CHEMICALLY MODIFIED LIGNIN AS REDUCING AGENT FOR ENZYMATIC HYDROLYSIS OF LIGNOCELLULOSIC BIOMASS

Abstract

The present invention relates to a method for increasing the rate of enzymatic hydrolysis of a polysaccharide substrate, said method comprising at least one step of: enzymatic hydrolysis of said substrate with a mixture of enzymes, said mixture comprising at least one enzyme selected from lytic polysaccharide monooxygenases; in the presence of chemically modified lignin, wherein during at least part of the time of said step of enzymatic hydrolysis, H.sub.2O.sub.2 is supplied to the reaction mixture comprising said substrate, said mixture of enzymes and said chemically modified lignin, either from an external source or by generation in situ.

Claims

1. A method for enzymatic hydrolysis of a polysaccharide substrate, said method comprising at least one step of: enzymatic hydrolysis of said substrate with a mixture of enzymes, said mixture of enzymes comprising at least one enzyme selected from lytic polysaccharide monooxygenases, wherein said at least one step of enzymatic hydrolysis occurs in the presence of chemically modified lignin, wherein during at least part of the time of said step of enzymatic hydrolysis, H.sub.2O.sub.2 is added to a reaction mixture comprising said substrate, chemically modified lignin and said mixture of enzymes.

2. The method according to claim 1, wherein the polysaccharide substrate comprises lignocellulosic biomass.

3. The method according to claim 1, wherein H.sub.2O.sub.2 is added directly to said reaction mixture comprising said substrate, chemically modified lignin and said mixture of enzymes.

4. The method according to claim 3, wherein H.sub.2O.sub.2 is added to the reaction mixture at a rate of 10 to 5,000 μmoles hydrogen peroxide per liter reaction mixture per hour.

5. The method according to claim 1 wherein a total amount of hydrogen peroxide added to the reaction mixture is 2 to 1,500 moles per ton of polysaccharide substrate.

6. The method according to claim 1, wherein an amount of chemically modified lignin present in the reaction mixture comprising the polysaccharide substrate is from 1 to 100 grams of dry weight chemically modified lignin per liter of reaction mixture.

7. The method according to claim 1, wherein the chemically modified lignin comprises spent sulfite liquor.

8. The method according to claim 7, wherein the spent sulfite liquor is from a sulfite pretreatment step of cellulosic biomass.

9. The method according to claim 8, wherein the spent sulfite liquor as formed during sulfite pretreatment of cellulosic biomass enters the hydrolysis step together with at least part of a cellulosic substrate, in the form of residual spent sulfite liquor left in lignocellulosic pulp after separating a liquid phase from a solid phase.

10. The method according to claim 7, wherein dry matter content of the spent sulfur liquor is 1.25 to 125 grams of dry weight spent sulfur liquor per liter of reaction mixture.

11. The method according to claim 1, comprising adding the chemically modified lignin to form the reaction mixture, wherein the step of adding the chemically modified lignin allows to lower an amount of enzymes vis-à-vis the same method not comprising said step of adding the chemically modified lignin, wherein the two methods as compared are otherwise the same and lead to essentially a same C6 sugar yield.

12. The method according to claim 11, wherein the amount of enzymes is lowered by 5%.

13. The method according to claim 1, comprising adding the chemically modified lignin to form the reaction mixture, wherein the step of adding the chemically modified lignin allows to increase a C6 sugar yield vis-à-vis the same method not comprising said step of adding the chemically modified lignin, wherein the two methods as compared are otherwise the same and use essentially a same amount and kind of enzymes.

14. The method according to claim 13, wherein the C6 sugar yield is increased by 5%.

15. The method according to claim 4, wherein H.sub.2O.sub.2 is added to the reaction mixture at a rate of 20 to 1,000 μmoles hydrogen peroxide per liter reaction mixture per hour.

16. The method according to claim 4, wherein H.sub.2O.sub.2 is added to the reaction mixture at a rate of 25 to 500 μmoles hydrogen peroxide per liter reaction mixture per hour.

17. The method according to claim 7, wherein the chemically modified lignin essentially consists of spent sulfite liquor.

Description

BRIEF DESCRIPTION OF THE FIGURE

[0040] FIG. 1 shows the C6 sugar yields of enzymatic hydrolysis experiments Example 1, 2, 3 and 4.

DETAILED DESCRIPTION OF THE INVENTION

Feedstock (Substrate)

[0041] In accordance with the present invention, there is no specific restriction in regard to the kind or the origin of the polysaccharide substrate. In preferred embodiments the polysaccharide substrate is or comprises cellulose. In principle, the raw material for the cellulose may be any cellulosic material, in particular wood, annual plants, corn stover, corn cobs, cotton, flax, straw, ramie, bagasse (from sugar cane), suitable algae, jute, sugar beet, citrus fruits, waste from the food processing industry or energy crops or cellulose of bacterial origin or from animal origin, e.g. from tunicates, or polysaccharides from other marine sources.

[0042] In a preferred embodiment, wood-based materials are used as raw materials/substrate, either hardwood or softwood or both (in mixtures). Further preferably softwood is used as a raw material, either one kind or mixtures of different soft wood types. Bacterial microfibrillated cellulose is also preferred, due to its comparatively high purity.

[0043] In embodiments of the present invention, the feedstock is in its native state or it has been subjected to at least one pretreatment step to facilitate enzymatic hydrolysis (e.g. acid hydrolysis, steam explosion, ammonia fiber explosion (AFEX), alkaline wet oxidation, Kraft pulping, sulfite pulping).

[0044] In accordance with the present invention, any feedstock containing polysaccharides may be used since LPMOs per definition are oxidoreductases capable of cleaving glycosidic bonds present in polysaccharides. In embodiments of the present invention, enzymatic hydrolysis is performed, in particular, on cellulose, hemicellulose, starch and polysaccharides from marine sources such as chitin and fucoidan. Any polysaccharide substrate is suitable as long as LPMO enzymes are active on that substrate.

Lignin

[0045] The chemically modified lignin preferably is a water-soluble lignin and can, for instance, be a sulfonated lignin, a carboxylated lignin, a hydrolysed carboxylated lignin, or an amine functionalized lignin.

[0046] In accordance with the present application, the term “lignin” relates to a biopolymer, respectively, a mixture of biopolymers, that is/are present in the support tissues of plants, in particular, in the cell walls providing rigidity to the plants. Lignin is a phenolic polymer, respectively, a mixture of a phenolic polymer. The composition of lignin depends on the plant and therefore varies depending on the plant it is derived from. Lignin in its native form, i.e., as present in the plant, is hydrophobic and aromatic.

[0047] In accordance with the present application, no restrictions exist in regards to the source of the lignin.

[0048] In accordance with the present application, the term “chemically modified lignin” is to be understood to relate to any lignin that is no longer present in its native form, but has been subjected to a chemical process. Processes for making chemically modified lignin are commonly known in the art.

[0049] The chemically modified lignin in accordance with the present invention is preferably water soluble and further preferably a sulfonated lignin. One preferred example of a sulfonated lignin is lignosulfonate. Lignosulfonate is obtained when lignin, respectively, lignin-containing cellulosic biomass, is subjected to sulfite cooking. Thus, lignosulfonate is the organic salt product recovered from digestion of wood (typically acid sulfite pulping with sulfurous acid). Preferred lignosulfonates can thus be described as water-soluble anionic polyelectrolyte polymers.

[0050] The term “lignosulfonate”, as used within the context of the present application, refers to any lignin derivative which is formed during sulfite pulping of lignin-containing material, such as, e.g., wood, in the presence of, for example, sulfur dioxide and sulfite ions, respectively, bisulfite ions.

[0051] For example, during the acidic sulfite pulping of lignin-based material, electrophilic carbon cations in the lignin are produced, which are a result of the acid catalyzed ether cleavage. Thus, lignin may react, via these carbo-cations, with the sulfite, respectively, bisulfite ions under the formation of lignosulfonate.

[0052] Another example of a chemically modified lignin is “Kraft lignin”. Kraft lignin is precipitated from Kraft alkaline pulping liquors, in particular from Kraft process pulp making during which the lignin has been broken down from its native form present in the wood pulp, representing molecular fractions of the original biopolymer. Kraft lignin can therefore be described as precipitated, unsulfonated alkaline lignin. Kraft lignin differs structurally and chemically from lignosulfonate, e.g., in that Kraft lignin is not watersoluble.

[0053] Kraft lignin can be further modified. The term “sulfonated lignin”, as used within the context of the present application, is to be understood as a lignin derivative in which sulfonic acid groups have been introduced. Thus, sulfonated lignin is characterized by the presence of —SO.sub.3.sup.−M.sup.+ groups, wherein M is a cation balancing the anionic charge of the —SO.sub.3.sup.− moiety and which is selected from alkali metal cation, in particular from Li.sup.+, Na.sup.+, or K.sup.+, Ca.sup.++, or Mg.sup.++, or ammonium cation NH.sub.4.sup.+, or mixtures thereof.

[0054] In one embodiment of the invention, the chemically modified lignin is sulfonated lignin obtained from Kraft lignin. In embodiments, sulfonated lignin may be obtained when Kraft lignin is treated with alkali sulfite and alkylaldehyde at elevated temperature and pressure.

[0055] In embodiments, sulfonated lignin is a lignosulfonate obtained by modifying a lignosulfonate, for instance, by subjecting it to ion exchange, preferably by reacting it with sodium sulfate.

Sulfite Pretreatment

[0056] In a preferred embodiment, cellulosic biomass is used as a substrate in the present process, in particular lignocellulosic biomass, which does not require mechanical (pre)treatment, and wherein sulfite pretreatment (“cooking”) is the only (pre)treatment.

[0057] Sulfite cooking may be divided into four main groups: acid, acid bisulfite, weak alkaline and alkaline sulfite pulping.

[0058] In the preferred pretreatment in accordance with the present invention, the cellulosic biomass is cooked with a sulfite, preferably a sodium, calcium, ammonium or magnesium sulfite under acidic, neutral or basic conditions. This pretreatment step dissolves most of the lignin as sulfonated lignin (lignosulfonate; water-soluble lignin), together with parts of the hemicellulose.

[0059] The fact that lignocellulosic pulp resulting from this one-step pretreatment is particularly low in impurities, in particular lignin, makes it easier to develop or adapt enzymes for the hydrolysis.

[0060] Sulfite pretreatment is preferably performed according to one of the following embodiments. Therein and throughout the present disclosure, the “sulfite pretreatment” is also referred to as “cook”: [0061] acidic cook (preferably SO.sub.2 with a hydroxide, further preferably with Ca(OH).sub.2, NaOH, NH.sub.4OH or Mg(OH).sub.2); [0062] bisulfite cook (preferably SO.sub.2 with a hydroxide, further preferably with NaOH, NH.sub.4OH or Mg(OH).sub.2); [0063] weak alkaline cook (preferably Na.sub.2SO.sub.3, further preferably with Na.sub.2CO.sub.3), and [0064] alkaline cook (preferably Na.sub.2SO.sub.3 with a hydroxide, further preferably with NaOH).

[0065] In regard to the sulfite pretreatment step (sulfite cooking), which is a preferred pretreatment to be implemented prior to the enzymatic hydrolysis in accordance with the present invention, the respective disclosure of WO 2010/078930 with the title “Lignocellulosic Biomass Conversion” as filed on Dec. 16, 2009 is incorporated by reference into the present disclosure.

Enzymatic Hydrolysis

[0066] In order to efficiently hydrolyze polysaccharides, it is important to maintain conditions that promote high total activity and ensure long term stability of the LPMO containing enzyme cocktail. Two relevant reaction parameters to consider are temperature and pH-value. Enzyme mixtures of fungal origin have an optimal performance at temperatures between 50-55° C. and within a pH interval of 5.0-5.5. However, other temperature and pH levels may be optimal, depending on the specific enzyme mixture used.

[0067] In order to ensure that temperature and pH are kept at their optimal levels, thorough mixing of the reaction mixture is preferred.

[0068] In accordance with the present invention, any LPMO containing enzyme mixture may be utilized. In preferred embodiments, in order to achieve synergy, enzyme mixtures contain endo-1,4-β-glucanases, exo-1,4-β-glucanases and β-glucosidases in optimized proportions. An enzyme loading sufficient to hydrolyze at least 70% of the substrate within 200 hours of reaction time is preferred.

[0069] The following summarizes the advantages that the method of the present invention is believed to have over the prior art.

[0070] The present invention provides a cost-effective method for enzymatic hydrolysis of polysaccharides in the presence of LPMO enzymes and H.sub.2O.sub.2, by: [0071] providing a low cost reducing agent; [0072] reducing the risk of oxidative enzyme deactivation.

[0073] LPMO enzymes enhance the activity of cellulolytic mixtures significantly by introducing cleavages into the polysaccharide chains, creating entry points for other enzymes. The other enzymes degrade the polysaccharides, exposing new surface areas for the LPMOs. This synergy makes it possible to minimize the enzyme loading in a enzymatic hydrolysis process, with maintained or even increased yield and productivity, thus improving the cost-efficiency of the process.

EXAMPLES

Example 1. Standard Enzymatic Hydrolysis of Spruce Pulp

[0074] This experiment shows enzymatic hydrolysis of spruce pulp under standard conditions, (as described in Example 5). The C6 sugar yield was 60% after 66 hours of hydrolysis (see FIG. 1, lowermost curve). Hydrogen peroxide can be generated by LPMO enzymes from oxygen in the head space through an “empty” cycle, and a limited reducing capacity is provided by residual insoluble lignin as naturally present in the spruce pulp. However, the LPMO activity is restricted and the C6 sugar yield remains low throughout the reaction.

Example 2. Standard Enzymatic Hydrolysis of Spruce Pulp with Added SSL

[0075] This experiment shows enzymatic hydrolysis of spruce pulp with 10 g/L of SSL dry matter added. The C6 sugar yield was 72% after 67 hours of hydrolysis (see FIG. 1, second curve from below). Hydrogen peroxide generated by LPMOs from head space oxygen together with the electrons provided by the reducing agent (chemically modified lignin) allows for increased LPMO activity, which is believed to explain the increased C6 sugar yield compared to Example 1.

Example 3. Enzymatic Hydrolysis of Spruce Pulp with Addition of H.SUB.2.O.SUB.2

[0076] This experiment shows enzymatic hydrolysis of spruce pulp, where 200 μmoles of hydrogen peroxide per liter reaction mixture per hour was continuously added after 20.5 hours of hydrolysis. The C6 sugar yield was 68% after 66 hours of hydrolysis (see FIG. 1, third curve from below). The C6 sugar yield increased compared to Example 1, which is explained by the availability of co-substrate (H.sub.2O.sub.2) and a limited reducing capacity provided by residual insoluble lignin in the spruce pulp, sufficient to yield some LPMO activity.

Example 4. Enzymatic Hydrolysis of Spruce Pulp with Added SSL and H.SUB.2.O.SUB.2 .Addition

[0077] This example (in accordance with the present invention) shows LPMO enhanced enzymatic hydrolysis of spruce pulp with 10 g/L of SSL dry matter present, where 200 μmoles hydrogen peroxide per liter reaction mixture per hour was continuously added after 20.5 hours of hydrolysis. The C6 sugar yield was 87% after 66 hours of hydrolysis (see FIG. 1, uppermost curve). The C6 sugar yield increased significantly compared to Example 1, 2 and 3, which is believed to be due to the combined availability of co-substrate (H.sub.2O.sub.2) and reducing agent (chemically modified lignin).

Materials and Methods

Substrate, Additive and Enzymes

[0078] Sulfite-pulped Norway spruce (Picea abies) and SSL was obtained from a commercial scale sulfite pulp mill (Borregaard AS, Norway). Commercial cellulase mixture Cellic® CTec3 was obtained from Novozymes A/S, Denmark.

Experimental Conditions

[0079] Enzymatic hydrolysis experiments were conducted in a 3.6 L bioreactor (Labfors 5 BioEtOH reactor, Infors-HT, Bottmingen, Switzerland) with 1.8 L working volume, a substrate loading of 12% (w/w) dry matter (DM) of sulfite-pulped Norway spruce and an enzyme loading of 4% (w enzyme/w substrate) of commercial cellulase cocktail Cellic® CTec3. The temperature was 50° C., pH 5 was maintained by automatic addition of 1 M NaOH and the stirring rate was 250 rpm. Hydrolysis reactions were started up as follows. All liquids, including SSL if applicable, but except enzymes, were added to the reactor together with approximately ⅓ of the wet pulp. The reactor was then heated to 50° C. and pH adjusted to approximately 5.1 using 7.5N NaOH. Once the pH and temperature were correct and stable, the enzyme mixture was added.

[0080] The reactor was then left for about 5 minutes to allow for the enzymes to blend in before the rest of the pulp was added. The reactor was then left over night for liquefaction. Automatic pH control was started after liquefaction. In case H.sub.2O.sub.2 was added, H.sub.2O.sub.2 feeding was started 20.5 hours after initiation of the reaction, after liquefaction, to ensure sufficient mixing. H.sub.2O.sub.2 was delivered continuously using a Masterflex L/S Standard Digital peristaltic pump (Cole-Parmer, Vernon Hills, USA); the H.sub.2O.sub.2 feed rate was 200 μmoles hydrogen peroxide per liter reaction mixture h.sup.−1. SSL was added to a concentration of 10 g/L of dry matter, in experiments where SSL was added.

Sample Preparation

[0081] 1.5-2 ml samples were withdrawn at various times throughout the reaction. The samples were centrifuged and kept in the freezer until analysis. Samples for analysis of sulfite and sulfate were diluted in a formaldehyde-solution and kept in a refrigerator until analysis.

Analysis of Sugars and Yield Calculations

[0082] The sugar monomers were analyzed using Agilent HPLC with RI detector, on a Bio-Rad Aminex HPX-87P cation exchange column using MQ-water as mobile phase. The samples were diluted with MilliQ-water and filtered before analysis. C6 sugar yields were calculated according to Zhu, Y. Et al., «Calculating sugar yields in high solids hydrolysis of biomass». Bioresource Technology 2011, 102, 2897-2903.

List of Abbreviations Used

[0083] AscA Ascorbic acid [0084] GMC Glucose-methanol-choline [0085] H.sub.2O.sub.2 Hydrogen peroxide [0086] LPMO Lytic polysaccharide monooxygenase [0087] SSL Spent sulfite liquor [0088] LS Lignosulfonate [0089] PEG Polyethylene glycol [0090] V—TiO.sub.2 Vanadium-doped titanium dioxide