# US 11,999,985 B2 · Cellulosic enzyme recycling from separation of saccharified biomass

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- Number: US 11,999,985 B2
- Document ID: US11999985B2
- Status: Granted
- Kind: B2
- Application number: 17/957,633
- Filed: 2022-09-30
- Published: 2023-04-06
- Granted: 2024-06-04
- Earliest claimed: 2013-03-15
- Assignees: Edeniq, Inc.
- Applicants: Edeniq, Inc.
- Inventors: Deepak Singh; Sandra Jacobson; Kris Ramos; Prachand Shrestha
- Web page: https://patentgrep.com/patent/US11999985B2

## 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}
- C07H 3/04: Disaccharides
- C12P 7/10: substrate containing cellulosic material
- C12P 19/02: Monosaccharides (2-ketogulonic acid C12P7/60)
- C12P 19/12: Disaccharides
- C13K 1/02: obtained by saccharification of cellulosic materials (manufacture of fodder A23K10/32)
- C13K 13/002: {Xylose}
- 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)}
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C13K: SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES (chemically synthesised sugars or sugar derivatives C07H; polysaccharides, e.g. starch, derivatives thereof C08B; malt C12C; fermentation or enzyme-using processes for preparing compounds containing saccharide radicals C12P19/00; production of sucrose C13B)
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The present disclosure provides methods for generating sugars from a cellulosic biomass. The methods combine treatment of the biomass using a high-shear milling device and saccharification of the biomass to partially hydrolyze the biomass. The biomass can be saccharified either after or simultaneously with the high-shear milling treatment. The partially hydrolyzed biomass is then separated into a solids stream with saccharification enzymes, and a liquid stream with sugars. The solids stream and associated enzymes are further incubated under saccharification conditions to produce additional sugars, or are recycled and added to fresh biomass, which is saccharified under high-shear milling conditions. The methods result in improved conversion of cellulosic biomass to glucose.

## Claims

### Claim 1

A method for generating sugars from biomass, comprising:
(a) hydrothermally pretreating the biomass at a temperature from about 150° C. to about 210° C. with a high shear/milling mixing device comprising a rotor and a stator, wherein the high shear/milling mixing device has a gap setting between the rotor and stator of between 0.1 and 2.2 millimeters, thereby reducing the size of biomass particles in the biomass, wherein the pretreating results in production of less than about 10% by weight fermentable sugars;
(b) contacting the biomass with a hydrolytic or saccharification enzyme to hydrolyze components of the biomass to sugars, thereby producing a mixture of solids and a liquid comprising sugars;
(c) separating the mixture into a liquid stream comprising sugars and furfural, and a solids stream comprising solids and the hydrolytic or saccharification enzyme, wherein the activity of the hydrolytic or saccharification enzyme from step (b) is increased;
(d) incubating the solids stream under conditions suitable to hydrolyze components of the solids to sugars, thereby producing additional sugars;
(e) separating the liquid stream produced in (c) into a second liquid stream comprising sugars and a second solid stream comprising solids; and
(f) incubating the second solid stream under conditions suitable to hydrolyze components of the solids to sugars, thereby producing additional sugars.

### Claim 2 (depends on 1)

The method of claim 1, wherein prior to the pretreatment step (a), the biomass is mixed with water to provide a biomass/water mixture.

### Claim 3 (depends on 1)

The method of claim 1, wherein the biomass comprises glucan and at least 80% of the glucan in the biomass is hydrolyzed to glucose in about 6 to about 24 hours.

### Claim 4 (depends on 1)

The method of claim 1, wherein the biomass is a lignocellulosic biomass.

### Claim 5 (depends on 1)

The method of claim 1, wherein the biomass comprises at least about 10% solids w/w prior to the contacting step.

### Claim 6 (depends on 1)

The method of claim 1, wherein the biomass is a pretreated biomass.

### Claim 7 (depends on 1)

The method of claim 1, wherein the conditions in step (a) produce a biomass particle size wherein at least about 80% of the particles have a particle size of from about 2 to about 200 microns.

### Claim 8 (depends on 1)

The method of claim 1, wherein the mixture is separated using a mechanical device, a filter, a membrane, or a tangential flow filtration device.

### Claim 9 (depends on 8)

The method of claim 8, wherein the mechanical device is a centrifuge, a press, or a screen.

### Claim 10 (depends on 1)

The method of claim 1, wherein the separating step occurs at about 2 to about 4 hours after the contacting step.

### Claim 11 (depends on 1)

The method of claim 1, wherein the separating step occurs at about 4 to about 6 hours after the contacting step.

### Claim 12 (depends on 3)

The method of claim 3, wherein the separating step occurs when about 30% to about 60% of the glucan is converted.

### Claim 13 (depends on 1)

The method of claim 1, wherein the incubating step is from about 8 to about 20 hours.

### Claim 14 (depends on 3)

The method of claim 3, wherein the amount of glucan hydrolyzed is at least 10% greater than the amount of glucan hydrolyzed when compared to a method that does not comprise the separating step.

### Claim 15 (depends on 1)

The method of claim 1, further comprising contacting the solids from step (c) with additional biomass in a batch, semi-batch, or continuous process.

### Claim 16 (depends on 15)

The method of claim 15, wherein the additional biomass comprises additional catalyst.

### Claim 17 (depends on 1)

The method of claim 1, wherein the incubating step occurs under conditions of high shear agitation.

### Claim 18 (depends on 1)

The method of claim 1, wherein the sugars from the liquid stream in step (b) and/or the additional sugars from step (c) are processed into ethanol, biofuels, biochemicals, or other chemical products.

### Claim 19 (depends on 1)

The method of claim 1, wherein the hydrolyzed components from step (d) contain decreased concentrations of furfural compared to biomass from step (b) that is not treated under the conditions as in step (c).

### Claim 20 (depends on 16)

The method of claim 16, wherein the additional catalyst comprises additional enzyme and/or a non-enzymatic catalyst.