# US 2014/0154746 A1 · Methods for Improvement of Enzymatic Hydrolysis of Lignocellullosic Material

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- Number: US 2014/0154746 A1
- Document ID: US20140154746A1
- Status: Published application
- Kind: A1
- Application number: 14/123,556
- Filed: 2012-06-11
- Published: 2014-06-05
- Earliest claimed: 2011-06-30
- Assignees: Sekab E-Technology AB
- Applicants: Sekab E-Technology AB
- Inventors: Leif Jönsson; Björn Alriksson; Venkata Prabhakar Soudhama
- Web page: https://patentgrep.com/patent/US20140154746A1

## CPC

- C12P 19/14: produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase}
- C08B 1/00: Preparatory treatment of cellulose for making derivatives thereof {, e.g. pre-treatment, pre-soaking, activation}
- C12P 7/10: substrate containing cellulosic material
- C12P 19/02: Monosaccharides (2-ketogulonic acid C12P7/60)
- C12P 19/04: Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
- C12P 19/16: produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin (non-biological hydrolysis of starch C08B30/00)
- C12P 2201/00: Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
- C12P 2203/00: Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
- Y02E 50/10: Biofuels, e.g. bio-diesel
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C08B: POLYSACCHARIDES; DERIVATIVES THEREOF (polysaccharides containing less than six saccharide radicals attached to each other by glycosidic linkages C07H; fermentation or enzyme-using processes C12P19/00; sugar industry C13; production of cellulose D21)
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The present invention relates to a method of enzymatic hydrolysis of a lignocellulosic material, comprising the steps of: a) pretreating the lignocellulosic material to obtain a slurry having a pH of less than 6; b) adding NaOH, Ca(OH)2 and/or CaO to the slurry to increase its pH to at least 8, said addition being carried out at a slurry temperature of at least 60° C.; c) reducing the pH of the slurry to below 7; and optionally cooling the slurry from step b) to a temperature below 60° C.; and d) adding hydrolytic enzymes to the slurry from c) and allowing the slurry to hydrolyze wherein no washing of the slurry is performed prior to step d)

## Claims

### Claim 1

A method of enzymatic hydrolysis of a lignocellulosic material, comprising the steps of:
a) pretreating the lignocellulosic material to obtain a slurry having a pH of less than 6;
b) adding NaOH, Ca(OH)2 and/or CaO to the slurry to increase its pH to at least 8, said addition being carried out at a slurry temperature of at least 60° C.;
c) reducing the pH of the slurry to below 7 and optionally cooling the slurry from step b) to a temperature below 60° C.; and
d) adding hydrolytic enzymes to the slurry from step c) and allowing the slurry to hydrolyze,
wherein no washing of the slurry is performed prior to step d).

### Claim 2

A method of enzymatic hydrolysis of a lignocellulosic material, comprising the steps of:
a) pretreating the lignocellulosic material using SO2 and/or sulfurous acid to obtain a slurry having a pH of less than 5;
b) adding Ca(OH)2 and/or CaO to the slurry to increase its pH to at least 8;
c) reducing the pH of the slurry from step b) to below 7; and
d) adding hydrolytic enzymes to the slurry from c) and allowing the slurry to hydrolyze,
wherein no washing of the slurry is performed prior to step d).

### Claim 3

A method of producing at least one target molecule comprising the steps of:
a) pretreating a lignocellulosic material to obtain a slurry having a pH of less than 6;
b) adding NaOH, Ca(OH)2 and/or CaO to the slurry to increase its pH to at least 8;
c) reducing the pH of the slurry to below 7;
d) adding hydrolytic enzymes to the slurry from c) and subjecting the slurry to enzymatic hydrolysis to obtain an at least partly hydrolyzed slurry; and
e) utilizing the at least partly hydrolyzed slurry from step d) as a substrate in a fermentation process for production of at least one fermentation product,
wherein the enzymatic hydrolysis in step d) and the fermentation process in step e) are performed separately and wherein no washing of the slurry is performed prior to step d).

### Claim 4 (depends on 1)

A method according to claim 1, wherein said method is for production of at least one target molecule from the lignocellulosic material and further comprising a step e) of utilizing the at least partly hydrolyzed slurry from step d) as a substrate for the production of at least one target molecule.

### Claim 5 (depends on 4)

A method according to claim 4 wherein the at least one target molecule is a fermentation product, and the step e) of utilizing the at least partly hydrolyzed slurry comprises subjecting the at least partly hydrolyzed slurry to fermentation.

### Claim 6 (depends on 5)

A method according to claim 5 wherein the enzymatic hydrolysis in step d) and the fermentation in step e) are performed separately in a separate hydrolysis and fermentation process.

### Claim 7 (depends on 5)

A method according claim 5 wherein the enzymatic hydrolysis in step d) and the fermentation in step e) is performed simultaneously in a simultaneous saccharification and fermentation process

### Claim 8 (depends on 3)

A method according to claim 3, wherein the fermentation is performed by a fermenting organism.

### Claim 9 (depends on 3)

A method according to claim 3, wherein the fermentation product is selected from alcohols, acids, alkanes, alkenes, aromatics, aldehydes, ketones, biopolymers, proteins, peptides, amino acids or vitamins.

### Claim 10 (depends on 1)

A method according to claim 1, wherein the addition of hydrolytic enzymes in step d) comprises the addition of at least one glycosidase.

### Claim 11 (depends on 1)

A method according to claim 1, wherein the lignocellulosic material is a wood material or an agricultural residue.

### Claim 12 (depends on 1)

A method according to claim 1, wherein the dry matter content of the slurry in step a) is in the range of from 5 to 40% (w/v).

### Claim 13 (depends on 1)

A method according to claim 1, wherein at least 90% of the liquid in the pretreated slurry from step a) is present during the enzymatic hydrolysis step d).

### Claim at position 14

14-15. (canceled)

### Claim 16 (depends on 10)

A method according to claim 10, wherein the glycosidase is a cellulose-hydrolyzing glycosidase, a hemicellulose hydrolyzing glycosidase and/or a starch hydrolyzing glycosidase.

### Claim 17 (depends on 12)

A method according to claim 12, wherein the dry matter content of the slurry in step a) is in the range of from 8 to 30% (w/v).

### Claim 18 (depends on 12)

A method according to claim 12, wherein the dry matter content of the slurry in step a) is in the range of from 12 to 20% (w/v).