FORM 2
THE PATENT ACT 1970
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION
METHOD FOR EFFICIENT ENZYMATIC HYDROLYSIS OF LIGNOCELLULOSIC BIOMASS
2. APPLICANT
(a) NAME: PRAJ Industries Limited
(b) NATIONALITY: Indian Company
(b) ADDRESS: PRAJ Tower, 274-275, Bhumkar
Chowk-Hinjewadi Road, Hinjewadi, Pune- 411057, INDIA
3. PREAMBLE TO THE DESCRIPTION
The following specification describes the invention and the manner in
which it is to be performed.

4. DESCRIPTION
FIELD OF INVENTION
The invention relates to a method for efficient enzymatic hydrolysis of lignocellulosic biomass and more particularly, it relates to efficient enzymatic hydrolysis of cellulosic part of lignocellulosic biomass like corncob, corn stover, sugarcane/ beet bagasse or any similar lignocellulosic materials using surfactants, to prepare ethanol or other bio-chemicals.
BACKGROUND
Due to the future limitations on the availability of fossil fuels particularly crude oil, the use of alternate fuels such as ethanol in motor vehicles is promoted worldwide. Ethanol is primarily produced by microbial fermentation of sugars. The three main groups of raw materials for production of ethanol by fermentation are sugars, starch, and lignocelluloses. A disadvantage of using the sugar/ starch-based materials for ethanol production is that many of these raw materials are considered a human food resource and will therefore be too expensive to use for fuel ethanol production. A disadvantage of using the lignocellulose-based biomass/ materials for ethanol production is that the hydrolysis cost is high due to high costs of energy and hydrolytic enzymes.

The largest potential feedstock for ethanol is lignocellulosic biomass/ materials (LCM), which includes materials such as agricultural residues (corn stover, crop straws, husks and bagasse), herbaceous crops (alfalfa, switchgrass), short rotation woody crops, forestry residues, waste paper and other wastes (municipal and industrial). Bioethanol production from these feedstocks could be an attractive alternative for disposal of these residues. Lignocellulosic feedstocks do not interfere with food security and are important in terms of energy security reason as well as environmental concern. It is mostly used inefficiently as an energy source or fed to animals; however, a large part is wasted as such without any use.
LCM constitutes a major portion of plant dry matter and has three major components such as cellulose, hemicellulose and lignin. Lignin and hemicetlulose can form lignin-carbohydrate. bonds, which result in complexes that provide a hydrolysis-resistant protecting sheet around the cellulose.
In the production of bio-ethanol from lignocellulosic biomass, dilute acid treatment is the most preferred pre-treatment as it provides advantages of low cost of acids, ease of unit operation and good separation of C5 sugars. However, the disadvantage of this method is that the C6 enzymatic hydrolysis efficiency is low (about 50 %) at economic amounts of cellulases. This is because of presence of

soluble inhibitors and lignin in the substrate that inhibit the activity of cellulases and hence more amounts of cellulases are required. Lignin present in the cake/ slurry irreversibly binds to the enzyme thus lowering the performance. Therefore, there is a challenge to overcome the lignin inhibition without loss of sugars.
There are methods, which enhance enzymatic hydrolysis of acid treated biomass slurry/ cake by adding surfactants or excess enzymes. In addition, there are methods of over-liming and detoxification using resins and charcoals to enhance enzymatic hydrolysis. Nevertheless, these methods are expensive and may result in delignification and loss of C5 sugars like xyiose. Therefore, it is desirable to provide an improved and economical method to reduce enzyme-lignin hindrance without the loss of lignin and sugars. Therefore, there is a need to provide an improved and economical method to reduce enzyme-lignin hindrance without loss of lignin and sugars. This is achieved by introducing a new improved method of present invention to enhance enzymatic hydrolysis without loss of lignin and sugars.
DETAILED DESCRIPTION OF THE INVENTION
In one embodiment of the invention, lignocellulosic biomass is used as a feedstock and its mixture is prepared in water. Then, the mixture is pre-treated with one or more acids at a desired temperature for a

specific time period to obtain a first stream. The pH of the first stream is adjusted with a base to obtain a second stream. Said second stream is treated with a betaine class of surfactants at a specific temperature for a specific time period to obtain a third stream. Then, said third stream is contacted with one or more of cellulolytic enzymes at specific temperature for a specific time period to obtain a sugar rich final stream. Said final stream is further subjected to fermenting yeast to obtain ethanol.
Present invention discloses an improved process for enzymatic hydrolysis of cellulosic part of lignocellulosic materials like corncob, corn stover, sugarcane/ beet bagasse or any similar lignocellulosic biomass.
In one embodiment of the disclosed invention, lignocellulosic biomass - bagasse is used as a feedstock and is further subjected to mechanical shearing for size reduction to form particulate material. This particulate material is soaked in water to form slurry and introduced continuously into a plug-screw type hydrolyser. In the hydrolyser, said slurry is mixed with an admixture of organic acid such as oxalic acid and mineral acid such as sulphuric acid. The resultant reaction is allowed to take place at temperature of about 140 °C to about 210 °C and at pressure of about 5.5 bar (absolute) for a period of about 5 minutes to about 120 minutes. This results in

formation of a first stream. The pH of said first stream is adjusted between about 4 and about 6 with a base such as sodium hydroxide to form a second stream. Next, amphoteric surfactants like coco-amido-propyl-betaine or coco-amido-propyl-hydroxyl-sulpho-betaine is added to the second stream and reaction is allowed to take place at temperature of about 50 °C for a period of about 60 minutes to form a third stream. In this slurry, cellulose is present as insoluble solids. Then this surfactant treated slurry is subjected to enzymatic hydrolysis with one or more of cellulases at temperature of about 40 °C to about 80 °C for a period of about 36 hours to about 120 hours to form a final stream. The enzymatic hydrolysis efficiency obtained at the end of hydrolysis is found to have increased between about 10 to about 20 percent points after treatment by the betaines, over the efficiency obtained without use of a surfactant.
Examples provided below give wider utility of the invention without any limitations as to the variations that may be appreciated by a person skilled in the art. A non-limiting summary of various experimental results is given in the examples, which demonstrate the advantageous and novel aspects of the process of using LCM for the preparation of ethanol.

EXAMPLE 1
A batch of about 130 Kg of bagasse having total solids of about 92 % by weight, cellulose of about 36 % by weight, hemicelluloses of about 21 % by weight and lignin of about 19 % by weight was used as a feedstock. It was subjected to mechanical shearing for size reduction to less than 40 mm particles affording about 120 Kg of the particulate material. This particulate material was soaked in water for about 30 min. Then about 400 Kg slurry containing about 30% by weight total solids was prepared and continuously introduced into a plug screw type hydrolyser. Here the slurry was mixed with about 160 litres of an admixture of oxalic and sulphuric acids. This admixture of mixed acids contained about 1.0 % by weight oxalic acid and about 1.5 % by weight sulphuric acid on dry biomass weight basis [total 2.5 % acid on dry biomass weight basis]. The resultant reaction mixture was then subjected to hydrolysis in a hydrolyser at a temperature of about 150 °C and pressure of about 5.5 bar [absolute] for a period of about 24 minutes at pH of about 1.2. At the end of this pre-treatment final slurry of about 560 Kg contained about 20 % of total solids with about 13 % of total insoluble solids. This slurry was then diluted with water to form diluted slurry containing about 16 % of total solids with about 10.5 % of total insoluble solids. The pH of this diluted slurry was about 1.4. In first step, about 3.5 Kg of NaOH was added to said diluted slurry to achieve the pH of about 5. In this slurry, cellulose form about 55 % of total insoluble solids. This slurry was subject to

enzymatic hydrolysis by addition of about 30 mg of mix of cellulases per 1 g of cellulose present in the slurry. Enzyme hydrolysis was carried out for 120 hr at 50 °C. Cellulose hydrolysis efficiency observed with bagasse was 50 % at the end of hydrolysis.
EXAMPLE 2
A batch of about 130 Kg of bagasse having total solids of about 92 % by weight, cellulose of about 36 % by weight, hemicelluloses of about 21 % by weight and lignin of about 19 % by weight was used as a feedstock. It was subjected to mechanical shearing for size reduction to less than 40 mm particles affording about 120 Kg of the particulate material. This particulate material was soaked in water for about 30 min. Then about 400 Kg slurry containing about 30% by weight total solids was prepared and continuously introduced into a plug screw type hydrolyser. Here the slurry was mixed with about 160 litres of the admixture of oxalic and sulphuric acids. This admixture of mixed acids contained about 1.0 % by weight oxalic acid and about 1.5 % by weight sulphuric acid on dry biomass weight basis [total 2.5 % acid on dry biomass weight basis]. The resultant reaction mixture was then subjected to hydrolysis in a hydrolyser at a temperature of about 150 °C and pressure of about 5.5 bar [absolute] for a period of about 24 minutes at pH of about 1.2. At the end of this pre-treatment final slurry of about 560 Kg contained about 20 % of total solids with about 13 % of total insoluble solids. This slurry was diluted to 11% total

insoluble solids and further subjected to enzymatic hydrolysis which contain about 55 % cellulose, about 3 % xylan and about 30 % lignin on dry solid basis. The pH of the slurry of bagasse was adjusted to 5.3 using sodium hydroxide. Next, 6.55 kg (90 mg/g of TIS) of coco-amido-propyl-hydroxyl-sulpho-betaine was mixed with slurry and mixed with moderate agitation at 50°C for 2 hr. Then about 1.2 kg ((30 rng/g of cellulose) of enzyme cocktail was added to bagasse slurry. Enzyme hydrolysis was carried out for 120 hr at 50°C. Cellulose hydrolysis efficiency observed on the treatment with coco-amido-propyl-hydroxyl-sulpho-betaine was 64 % at the end of hydrolysis.
EXAMPLE 3
A batch of about 130 Kg of bagasse having total solids of about 92 % by weight, cellulose of about 36 % by weight, hemicelluloses of about 21 % by weight and lignin of about 19 % by weight was used as a feedstock. It was subjected to mechanical shearing for size reduction to less than 40 mm particles affording about 120 Kg of the particulate material. This particulate material was soaked in water for about 30 min. Then about 400 Kg slurry containing about 30% by weight total solids was prepared and continuously introduced into a plug screw type hydrolyser. Here the slurry was mixed with about 160 litres of the admixture of oxalic and sulphuric acids. This admixture of mixed acids contained about 1.0 % by weight oxalic acid and about 1.5 % by

weight sulphuric acid on dry biomass weight basis [total 2.5 % acid on dry biomass weight basis]. The resultant reaction mixture was then subjected to hydrolysis in a hydrolyser at a temperature of about 150 °C and pressure of about 5.5 bar [absolute] for a period of about 24 minutes at pH of about 1.2. At the end of this pre-treatment final slurry of about 560 Kg contained about 20 % of total solids with about 13 % of total insoluble solids. This slurry was diluted to 11% total insoluble solids and further subjected to enzymatic hydrolysis which contain about 55 % cellulose, about 3 % xylan and about 30 % lignin on dry solid basis. The pH of the slurry of bagasse was adjusted to 5.3 using sodium hydroxide. Next, about 6.55 kg (90 mg/g of cellulose) of coco-amido-propyl-betaine was mixed with slurry and mixed with moderate agitation at 50 °C for 2 hr. Then about 1.2 kg ((30 mg/g of cellulose) of enzyme cocktail was added to bagasse slurry. Enzyme hydrolysis was carried out for 120 hr at 50 °C. Cellulose hydrolysis . efficiency observed with coco-amido-propyl-betaine was 65 % at the end of hydrolysis.
EXAMPLE 4
As listed in TABLE 1 of enzymatic hydrolysis after treatment with the amphoteric surfactant coco-amido-propyl-betaine of acid-treated bagasse shows remarked increase in the efficiency of conversion of cellulose to glucose by the enzymes at various dosages. Conversion efficiencies were measured for low and high enzyme dosages and

controls were used for the comparison to show the pronounced effect of coco-amido-propyl-betaine on the substrate.
TABLE 1:

No. Dose of enzyme (mg/g of cellulose) Dose of coco-amido-propyl-betaine (mg/g of cellulose) Enzyme hydrolysis
(%)
1 30 0 50
2 30 30 51
3 30 60 53
4 30 90 65
5 60 0 70
6 60 30 80
7 60 60 87
8 60 90 88
9 90 0 75
10 90 30 82
11 90 60 88
12 90 90 90
EXAMPLE 5
As listed in TABLE 2 of enzymatic hydrolysis after treatment with the amphoteric surfactant coco-amido-propyl-hydroxyl-sulpho-betaine of acid-treated bagasse shows remarked increase in the efficiency of conversion of cellulose to glucose by the enzymes at various dosages. Conversion efficiencies were measured for low and high enzyme dosages and controls were used for the comparison to show the pronounced effect of coco-amido-propyl- hydroxyl-sulpho-betaine on the substrate.

TABLE 2:

No. Dose of enzyme (mg/g of cellulose) Dose of coco-amido-propyl-hydrol-sulpho- betaine (mg/g of cellulose) Enzyme hydrolysis
(%)
1 30 0 50
2 30 30 52
3 30 60 56
4 30 90 64
5 60 0 70
6 60 30 72
7 60 60 77
8 60 90 79
9 90 0 75
10 90 30 91
11 90 60 92
12 90 90 94
While the invention has been particularly shown and described with reference to embodiments listed in examples, it will be appreciated that several of the above disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also that various presently unforeseen and unanticipated alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims. Although the invention has been described with reference to specific preferred embodiments, it is not intended to be limited thereto, rather those having ordinary skill in the art will recognize that variations and modifications may be made therein

which are within the spirit of the invention and within the scope of the claims.

5. CLAIMS
WE CLAIM:
1. A process for the preparation of glucose from a dry
lignocellulosic biomass comprising:
(a) converting said biomass to a particulate matter;
(b) preparing a slurry of said particulate matter in water;
(c) treating said slurry with an acid at a desired temperature for a desired time period to obtain a first stream;
(d) adjusting pH of said first stream with a base to obtain a second stream;
(e) adding an amphoteric surfactant to said second stream to obtain a third stream; and
(f) contacting said third stream with a mixture of cellulolytic enzymes at desired temperature for a desired time period to obtain a final stream.
2. The process of claim 1, wherein said:
(a) slurry comprises about 10 % to about 20% total insoluble solids;

(b) acid is oxalic acid or sulphuric acid or a combination thereof;
(c) desired temperature to obtain said first stream ranges from about 140° C to about 210° C;
(d) desired time period to obtain said first stream ranges from about 5 minutes to about 120 minutes; and
(e) pH of said first stream is adjusted to between about 4
and about 6.
3. The process of claim 1, wherein said surfactant is coco-amido-propyl-betaine or coco-amido-propyl-sulpho-betaine or a combination thereof.
4. The process of claim 1, wherein amount of said surfactant is between 20 mg and 100 mg per gram of cellulose.present in said second stream.
5. The process of claim 1, wherein said enzymes are used between 10 mg and 100 mg per gram of cellulose present in said second stream.
6. The process of claim 1, wherein said desired temperature to obtain said final stream ranges from about 40° C to about 80° C.

7. The process of claim 1, wherein said desired time period to
obtain said final stream ranges from about 50 hours to about 120
hours.
8. . The process of claim 1, wherein said final stream comprises
glucose derived from enzymatic hydrolysis of cellulose present in said
biomass.
9. The process of claim 1wherein efficiency of said cellulolytic enzymes to hydrolyse cellulose is increased between about 10 % and about 20%.
10. The process of claim 1, wherein said lignocellulosic biomass is bagasse.