is independently a monovalent, divalent or trivalent 1 -6 carbon alkane, alkene or alkyne, linear, cyclic or branched.
27. The method of Claim 26 wherein R is independently methyl, ethyl, propyl or butyl.
28. The method of Claim 26 wherein the alkylamine is methylamine.
29. The method of Claims 1 or 18 where the sulfide salt is selected from the group consisting of alkali metal and alkaline earth sulfides (e.g. Na2S), alkali metal and alkaline earth hydrosulfides (e.g.
NaHS), alkali metal and alkaline earth polysulfides (e.g., Na2Sn), alkali metal and alkaline earth hydropolysulfides (e.g., NaHSn), ammonium sulfide, hydrosulfide, polysulfides, and hydropolysulfides, alkylammonium sulfide, hydrosulfide, polysulfides, and hydropolysulfides.
30. The method of Claims 1 and 18 wherein the temperature of step (c) is from about 165 0C to about 195 0C.
ORGANIC SOLVENT PRETREATMENT OF BIOMASS TO ENHANCE ENZYMATIC SACCHARIFICATION
The application claims the benefit of U.S. Provisional Application No. 61/139163, filed December 19, 2008, the disclosure of which is hereby incorporated in its entirety.
FIELD OF THE INVENTION Methods for producing readily saccharifiable carbohydrate- enriched lignocellulosic biomass are provided and disclosed. Specifically, pretreated biomass is prepared through simultaneous fragmentation and selective extraction of lignin in an organic solvent solution at elevated temperatures in the presence of a sulfide salt such as ammonium sulfide at an alkaline pH. Optionally, one or more alkylamine and various nucleophiles may be added to the biomass pretreatment solution. The remaining carbohydrate-enriched solids in the pretreated biomass may then be subjected to enzymatic sacchahfication to obtain fermentable sugars, which may be subjected to further processing for the production of target products.
BACKGROUND OF THE INVENTION
Cellulosic and lignocellulosic feedstocks and wastes, such as agricultural residues, wood, forestry wastes, sludge from paper manufacture, and municipal and industrial solid wastes, provide a potentially large renewable feedstock for the production of chemicals, plastics, fuels and feeds. Cellulosic and lignocellulosic feedstocks and wastes, composed of cellulose, hemicellulose, pectins and of lignin are generally treated by a variety of chemical, mechanical and enzymatic means to release primarily hexose and pentose sugars, which can then be fermented to useful products.
Pretreatment methods are often used to make the polysaccharides of lignocellulosic biomass more readily accessible to cellulolytic enzymes. One of the major impediments to cellulolytic enzyme digest is the presence of lignin, a barrier that limits the access of the enzymes to their substrates, and a surface to which the enzymes bind non-productively. Because of the significant costs associated with enzymatic saccharification, it is desirable to minimize the enzyme loading by either inactivation of the lignin to enzyme adsorption or its outright extraction. Another challenge is the inaccessibility of the cellulose to enzymatic hydrolysis either because of its protection by hemicellulose and lignin or by its crystal unity. Pretreatment methods that attempt to overcome these challenges include: steam explosion, hot water, dilute acid, ammonia fiber explosion, alkaline hydrolysis (including ammonia recycled percolation), oxidative delignification and organosolv.
Organosolv methods, as previously practiced for the treatment of lignocellulose biomass, for either the production of pulp or for biofuels applications, while generally successful in lignin removal, have suffered from poor sugar recoveries, particularly of xylose. For example, the use of slightly acidic ethanol-water mixtures (e.g., EtOH 42 weight%) at elevated temperature to remove lignin from lignocellulosic biomass (Kleinert, T. N., Tappi 57: 99-102, 1974) resulted in substantial loss of carbohydrate. Dilute acid hydrolysis at 95 0 C followed by organic solvent extraction and enzymatic saccharification (Lee, Y-H. et al., Biotech. Bioeng., 29: 572-581 , 1987) resulted in substantial loss of hemicellulose during hydrolysis, additional carbohydrate loss upon organic solvent extraction and poor yield (-50% of total carbohydrate) upon enzymatic saccharification of residue. Use of aqueous organic solvent containing ammonia at elevated temperatures to treat lignocellulosic biomass (Park J. -K. and Phillips, J. A., Chem. Eng. Comm., 65: 187-205, 1988) required the use of a high liquid to solids ratio in pretreatment and resulted in substantial loss of hemicellulose and poor enzymatic saccharification of cellulose. Treatment of biomass with gaseous water and methylamine followed by extraction with organic solvent and then extraction with water, required three steps and resulted in a substantial loss of carbohydrate (Siegfried, P. and Gόtz, R., Chem. Eng. Technol., 15: 213-217, 1992). Treatment with polyamines or ethylamine in water-aliphatic alcohol mixtures plus catalyst at elevated temperature required high liquid/solids ratio and low concentrations of alcohol led to poor sugar recovery, particularly of xylan (U.S. patent 4,597,830A). Thioglycolate in aqueous alkaline solution used to treat lignocellulosic biomass at elevated temperature, followed by a hot water wash required use of alkali-metal or alkaline-earth hydroxides. This method requires the costly disposal of inorganic ions, high weight% thioglycolate, and use of large volumes of water (U.S. patent 3490993). Treatment with organic solvent-water mixtures in the presence of sulfide/bisulfide at elevated temperatures required a high solvent/solids ratio and elevated sulfur content and resulted in a substantial loss of carbohydrate, (U.S. patent 4,329,200A).
Additional shortcomings of previously applied methods include, separate hexose and pentose streams (e.g. dilute acid), inadequate lignin extraction or lack of separation of extracted lignin from polysaccharide, particularly in those feedstocks with high lignin content (e.g., sugar cane bagasse, softwoods), need to dispose of waste products (e.g., salts formed upon neutralization of acid or base), and poor recoveries of carbohydrate due to breakdown or loss in wash steps. Other problems include the high cost of energy, capital equipment, and pretreatment catalyst recovery, and incompatibility with saccharification enzymes.
One of the major challenges of biomass pretreatment is to maximize the extraction or chemical neutralization (with respect to nonproductive binding of cellulolytic enzymes) of the lignin while minimizing the loss of carbohydrate (cellulose plus hemicellulose) via low-cost, efficient processes. The higher the selectivity, the higher the overall yield of monomeric sugars following combined pretreatment and enzymatic saccharification.
There is therefore a need to develop a single step process using substantially lower concentrations of sulfur and recyclable base in the form of ammonia or alkylamines as opposed to the use of alkali metal hydroxides which are not amenable to either recycle or disposal. The current disclosure addresses this need. In this disclosure, a sulfide salt such as ammonium sulfide, in an organic solvent-mediated process and at alkaline pH, and optionally various nucleophiles followed by selective extraction of lignin at elevated temperatures is used. Surprisingly, this cost-effective process resulted in significantly improved lignin fragmentation and extraction and high carbohydrate retention.
SUMMARY OF THE INVENTION The present invention provides methods for producing readily saccharifiable carbohydrate-enriched biomass and for selectively extracting lignin from lignocellulosic biomass while nearly quantitatively retaining carbohydrate. The methods include treating lignocellulosic biomass with an organic solvent, such as EtOH in H 2 O, and one or more sulfide (hydrosulfide) salt under alkaline conditions at elevated temperatures in a single step. In another embodiment the solvent solution further comprises one or more alkylamines. Following pretreatment, the biomass may be further treated with a sacchahfication enzyme consortium to produce fermentable sugars. These sugars may be subjected to further processing for the production of target products.
Accordingly, the invention provides a method for producing carbohydrate-enriched biomass comprising: (a) providing lignocellulosic biomass comprising lignin;
(b) suspending the biomass of (a) in an organic solvent solution comprising water and one or more sulfide salt under alkaline conditions whereby a biomass-solvent suspension is formed;
(c) heating the biomass-solvent suspension to a temperature of about 100 0 C to about 220 0 C for about 5 minutes to about 5 hours whereby lignin is fragmented and is dissolved in the suspension; and
(d) filtering free liquid whereby the dissolved lignin is removed and whereby readily carbohydrate-enriched biomass is produced.
In another embodiment the invention provides a method of simultaneous fragmentation and selective extraction of lignin from lignocellulosic biomass comprising: (a) providing:
1 ) an amount of lignocellulosic biomass;
2) a multi-component organic solvent solution comprising from about 40% to about 70% ethanol in water;
3) one or more sulfide salt(s);and
4) one or more alkylamine(s) under alkaline conditions;
(b) contacting said biomass with the multi-component solvent solution of (a) whereby a solvent-biomass mixture is produced;
(c) placing the solvent-biomass mixture in a sealed pressure vessel whereby the mixture of (b) is heated from about 100 0 C to about 220 0 C for about 5 minutes to about 5 hours whereby lignin is fragmented and dissolved in the solvent; (d) removing the dissolved lignin of (c) by filtration; and
(e) washing the residual biomass with organic solvent, whereby substantially lignin-free biomass is produced. Particularly suitable one or more sulfide (hydrosulfide) salt includes ammonium sulfide (hydrosulfide). In another aspect, one or more alkylamine or an amount of ammonia is present in the solvent solution.
Particularly suitable feedstocks for use in the methods of the invention include but are not limited to switchgrass, waste paper, sludge from paper manufacture, corn fiber, corn cobs, corn husks, corn stover, grasses, wheat, wheat straw, hay, barley, barley straw, rice straw, sugar cane bagasse, sugar cane straw, yellow poplar, sorghum, soy, components obtained from processing of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure and combinations thereof.
Particularly suitable alkylamines include those selected from the group consisting of R-NH 2 , R 2 -NH, R 3 N, (H 2 N-R-NH 2 ), (H 2 N-R(NH 2 ) 2 ), (HO-R-NH 2 ), ((HO) 2 -R-NH 2 ), (HO-R-(NH 2 ) 2 ), (HS-R-NH 2 ), ((HS) 2 -R-NH 2 ), (HS-R-(NH 2 ) 2 ) and (H 2 N-R(OH)(SH) and combinations thereof, wherein R is independently a monovalent, divalent or trivalent 1-6 carbon alkane, alkene or alkyne, linear, cyclic or branched.
BRIEF DESCRIPTION OF THE FIGURES Figure 1 - Figure 1 shows the UV absorbance spectra of filtrates (diluted 1 :5000 with 70% EtOH in H 2 O (v/v)) following pretreatment at 187 0 C for 1 hour in 70% EtOH in H 2 O (v/v) plus 14% methylamine (w/w biomass) with or without 2% or 6% (NH 4 ) 2 S.
DETAILED DESCRIPTION OF THE INVENTION Applicants specifically incorporate the entire content of all cited references in this disclosure. Unless stated otherwise, all percentages, parts, ratios, etc., are by weight. Trademarks are shown in upper case. Further, when an amount, concentration, or other value or parameter is given as either a range, preferred range or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the invention be limited to the specific values recited when defining a range.
The present invention provides a process for the treatment of biomass in order to produce readily saccharifiable carbohydrate-enriched biomass to enhance the subsequent enzymatic sacchahfication step. A process involving a pretreatment step wherein lignin is simultaneously fragmented and extracted using an organic solvent under alkaline conditions at elevated temperatures in the presence of one or more sulfide salt is employed. Additional nucleophiles may be employed for further benefit. The treated biomass is then filtered and washed to remove solubilized lignin, acetic acid, acetamides, alkylamides and excess reagent and then digested with a saccharification enzyme consortium to produce readily fermentable sugars. The sugars may then be further processed to one or more target product. The removed lignin may also be further processed and utilized for other purposes (such as burning for energy) to increase efficiency.
The following definitions are used in this disclosure: "Room temperature" and "ambient" when used in reference to temperature refer to any temperature from about 15 0 C to about 25 0 C.
"Fermentable sugars" refers to a sugar content primarily comprising monosaccharides and some disacchahdes that can be used as a carbon source by a microorganism (and some polysaccharides may be present)) in a fermentation process to produce a target product. "Readily fermentable sugars" means that additional costly processing is not necessary and/or that a fermentative microorganism can be contacted with the resulting sugars with minimal impediments from inhibitors or other components that may adversely affect fermentation. "Lignocellulosic" refers to material comprising both lignin and cellulose. Lignocellulosic material may also comprise hemicellulose. In the processes described herein, lignin is dissolved and substantially removed from the lignocellulosic biomass to produce a carbohydrate- enriched biomass. "Dissolved lignin" as referred to herein means the lignin that is dissolved in an organic solvent solution.
"Al lignin" refers to acid-insoluble lignin.
"Autohydrolysis" refers to the hydrolysis of biomass in the presence of solvent (water or organic solvent plus water) plus heat with no further additions, such as without exogenous acid or base or hydrolytic enzyme addition.
"Cellulosic" refers to a composition comprising cellulose. "Target product" refers to a chemical, fuel, or chemical building block produced by fermentation. Product is used in a broad sense and includes molecules such as proteins, including, for example, peptides, enzymes and antibodies. Also contemplated within the definition of target product are ethanol and butanol. "Dry weight of biomass" refers to the weight of the biomass having all or essentially all water removed. Dry weight is typically measured according to American Society for Testing and Materials (ASTM) Standard E1756-01 (Standard Test Method for Determination of Total Solids in Biomass) or Technical Association of the Pulp and Paper Industry, Inc. (TAPPI) Standard T-412 om-02 (Moisture in Pulp, Paper and Paperboard).
"Selective extraction" means removal of lignin while substantially retaining carbohydrates.
"Solvent solution" and "an organic solvent solution", as used herein, is an organic solvent mixture in water that includes any organic liquid that dissolves a solid, liquid, or gaseous solute, resulting in a solution. The most suitable solvent solutions for this invention are organic solvents such as ethanol, methanol, n-propanol, isopropanol, n-butanol, 2-butanol, isobutanol, t-butanol, pentanol and hexanol and diols with the same number of carbons. They can also include aprotic solvents. The solvent solutions can include additional components in mixture with the solution, e.g, the solvent solution may include one or more nucleophile.
"Biomass" and "lignocellulosic biomass" as used herein refer to any lignocellulosic material, including cellulosic and hemi-cellulosic material, for example, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, yard waste, wood, forestry waste and combinations thereof, and as further described below. Biomass has a carbohydrate content that comprises polysaccharides and oligosaccharides and may also comprise additional components, such as protein and/or lipid. "Highly conserved" as used herein refers to the carbohydrate content of the lignocellulosic material after the processing steps described herein. In an embodiment of the invention, the highly conserved carbohydrate content provides for sugar yields after saccharification that are substantially similar to theoretical yields with minimal loss of sugar yield from the processes described herein. In an embodiment of the invention, highly-conserved with reference to carbohydrate content refers to the conservation of greater than or equal to 85% of the biomass carbohydrate as compared to biomass prior to pretreating as described herein.
"Preprocessing" as used herein refers to processing of lignocellulosic biomass prior to pretreatment. Preprocessing is any treatment of biomass that prepares the biomass for pretreatment, such as mechanically milling and/or drying to the appropriate moisture contact.
"Biomass-solvent suspension" refers to a mixture of biomass and solvent. The biomass-solvent solution may comprise additional components such as one or more sulfide salt, one or more alkylamine, etc. "Saccharification" refers to the production of fermentable sugars from primarily polysaccharides by the action of hydrolytic enzymes. Production of fermentable sugars from pretreated biomass occurs by enzymatic saccharification by the action of cellulolytic and hemicellulolytic enzymes. "Pretreating biomass" or "biomass pretreatment" as used herein refers to subjecting native or preprocessed biomass to chemical or physical action, or any combination thereof, rendering the biomass more susceptible to enzymatic saccharification or other means of hydrolysis prior to saccharification. For example, the methods claimed herein may be referred to as pretreatment processes that contribute to rendering biomass more accessible to hydrolytic enzymes for saccharification.
"Pretreatment filtrate" means the free liquid that is in contact with the biomass following pretreatment and which is separated by filtration.
"Pretreated Biomass" as used herein refers to native or preprocessed biomass that has been subjected to chemical or physical action, or any combination thereof, rendering the biomass more susceptible to enzymatic saccharification or other means of hydrolysis prior to saccharification.
"Air-drying the filtered biomass" can be performed by allowing the biomass to dry through equilibration with the air of the ambient atmosphere.
"Readily sacchahfiable biomass" means biomass that is carbohydrate-enriched and made more amenable to hydrolysis by cellulolytic or hemi-cellulolytic enzymes for producing monomehc and oligomeric sugars, i.e., pretreated biomass as described herein.
"Carbohydrate-enriched" as used herein refers to the biomass produced by the process treatments described herein. In one embodiment the readily saccharifiable carbohydrate-enriched biomass produced by the processes described herein has a carbohydrate concentration of greater than or equal to 85% of the dried biomass by weight, while having removed 75% or greater of the starting biomass lignin content based on dry weight. "Heating the biomass suspension" means subjecting the biomass suspended in a solvent to a temperature greater than ambient or room temperature. Temperatures relevant to the present pretreatments are from about 100 to about 22O 0 C, or from about 140 to about 18O 0 C, or any temperature within or approximately these ranges. "Filtering free liquid under pressure" means removal of unbound liquid through filtration, with some pressure difference on opposite faces of the filter
"Alkaline" or "under alkaline conditions" means a pH of the biomass-solvent suspension equal to or greater than the pKas of the nucleophiles present such that these are substantially deprotonated and more highly reactive than in their protonated states. These nucleophiles would include alkylamines, and ammonia, thiols, polysulfides and hydrosulfide (if present).
"Divalent alkane" means a linear, branched or cyclic alkane with two open valences.
For the purposes of this invention, an organic solvent solution comprising a sulfide salt refers to the use of compounds comprising sulfide (S = ) or polysulfide (S n S = , where n is an integer) ions such as ammonium sulfide, compounds that release sulfides or polysulfides upon disproportionation of elemental sulfur under alkaline conditions at elevated temperature or in which oxides of sulfur are combusted in the presence of electron-rich sources such as lignin to produce sulfides (as in the Kraft process). If alkylamines are present in the organosolv solution, then alkylammonium salts of sulfides are produced that perform similarly to ammonium sulfide. Inorganic salts of sulfide or polysulfide (e.g. Na2S or Na 2 S n ) may also be used in the organic solvent solution.
"Alkylamine" means an alkane containing an -NH 2 group in place of one, two or three H atoms; e.g., monomethylamine, dimethylamine, trimethylamine, ethylamine, isopropyl- amine, ethylhexylamine, cyclohexylamine, and as further defined below.
"Air-dried sample" means a pretreated sample which had been allowed to air-dry at ambient temperature and pressure to the point where its moisture content was in equilibrium with that of the ambient air, typically ≥85% dry matter.
"Substantially lignin-free biomass" means a pretreated sample in which about >75% of the lignin is removed.
"Dry biomass" means biomass with a dry matter content of >85%. Methods for drying the biomass include exposure at ambient temperature to vacuum or flowing air at atmospheric pressure and or heating in an oven or a vacuum oven.
"Multi-component solvent" means a solvent containing organic solvent, water, and reagents capable of chemical attack on the lignin. "Pressure vessel" is a sealed vessel that may be equipped or not with a mechanism for agitation of a biomass/solvent suspension, in which a positive pressure is developed upon heating the lignocellulosic biomass.
"Nucleophile" is a chemical reagent capable of forming a covalent bond with its reaction partner by contributing both of the bonding electrons.
"Hydrolysate" refers to the liquid in contact with the lignocellulose biomass which contains the products of hydrolytic reactions acting upon the biomass (either enzymatic or not), in this case monomeric and oligomeric sugars. "Organosolv" means a mixture of organic solvent and water which is typically in contact with biomass and in which the lignin or its fragments are soluble. "Enzyme consortium" or "sacchahfication enzyme consortium" is a collection of enzymes, usually secreted by a microorganism, which in the present case will typically contain one or more cellulases, xylanases, glycosidases, ligninases and esterases. "Monomeric sugars" or "simple sugars" consist of a single pentose or hexose unit, e.g., glucose, xylose and arabinose.
"Delignification" is the act of removing lignin from lignocellulosic biomass. In the context of this application, delignification means fragmentation and extraction of lignin from the lignocellulosic biomass using an organic solvent under alkaline conditions at elevated temperatures in the presence of alkylamines and optionally various nucleophiles.
"Fragmentation" is a process in which lignocellulosic biomass is treated with organic solvent under alkaline conditions breaking the lignin down into smaller subunits.
"Selective extraction" is a process by which fragmented lignin is dissolved by treatment with an organic solvent under alkaline conditions leaving behind the polysaccharide.
"Simultaneous fragmentation and selective extraction" as used herein refers to a fragmentation reaction performed in organic solvent such that the lignin fragments go into solution as soon as they are released from the bulk biomass.
Methods for pretreating lignocellulosic biomass to produce readily saccharifiable biomass are provided. These methods provide economical processes for rendering components of the lignocellulosic biomass more accessible or more amenable to enzymatic saccharification. The pretreatment can be chemical, physical or biological, or any combination of the foregoing. In this disclosure the pretreatment is performed in the presence of nucleophiles, specifically in the presence of a sulfide salt such as ammonium sulfide under alkaline conditions. Additional nucleophiles may also be present, such as NH 3 , one or more alkylamines, sulfide reagents, or combinations thereof. The presence of an organic solvent and alkaline conditions assists lignin fragmentation and removal and carbohydrate recovery.
In addition, the methods described in the present disclosure minimize the loss of carbohydrate during the pretreatment process and maximize the yield of solubilized (monomehc + oligomeric) sugars in saccharification.
As disclosed above the methods described herein include pretreating lignocellulosic material, with a solvent solution comprising the components described below, to produce a readily sacchahfiable carbohydrate-enriched biomass. Solvents
The methods described herein include use of an organic solvent for pretreating biomass and specifically for fragmentation and extraction of lignin. Solvents useful in the present methods are frequently referred to in the art as Organosolv (e.g., E. Muurinen (2000) Organosolv Pulping, A review and distillation study related to peroxyacid pulping Thesis, University of OuIu, pp. 314; S. Aziz, K. Sarkanen, Tappi J., 72/73: 169- 175, 1989; A. K. Varsheny and D. Patel, J. Sci. Ind. Res., 47: 315-319, 1988; A. A. Shatalov and H. Pereira, BioResources 1 : 45-61 , 2006; T. N. Kleinert, Tappi J., 57: 99-102, 1979; Practice of organosolv technology for biofuels, derived from Kleinert, which has advanced to the pilot scale using EtOH/H 2 O has been described (WO 20071051269), and X. Pan, N. Gilkes, J. Kadla, K. Pye, S. Saka, D. Gregg, K. Ehara, D. Xie, D. Lam, and J. Saddler, Biotechnol. Bioeng., 94: 851 -861 , 2006. While still at lab scale, use of acetone/H 2 O is described in U.S. patent 4,470,851. Further details on pretreatment technologies related to use of solvents and other pretreatments can be found in Wyman et al., (Bioresource Tech., 96: 1959, 2005); Wyman et al., (Bioresource Tech., 96: 2026, 2005); Hsu, ("Pretreatment of biomass" In Handbook on Bioethanol: Production and Utilization, Wyman, Taylor and Francis Eds., p. 179-212, 1996); and Mosier et al., (Bioresource Tech., 96: 673, 2005). Solvents are used herein for pretreating biomass to remove lignin. Delignification is typically conducted at temperatures of 165 - 225 0 C, at liquid to biomass ratios of 4:1 to 20:1 , at liquid compositions of 50% organic solvent (v/v), and at reaction times between 0.5 -12 hours. A number of mono- and polyhydroxy-alcohols have been tested as solvents. Ethanol, butanol and phenol have been used in these reactions (Park, J. K., and Phillips, J. A., Chem. Eng. Comm., 65: 187-205, 1988).
The organosolv or organic solvent solution pretreatment in the present methods may comprise a mixture of water and an organic solvent at selected condition parameters that include temperature, time, pressure, solvent-to-water ratio and solids-to-liquid ratio. The solvent can comprise, but is not limited to, alcohols and aprotic solvents (solvents that do not have a hydrogen atom bound to an oxygen as in a hydroxyl group or a nitrogen as in an amine group or a sulfur as in a thiol group, e.g., ketones). The alcohols may include methanol, ethanol, propanol, butanol, pentanol and hexanol and isomers thereof and diols with the same number of carbon atoms, such as 1 ,2-ethanediol, 1 ,2-propandiol, 1 ,3-propanediol, 1 ,3-hexanediol.
The concentration of the solvent in solution (i.e. water) in the present invention is from about 2 to about 90% (v/v), or from about 10% to about 85% or from about 20% to about 80% or from about 30% to about 80% or more preferably from about 40% to about 70% (v/v). Specifically, for purposes of an embodiment of the methods herein, EtOH in H 2 O mixtures from about 0% - 80% (v/v) ethanol concentrations were examined and solutions containing 40-70% (v/v) EtOH were found to be most effective. Sulfide or polvsulfide salts
According to the present method, ammonium sulfide is added to the alkaline organic solvent mixture in the presence of ammonia or alkylamines increasing lignin fragmentation and extraction, and resulting in an increased accessibility of the carbohydrate-enriched biomass to enzymatic saccharification. In the present invention, concentrations of ammonium sulfide from 0.5% to 15% (w/w biomass) could be used. More specifically concentrations of 1 % to 6% (w/w biomass) are more useful. Even more specifically concentrations of 2% to 4% (w/w biomass) would be most useful. Alkylammonium sulfides function similar to ammonium sulfide, with the added advantage that alkylamines are better nucleophiles than ammonia. In addition ammonium and alkylammonium polysulfides are expected to behave like the sulfides. Inorganic ions of sulfides and polysulfides also work under organosolv conditions to fragment and extract lignin. Additional components of the solvent solution
In one embodiment, alkylamines are used for pretreatment of biomass according to the present methods as components of the organic solvent solution. Alkylamines are strong bases owing to electron donation to the amine nitrogen by the alkyl chain carbons, and consist of primary amines (R-NH 2 ), secondary amines (R-N-R') and tertiary amines where R is an alkyl chain. Specifically R could be selected from a group consisting of a monovalent, divalent or trivalent 1 -6 carbon alkane, alkene or alkyne, linear, cyclic or branched. Examples of alkylamines include, mono, di- and tri-methylamine, mono, di- and tri-ethylamine, mono, di- and tri- propylamine, mono, di- and th-butylamine. Alkylamines include mono-, di- and tri-amines, alcohol amines (HO-R-NH 2 ), diolamines ((HO) 2 -R-NH 2 ), alcohol diamines (HO-R-(NH 2 ) 2 ), thiolamines (HS-R-NH 2 ), dithiolamines ((HS) 2 -R-NH 2 ), thioldiamines (HS-R-(NH 2 ) 2 ) and alcohol thiolamines (H 2 N- R(OH)(SH) where R is as defined.
Suitable alkylamines for this invention comprise: methylamine (MA), dimethylamine (DMA), trimethylamine (TMA), ethylamine, propylamine, and butylamine. The more suitable alkylamines for this invention include, but are not limited to MA and DMA. The concentration of the alkylamines according to the present method may be used from about 1 % to about 20 wt% of dry biomass. In accordance with the present methods alkylamines, especially MA and DMA, are highly active in a concentration ranges of from 10 to 14% relative to dry weight of biomass. In this concentration range there is sufficient alkylamine to assure that the pH of the solvent solution remains high and that the concentration of alkylamine is sufficient to assure continued lignin fragmentation as pretreatment occurs. The inorganic base could be used at various concentrations of at least from 0.5% to about 16% (wt% of dry biomass). More suitable are the concentrations from 1 % to 10%. Most suitable are the concentrations between 2% to 8% (wt% of dry biomass). Lignocellulosic Biomass
The lignocellulosic biomass pretreated herein includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Examples of biomass include, but are not limited to corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sugar cane straw, yellow poplar, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers and animal manure.
In one embodiment, the lignocellulosic biomass includes agricultural residues such as corn stover, wheat straw, barley straw, oat straw, rice straw, canola straw, and soybean stover; grasses such as switchgrass, miscanthus, cord grass, and reed canary grass; fiber process residues such as corn fiber, beet pulp, pulp mill fines and rejects and sugar cane bagasse; sugar cane straw and sorghum; forestry wastes such as yellow poplar, aspen wood, other hardwoods, softwood and sawdust; and post-consumer waste paper products; as well as other crops or sufficiently abundant lignocellulosic material. In another embodiment, biomass that is useful for the invention includes biomass that has a relatively high carbohydrate content, is relatively dense, and/or is relatively easy to collect, transport, store and/or handle.
In another embodiment of the invention, biomass that is useful includes corn cobs, corn stover, sugar cane bagasse, sugar cane straw, yellow poplar and switchgrass.
The lignocellulosic biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass could comprise a mixture of corn cobs and corn stover, or a mixture of stems or stalks and leaves.
In the present method, the biomass dry weight is at an initial concentration of at least about 9% up to about 80% of the weight of the biomass-solvent suspension during pretreatment. More suitably, the dry weight of biomass is at a concentration of from about 15% to about 70%, 15% to about 60%, or about 15% to about 50% of the weight of the biomass-solvent suspension. The percent of biomass in the biomass- solvent suspension is kept high to reduce the total volume of pretreatment material, decreasing the amount of solvent and reagents required and making the process more economical.
The biomass may be used directly as obtained from the source, or may be subjected to some preprocessing, for example, energy may be applied to the biomass to reduce the size, increase the exposed surface area, and/or increase the accessibility of lignin and of cellulose, hemicellulose, and/or oligosaccharides present in the biomass to organosolv pretreatment and to saccharification enzymes used, respectively, in the second and third steps of the method. Energy means useful for reducing the size, increasing the exposed surface area, and/or increasing the accessibility of the lignin, and the cellulose, hemicellulose, and/or oligosaccharides present in the biomass to the organosolv pretreatment and to saccharification enzymes include, but are not limited to, milling, crushing, grinding, shredding, chopping, disc refining, ultrasound, and microwave. This application of energy may occur before or during pretreatment, before or during saccharification, or any combination thereof.
Drying prior to pretreatment may occur as well by conventional means, such as exposure at ambient temperature to vacuum or flowing air at atmospheric pressure and or heating in an oven at atmospheric pressure or a vacuum oven. Pretreatment Conditions
Pretreatment of biomass with the solvent solution comprising one or more sulfide salt under alkaline conditions is carried out in any suitable vessel. Typically the vessel is one that can withstand pressure, has a mechanism for heating, and has a mechanism for mixing the contents. Commercially available vessels include, for example, the Zipperclave ® reactor (Autoclave Engineers, Erie, PA), the Jaygo reactor (Jaygo Manufacturing, Inc., Mahwah, NJ), and a steam gun reactor (described in General Methods Autoclave Engineers, Erie, PA). Much larger scale reactors with similar capabilities may be used. Alternatively, the biomass and organosolv solution may be combined in one vessel, then transferred to another reactor. Also biomass may be pretreated in one vessel, then further processed in another reactor such as a steam gun reactor (described in General Methods; Autoclave Engineers, Erie, PA).
The pretreatment reaction may be performed in any suitable vessel, such as a batch reactor or a continuous reactor. One skilled in the art will recognize that at higher temperatures (above 100 0 C), a pressure vessel is required. The suitable vessel may be equipped with a means, such as impellers, for agitating the biomass-organosolv mixture. Reactor design is discussed in Lin, K.-H., and Van Ness, H. C. (in Perry, R. H. and Chilton, C. H. (eds), Chemical Engineer's Handbook, 5 th Edition (1973) Chapter 4, McGraw-Hill, NY). The pretreatment reaction may be carried out either as a batch,or a continuous process.
Prior to contacting the biomass with solvent, vacuum may be applied to the vessel containing the biomass. By evacuating air from the pores of the biomass, better penetration of the solvent into the biomass may be achieved. The time period for applying vacuum and the amount of negative pressure that is applied to the biomass will depend on the type of biomass and can be determined empirically so as to achieve optimal pretreatment of the biomass (as measured by the production of fermentable sugars following sacchahfication).
The heating of the biomass with solvent is carried out at a temperature of from about 100 0 C to about 220 0 C, about 150 0 C to 200 0 C, or about 165 0 C to about 195 0 C. The heated solution may be cooled rapidly to room temperature. In still another embodiment, the heating of the biomass is carried out at a temperature of about 180 0 C. Heating of the biomass-solvent suspension may occur for about 5 minutes to about 5 hours, or for about 30 minutes to about 3 hours, or more preferably from about 1 to 2 hours.
For the pretreatment methods described herein, the temperature, pH, time of pretreatment and concentration of reactants such as the organic solvent and ammonium sulfide solutions, under alkaline conditions, and the concentration of one or more additional reagents, biomass concentration, biomass type and biomass particle size are related; thus these variables may be adjusted as necessary for each type of biomass to optimize the pretreatment processes described herein.
The pretreatment of biomass with the solvent solution, one or more alkylamine and one or more sulfide salts occurs under alkaline conditions at a pH that is equal to or greater than the pKa of the nucleophiles present. Under these high pH conditions at least 50% of the nucleophiles are in their deprotonated states. Deprotonation typically increases the reactivity of the nucleophiles. The nucleophiles present, in addition to alkylamine, can include ammonia, thiols, polysulfides, or hydrosulfide.
Following pretreatment at elevated temperature the biomass is filtered under pressure. The filtration may either be preceded or not by cooling. Following filtration, the biomass may be washed one or more times with hydrated organic solvent at elevated or at ambient temperature. It may then either be washed with water or dried to remove the organic solvent and then saccharified. Methods for drying the biomass were described above. To assess performance of the pretreatment, i.e., the production of readily sacchahfiable carbohydrate-enriched biomass and subsequent saccharification, separately or together, the theoretical yield of sugars derivable from the starting biomass can be determined and compared to measured yields. Pretreatment performance may be further assessed by relating how enzyme loadings affect target product yields in overall system performance. Further Processing Saccharification Following pretreatment, the readily saccharifiable carbohydrate- enriched biomass comprises a mixture of organic solvent, a sulfide salt and any additional components of the solvent solution such as alkylamines or ammonia; fragmented and extracted lignin; and polysaccharides. Prior to further processing, the ammonium or alkylammonium sulfides or polysulfides, and/or additional solvent components such as alkylamines or ammonia and lignin fragments may be removed from the pretreated biomass by filtration and washing the sample with EtOH in H 2 O (0% to 100% EtOH v/v) or water. The biomass may be washed with water to remove EtOH or be dried resulting in carbohydrate-enriched, readily saccharifiable biomass and the concentration of glucan, xylan and acid- insoluble lignin content of the said biomass may be determined using analytical means well known in the art. It is a real benefit of this invention that the pretreated biomass can be either washed with water or dried for saccharification. The readily saccharifiable biomass may then be further hydrolyzed in the presence of a saccharification enzyme consortium to release oligosaccharides and/or monosaccharides in a hydrolysate.
Surfactants such as Tween 20 or Tween 80 or polyoxyethylenes such as PEG 2000, 4000 or 8000 may be added to improve the saccharification process (U.S. patent 7,354,743 B2, incorporated herein by reference). The addition of surfactant (e.g., Tween 20) to the enzymatic saccharification often enhances the rate and yield of monomeric sugar release. It is likely that the surfactant coats any residual lignin, decreasing the non-productive binding of the enzyme to the lignin. An alternative approach is to either enhance the extraction of lignin in the pretreatment or to modify the lignin chemically such that less enzyme is lost to lignin adsorption.
Saccharification enzymes and methods for biomass treatment are reviewed in Lynd, L. R., et al., (Microbiol. MoI. Biol. Rev., 66:506-577, 2002). The saccharification enzyme consortium may comprise one or more glycosidases; the glycosidases may be selected from the group consisting of cellulose-hydrolyzing glycosidases, hemicellulose- hydrolyzing glycosidases, and starch-hydrolyzing glycosidases. Other enzymes in the sacchahfication enzyme consortium may include peptidases, lipases, ligninases and esterases.
The saccharification enzyme consortium comprises one or more enzymes selected primarily, but not exclusively, from the group "glycosidases" which hydrolyze the ether linkages of di-, oligo-, and polysaccharides and are found in the enzyme classification EC 3.2.1.x (Enzyme Nomenclature 1992, Academic Press, San Diego, CA with Supplement 1 (1993), Supplement 2 (1994), Supplement 3 (1995, Supplement 4 (1997) and Supplement 5 [in Eur. J. Biochem., 223:1 -5, 1994; Eur. J. Biochem., 232:1 -6, 1995; Eur. J. Biochem., 237:1 -5, 1996; Eur. J. Biochem., 250:1 -6, 1997; and Eur. J. Biochem., 264:610-650 1999, respectively]) of the general group "hydrolases" (EC 3.). Glycosidases useful in the present method can be categorized by the biomass component that they hydrolyze. Glycosidases useful for the present method include cellulose-hydrolyzing glycosidases (for example, cellulases, endoglucanases, exoglucanases, cellobiohydrolases, β- glucosidases), hemicellulose-hydrolyzing glycosidases (for example, xylanases, endoxylanases, exoxylanases, β-xylosidases, arabino- xylanases, mannases, galactases, pectinases, glucuronidases), and starch-hydrolyzing glycosidases (for example, amylases, α-amylases, β- amylases, glucoamylases, α-glucosidases, isoamylases). In addition, it may be useful to add other activities to the saccharification enzyme consortium such as peptidases (EC 3.4.x.y), lipases (EC 3.1.1.x and 3.1.4.x), ligninases (EC 1.11.1.x), and feruloyl esterases (EC 188.8.131.52) to help release polysaccharides from other components of the biomass. It is well known in the art that microorganisms that produce polysaccharide- hydrolyzing enzymes often exhibit an activity, such as cellulose degradation, that is catalyzed by several enzymes or a group of enzymes having different substrate specificities. Thus, a "cellulase" from a microorganism may comprise a group of enzymes, all of which may contribute to the cellulose-degrading activity. Commercial or noncommercial enzyme preparations, such as cellulase, may comprise numerous enzymes depending on the purification scheme utilized to obtain the enzyme. Thus, the saccharification enzyme consortium of the present method may comprise enzyme activity, such as "cellulase", however it is recognized that this activity may be catalyzed by more than one enzyme. Saccharification enzymes may be obtained commercially, in isolated form, such as Spezyme ® CP cellulase (Genencor International, Rochester, NY) and Multifect ® xylanase (Genencor). In addition, saccharification enzymes may be expressed in host microorganisms at the biofuels plant, including using recombinant microorganisms. One skilled in the art would know how to determine the effective amount of enzymes to use in the consortium and adjust conditions for optimal enzyme activity. One skilled in the art would also know how to optimize the classes of enzyme activities required within the consortium to obtain optimal saccharification of a given pretreatment product under the selected conditions.
Preferably the saccharification reaction is performed at or near the temperature and pH optima for the saccharification enzymes. The temperature optimum used with the saccharification enzyme consortium in the present method ranges from about 15 0 C to about 100 0 C. In another embodiment, the temperature optimum ranges from about 20 0 C to about 80 0 C and most typically 45-50° C. The pH optimum can range from about 2 to about 11. In another embodiment, the pH optimum used with the saccharification enzyme consortium in the present method ranges from about 4 to about 5.5. The saccharification can be performed for a time of about several min to about 120 hours, and preferably from about several minutes to about 48 hours. The time for the reaction will depend on enzyme concentration and specific activity, as well as the substrate used, its concentration (i.e., solids loading) and the environmental conditions, such as temperature and pH. One skilled in the art can readily determine optimal conditions of temperature, pH and time to be used with a particular substrate and saccharification enzyme(s) consortium. The saccharification can be performed batch-wise or as a continuous process. The saccharification can also be performed in one step, or in a number of steps. For example, different enzymes required for saccharification may exhibit different pH or temperature optima. A primary treatment can be performed with enzyme(s) at one temperature and pH, followed by secondary or tertiary (or more) treatments with different enzyme(s) at different temperatures and/or pH. In addition, treatment with different enzymes in sequential steps may be at the same pH and/or temperature, or different pHs and temperatures, such as using cellulases stable and more active at higher pHs and temperatures followed by hemicellulases that are active at lower pHs and temperatures.
The degree of solubilization of sugars from biomass following saccharification can be monitored by measuring the release of monosaccharides and oligosaccharides. Methods to measure monosaccharides and oligosaccharides are well known in the art. For example, the concentration of reducing sugars can be determined using the 1 ,3-dinitrosalicylic (DNS) acid assay (Miller, G. L., Anal. Chem., 31 : 426-428, 1959). Alternatively, sugars can be measured by HPLC using an appropriate column as described below. Fermentation To Target Products
The readily saccharifiable biomass produced by the present methods may be hydrolyzed by enzymes as described above to produce fermentable sugars which then can be fermented into a target product. "Fermentation" refers to any fermentation process or any process comprising a fermentation step. Target products include, without limitation alcohols (e.g., arabinitol, butanol, ethanol, glycerol, methanol, 1 ,3- propanediol, sorbitol, and xylitol); organic acids (e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, propionic acid, succinic acid, and xylonic acid); ketones (e.g., acetone); amino acids (e.g., aspartic acid, glutamic acid, glycine, lysine, serine, and threonine); gases (e.g., methane, hydrogen (H 2 ), carbon dioxide (CO 2 ), and carbon monoxide (CO)).
Fermentation processes also include processes used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry, and tobacco industry.
Further to the above, the sugars produced from saccharifying the pretreated biomass as described herein may be used to produce in general, organic products, chemicals, fuels, commodity and specialty chemicals such as xylose, acetone, acetate, glycine, lysine, organic acids (e.g., lactic acid), 1 ,3-propanediol, butanediol, glycerol, 1 ,2-ethanediol, furfural, polyhydroxy- alkanoates, cis, c/s-muconic acid, and animal feed (Lynd, L. R., Wyman, C. E., and Gerngross, T. U., Biocom. Eng. Biotechnol. Prog., 15: 777-793, 1999; and Philippidis, G. P., Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, D. C, 179- 212, 1996; and Ryu, D. D. Y., and Mandels, M., Cellulases: biosynthesis and applications, Enz. Microb. Technol., 2: 91 -102, 1980).
Potential coproduction of products may also be produced, such as multiple organic products from fermentable carbohydrate. Lignin-rich residues remaining after pretreatment and fermentation can be converted to lignin-dehved chemicals, chemical building blocks or used for power production.
Conventional methods of fermentation and/or saccharification are known in the art including, but not limited to, saccharification, fermentation, separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), simultaneous saccharification and cofermentation (SSCF), hybrid hydrolysis and fermentation (HHF), and direct microbial conversion (DMC).
SHF uses separate process steps to first enzymatically hydrolyze cellulose to sugars such as glucose and xylose and then ferment the sugars to ethanol. In SSF, the enzymatic hydrolysis of cellulose and the fermentation of glucose to ethanol is combined in one step (Philippidis, G. P., supra). SSCF includes the cofermentation of multiple sugars (Sheehan, J., and Himmel, M., Bioethanol, Biotechnol. Prog., 15: 817-827, 1999). HHF includes two separate steps carried out in the same reactor but at different temperatures, i.e., high temperature enzymatic saccharification followed by SSF at a lower temperature that the fermentation strain can tolerate. DMC combines all three processes (cellulase production, cellulose hydrolysis, and fermentation) in one step (Lynd, L. R., Weimer, P. J., van ZyI, W. H., and Pretohus, I. S., Microbiol. MoI. Biol. Rev.,, 66: 506-577, 2002). These processes may be used to produce target products from the readily saccharifiable biomass produced by the pretreatment methods described herein. Advantages of the Present Methods
Sulfides are among the best soft nucleophiles. By incorporating sulfides into an organic solvent process under alkaline conditions (due to the presence of alkali metal or alkaline earth hydroxide or ammonia or alkylamine or a combination thereof), the anionic sulfide (hydrosulfide), is primed to carry out substitution reactions on the aryl ethers of the lignin. The alkaline conditions also favor the formation of quinone methides from lignin. These are also readily attacked by sulfides. The presence of ammonia and/or alkylamines, in addition to raising the pH, supplements the sulfide nucleophilic chemistry in attacking the lignin, and likely protect the polysaccharide against peeling reactions, that result in monosaccharide release and loss at high pH. The use of alkylamines and/or ammonia as bases avoids the generation of an inorganic waste stream which would otherwise add to the financial and environmental cost of the process. The sulfides can also act as a reducing agents, promoting the reduction of quinone methides, eliminating β-aryl ethers as phenoxyl radicals and protecting sugar residues from oxidative reactions. The use of sulfides in the lignocellulosic biomass pretreatment process enhances lignin fragmentation and therefore increases the selectivity of lignin extraction with respect to carbohydrate, producing carbohydrate-enriched biomass that is highly susceptible to enzymatic saccharification. Methods described in this invention for pretreatment of the lignocellulosic biomass using an organic solvent-mediated fragmentation in the presence of one or more ammonium or alkylammonium sulfides and various nucleophiles in combination with selective extraction of lignin at elevated temperatures under alkaline conditions will provide a cost-effective process to obtain carbohydrate-enriched biomass for enzymatic saccharification. Such biomass then, produces very high yields of fermentable sugars (glucose, as well as xylose) for their bioconversion to value-added chemicals and fuels.
EXAMPLES PRETREATMENT OF BIOMASS TO OBTAIN READILY SACCHARIFIABLE CARBOHYDRATE-ENRICHED BIOMASS
The goal of the experimental work described below was to develop a pretreatment process for lignocellulose that maximized both lignin extraction and sugar retention and to produce a readily sacchahfiable carbohydrate-enriched biomass that may be further processed to result in a maximal monomeric sugar yield following enzymatic saccharification. The approach adopted was to selectively fragment and extract the lignin into a suitable solvent while retaining the sugars in the solids residue. The following experiments show the development of a solvent solution that combines the presence of nucleophiles like sulfide salts, alkylamines, NH 3 , and thiol for selective extraction of lignin. It was found that the combined presence of an organic solvent and a sulfide salt and optionally certain nucleophiles like alkylamine, NH 3 , and thiol reactants selectively fragmented and dissolved the lignin components of biomass providing for the generation of readily sacchahfiable carbohydrate-enriched biomass. Ground sugar cane bagasse, which was milled in a Wiley Knife mill, through a 1 mm sieve, was used in all Examples.
The following abbreviations are used in the Examples: "HPLC" is High Performance Liquid Chromatography, "C" is degrees Centigrade or Celsius; "%" is percent; "wt" is weight; "w/w" is weight for weight; "mL" is milliliter; "OD" is outer diameter; "ID" is internal diameter; "h" is hour(s); "rpm" is revolution per minute; "EtOH" is ethanol; "mg/g" is milligram per gram; "g/100 mL" is gram per 100 milliliters; "N" is normal; "g" is gram; "w/v" is weight per volume; "v/v" is volume for volume; "mm" is millimeter; "mL/min" is milliliter per minute; "min" is minutes; "mM" is millimolar. Materials
Sulfuric acid, ammonium hydroxide, acetic acid, acetamide, yeast extract, 2-morpholinoethanesulfonic acid (MES), potassium phosphate, glucose, xylose, tryptone, sodium chloride and citric acid, monomethyl and dimethylamine were obtained from Sigma-Aldrich (St. Louis, MO). Spezyme CP and Multifect CX12L were from Genecor (Genencor International, Palo Alto, CA) and Novozyme 188 was from Novozyme (Bagsvaerd, Denmark).
EFFECTIVE ETHANOL CONCENTRATION The purpose of this Example was to examine the effect of the concentration of solvent (e.g., ethanol) in water on the recovery of carbohydrate and on the solubilization/extraction of lignin in the absence of pH control. Bagasse (0.2 g, 95.78% dry matter) was suspended in 1.56 mL of an EtOH in water solution containing various concentrations (from 0 to 80%) of EtOH. The suspensions were loaded into type 316 stainless steel tubing (1/4 inches ID, 3/8 inches OD, 4 inches long) capped by Swagelock fittings (Penn Fluid System Technologies, Huntingdon Valley, PA). These were placed in a fluidized sand bath (Techne Model SBS-4, Techne Inc., Burlington, NJ) and heated at 180 0 C for 2h and cooled rapidly by plunging into a water bath at room temperature. The samples were removed from the tubes and filtered by centhfugation at 14,000 rpm using Spin-X filters (Costar, Corning Inc., Corning NY) at room temperature in a table top centrifuge (Spectrifuge 16M, Labnet International Inc., Edison, NJ) to remove the dissolved lignin. The retentate of each sample was washed (4x) with 0.5 mL of EtOH in H 2 O using the same EtOH concentration as used in the 180 0 C treatment (0- 80% EtOH in H 2 O). The samples were then allowed to air dry at room temperature (to -92% dry matter) and the glucan, xylan and acid-insoluble lignin contents of the residues determined using the National Renewable Energy Laboratory (NREL) procedure (Determination of Structural Carbohydrates and Lignin in Biomass - Version 2006, Amie Sluiter et al., available from the NREL website. Subsequent enzymatic saccharification
The air-dried sample prepared above was suspended in 50 mM citrate buffer, pH 4.6 at a -14% solids loading. The saccharification enzymes, e.g. Spezyme CP, Multifect CX12L and Novozyme 188 were added at concentrations of 6:3:6 mg/g cellulose, respectively. Also added were 1 % (g/100 ml_) Tween 20 and 0.01 % (w/v) NaN 3 , the latter to prevent microbial growth. Samples (-0.4 ml_) were placed in screw cap vials containing two 5 mm glass beads and incubated at 46 0 C on a rotary shaker run at 250 rpm. Aliquots were removed for analysis at 4h and at every 24h interval from the start and diluted 41.25-fold with 0.01 N H 2 SO 4 . The samples were then filtered through Spin-X filters and the filtrates were analyzed by HPLC (Agilent series 1100/1200, Agilent Technologies, Wilmington, DE). A BioRad HPX-87H Aminex column (Bio-Rad
Laboratories, Hercules, CA) was used to fractionate the released sugars using 0.01 N H 2 SO 4 as the mobile phase at a flow rate of 0.6 mL/min. The column was maintained at 60 0 C. A differential refractive index detector was used to detect the eluted sugars and was maintained at 55 0 C. The retention times for glucose, xylose and arabinose were 9.05, 9.72 and 10.63 min, respectively. Table 1 A outlines the percentages of glucan and xylan recovery and the percent change in acid insoluble (Al) lignin content after pretreatments at EtOH concentrations of 0% - 80%. Concentration of Bagasse was (0.2 g/1.56 mM) variable concentrations of EtOH were used at 180 0 C for 2h
TABLE 1A Glucan and xylan recovery following pretreatment according to Example 1
Results shown in Table 1A indicate that lignin extraction increased with increasing EtOH content presumably because the solubility of lignin increased with increasing EtOH concentration. However, the amount of lignin extracted remained modest even at high ethanol concentrations.
Hemicellulose hydrolysis and the solubility of xylose oligomers decreases with increasing EtOH, increasing the recovery of xylan and xylose oligomers in the residue. The amount of acetate liberated by the pretreatment also decreased with increasing EtOH content, consistent with decreasing auto hydrolysis of the biomass at increasing EtOH concentration.
Table 1 B shows the glucose and xylose yields after 96h of enzymatic saccharification following pretreatment at different EtOH concentrations. The saccharification of cellulose increased when the concentration of EtOH in pretreatment was increased from 0 to 20%, ,but then declined with higher pretreatment concentrations of EtOH. A likely decrease in partial hydrolysis of lignin and cellulose (increase in degree of polymerization, of cellulose which lowered the glucose yield on subsequent saccharification- Table 1 B) was observed at concentrations of more than 20% EtOH.
TABLE 1 B Monomeric glucose and xylose yields following enzymatic saccharification for 96h, pretreated as described in Example 1
The monomeric sugar recoveries (Table 1 B), particularly of xylose, were quite poor at the lower EtOH concentrations. At low EtOH concentration, the acidic conditions, produced at high temperatures by hydrolysis of the acetyl groups of the hemicellulose, hydrolyze the hemicellulose. The solubilized xylose and some glucose is lost in the filtration and washes that follow the pretreatment. At higher EtOH concentrations there is less partial hydrolysis of the cellulose, hemicellulose and lignin which lowers the saccharification yield. The behavior at the low and high ethanol concentrations together produce low overall yields of monomeric glucose and xylose.
EFFECT OF ALKALINE ORGANIC SOLVENT SOLUTION PRETREATMENT ON LIGNIN EXTRACTION
The purpose of this Example was to examine the effect of raising the pH of an organic solvent solution pretreatment at different EtOH in H 2 O ratios on carbohydrate retention and lignin extraction and on monomeric sugar during subsequent enzymatic saccharification. Given that autohydrolysis lowers the pH, hydrolyzes xylan, and promotes the loss of xylose, the pH of the pretreatment was elevated by the addition of NaOH. The effect of higher pH on xylose recovery is demonstrated below. Sugar cane bagasse (0.25 g, 95.78% dry matter) was suspended in 1.75 mL of a solvent containing EtOH (20-80% in water) and 8% NaOH (w/w biomass) plus 1 mg anthraquinone (AQ, a catalyst for lignin fragmentation). The initial pH of this solution was -13.7. As described in Example 1 , the suspensions were loaded into type 316 stainless steel tubing, capped, treated at 168 0 C for 140 min and cooled in room-temperature water. The samples were removed from the pressure vessels, filtered, washed, air- dried and analyzed all as described above in Example 1. The glucan, xylan, arabinan contents and change in lignin content following pretreatment are shown in Table 2A.
Subsequent enzymatic saccharification was carried out as described in Example 1 except that the Spezyme:Multifect:Novozymes 188 ratio was 12:6:1.2 mg/g dry solids in the presence of 1 % Tween 20 (w/v). Table 2B shows the monomehc sugar yields after 96h of enzymatic saccharification of biomass previously pretreated at the different EtOH concentrations.
Glucan, xylan and arabinan yields following pretreatment according to
Table 2B Monomeric glucose and xylose yields following enzymatic saccharification for 96h, pretreated as described in Example 2
As can be seen in Tables 2A and 2B, the alkaline conditions of this experiment substantially increased the retention of xylan in the pretreatment compared to the autohydrolysis experiments of Example 1. This effect was most pronounced at low EtOH concentrations. The NaOH prevented the solution from becoming acidic (final pH -10.7) and therefore protected the hemicellulose from acid-catalyzed hydrolysis. In addition, significantly more lignin was extracted, presumably through base catalyzed fractionation of the lignin. The overall monomehc sugar yields following sacchahfication were substantially higher than those observed in Example 1. The higher sugar recovery and the greater lignin extraction in the pretreatment, increased the yields of the subsequent enzymatic saccharification. The xylose and glucose saccharification yields peaked at ~ 45% EtOH as a consequence of two opposing processes, i.e., the increasing extraction of lignin at higher EtOH which tends to increase the sugar yields, and the decreasing partial hydrolysis of hemicellulose and of lignin as the EtOH concentration is further increased. It is likely that the formation of quinone methides, which could repolymehze or react with sugars, and "peeling' and alkaline scission reactions of polysaccharide all together contribute to limit the overall sugar yields.
PRETREATMENT OF BIOMASS USING AMMONIUM SULFIDE DURING
The purpose of this Example was to study the effect of ammonium sulfide on biomass pretreatment. Pretreatment was performed as in Example 1 except that sugar cane bagasse (0.375 g, 95.78% dry matter) was suspended in 1.125 mL of solvent (70% EtOH in H 2 O (v/v)) containing 14% MA (w/w biomass) plus 2% or 6% (NH 4 ) 2 S (w/w biomass). The suspensions were loaded into type 316 stainless steel pressure vessels (3/16 inches ID, 1/4 inches OD, 4 inches long), capped and treated as described above in Example 1 , except that solids loading was higher and the samples were heated at 168 0 C, for 140 min. The subsequent enzymatic saccharification was performed for 96 h as described in Example 1 except that the Spezyme:Multifect:Novozymes 188 ratio was 6.68:3.34:1.67 mg/g dry solids in the presence and absence of 1 % Tween 20 (w/v) at a solids loading of 14% (w/w). The saccharification yields on material pretreated as described in the presence and absence of 2% and 6% ammonium sulfide (w/w biomass) are shown in Table 3.
TABLE 3 Yields of glucan and xylan following pretreatment according to Example 3
The comparison of the enzymatic saccharifications in the absence of Tween 20 following pretreatment with 70% EtOH in H 2 O (v/v) plus 14% MA (w/w biomass) containing either 0%, 2% or 6% (NH 4 ) 2 S (w/w biomass) showed that (NH 4 ) 2 S when present in the pretreatment promoted subsequent enzymatic saccharification. The enhanced saccharification was likely associated with an increased fragmentation and extraction of the lignin (Figure 1 ), through reactions similar to those that occur with thioglycolic acid. Table 3 shows that the effect of the 2% (NH 4 ) 2 S pretreatment was quite marked for the saccharification of xylan to xylose and for glucan to glucose. The HPLC chromatographic profiles of the hydrolysates indicated, as in the case of thioglycolic acid, that the enhanced extraction of the lignin by (NH 4 ) 2 S in the pretreatment with no surfactant in the saccharification behaved similarly to the addition of surfactant in the saccharification but without (NH 4 ) 2 S in the pretreatment. The surfactant coats the residual lignin while the (NH 4 ) 2 S reduces the amount of residual lignin. The presence of 6% (NH 4 ) 2 S to the pretreatment produces an additional boost in the saccharification yield, well above that of the sample saccharified with Tween 20, but pretreated in the absence of (NH 4 ) 2 S.
AMMONIUM SULFIDE ENHANCED LIGNIN EXTRACTION Pretreatment was performed as in Example 3 except that the 70% EtOH in H 2 O (v/v) solvent in which the bagasse was suspended contained 14% MA (w/w biomass) with no additions and with 2% or 6% (NH 4 ) 2 S (w/w biomass). The samples were heated at 187 0 C for 1 h. Figure 1 shows the UV absorbance spectra of the filtrates following pretreatment, diluted 5000-fold with 70% EtOH in H 2 O (v/v). The addition of 2% and 6% (NH 4 ) 2 S to the 70% EtOH/H 2 O plus MA showed a very large enhancement in the UV absorption of the filtrate following pretreatment, indicating an increase in the extracted lignin. The enhancement of the lignin extraction by inclusion of (NH 4 ) 2 S in the pretreatment is consistent with the significant enhancement of the subsequent enzymatic saccharification (Table 3).