FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, rule 13)
1. Title of the inventionA PROCESS FOR PRETREATIMENT OF LIGNOCELLULOSIC BIOIVIASS.
2. Applicant(s)
Name Nationality Address
TATA CHEMICALS LIMITE'D INDIA BOMBAY HOUSE, 24 HOMI MODI STREET, MUMBAI-400001


3. Preamble to the description
COMPLETE SPECIFICATIONThe following specification particularly describes the invention and the manner in which it isto be performed.

The invention relates to the pretreatment of biomass. More particularly the invention relates to the pretreatment of lignocellulosic biomass for the separation of cellulose from other constituents of cellulosic biomass.
DESCRIPTION OF RELATED ART
Naturally cellulosic materials are typically referred to as "biomass". Many types of biomass including wood, paper, agricultural residues, herbaceous crops, stalks, stovers, leaves and other materials of plant origin have been considered as feedstock. These biomass materials primarily consist of cellulose, hemicelluloses and lignin bound together in a complex structure. Successful commercial use of biomass depends on the separation of cellulose from the other constituents.
Lignocellulosic materials have three major components: cellulose, hemicellulose and lignin. Cellulose is a linear polysaccharide built up to gluco-glycosidic bonds. It has a relatively well-ordered structure, is pseudo-crystalline, and has a high enough chain length to be insoluble in water or dilute acids and alkali at room temperature. Cellulose is the major structural component of the cell wall and can be isolated as fibre.
Hemicelluloses are non-cellulosic polysaccharides. They are built up mainly of sugars other than glucose, are generally poorly ordered and non-crystalline, and have a much lower chain length than cellulose. The hemicelluloses occur in intimate association with the cellulose in certain layers of the cell wall, as well as in close association with the lignin between cells.
Lignin is an aromatic polymer, phenolic in nature, and built up from phenylpropane units. It has no systematic structure. Lignin occurs mainly as an encrusting agent between the fibres and on the outer layers of the cell wall.


The cellulose in lignocellulosic material is poorly accessible to micro-organisms, to enzymes and the like. That is, the micro-organisms can not easily react with the cellulose. This is due to its close association with lignin and hemicellulose in the cell structure, and to its highly cross-linked and crystalline molecular structure. To improve the accessibility, one must rupture the cell and break the close association with lignin.
Many techniques have been used to increase the chemical and biochemical reactivity of cellulose. A number of factors influence this reactivity. They include particle size and fiber surface area, lignin content, cellulose crystallinity, etc. The prior art techniques used to accomplish this generally can be characterized as either physical or chemical in nature. The physical treatments include ball-milling to very small mesh sizes, two-roll milling and attrition milling. These physical treatments are effective in producing a highly reactive and accessible cellulose component from the material. However, the power requirements for size reduction are so large that such treatments are generally considered prohibitively costly.
Chemical treatments on the other hand usually involve the use of strong bases such as sodium hydroxide, strong acids, and various other cellulose swelling and dissolving agents such as certain transition metal complex cellulose solvents. Sulfuric acid, NaOH, NH3, Lime, ethanol and steam have been shown to improve the digestibility of biomass. Such chemical agents are expensive and therefore must be recovered to make these processes economically viable. Furthermore, many of these chemicals are toxic to or otherwise inhibit the biological processes associated with alcohol fermentation or the digestive processes of ruminant animals such as cattle, sheep, etc. Therefore, removal of these chemical agents from the treated cellulose-containing material must be very complete. Such requirements further increase the expense of these prior art chemical treatments.


There remains a pressing need for a process to provide low cost cellulose for subsequent conversion to glucose sugar by enzymatic hydrolysis. However, the presence of lignin in cellulosic biomass increases dramatically the amount of enzyme needed, thereby imposing high conversion costs. Economics demand a process by which cellulose can be produced at lowest possible rates. Mainstream scientific and engineering efforts to utilize lignocellulosic biomass have been unable to achieve this goal. It is therefore desirable to identify a process that solves or avoids the problems of cost, chemical wastes or the clean separation of lignocellulosic components and the unwanted degradation of said components.
SUMMARY
The invention relates to a process for the pretreatment of lignocellulosic biomass for the separation of cellulose from other constituents of cellulosic biomass characterized by an alkali pretreatment of the biomass comprising adding crushed refined soda or trona soda and water to the biomass for a predetermined residence period.
The invention provides for a process for the pretreatment of lignocellulosic biomass comprising adding trona soda or crushed refined soda to the lignocellulosic biomass in an amount ranging from 0.1% to 20 % wt of the dry biomass, adding a predetermined amount of water to the mixture of lignocellulosic biomass to obtain a feedstock, holding the feedstock for a residence time and adding steam to the feedstock to heat the feedstock to a temperature in the range of 150 to 250 ° C to obtain pretreated biomass.
The invention provides for a process for the pretreatment of lignocellulosic biomass comprising adding trona soda or crushed refined soda to the lignocellulosic biomass in an amount ranging from 0.1% to 20 % wt of the dry biomass, adding a predetermined amount of


water to the mixture of lignocellulosic biomass to obtain a feedstock, holding the feedstock for a residence time and adding steam to the feedstock at a high pressure to heat the feedstock to a temperature in the range of 150 to 250 ° C to obtain pretreated biomass.
The invention provides for a process of preparing ethanol from lignocellulosic biomass comprising pretreating the lignocellulosic biomass with trona soda or crushed refined soda, the trona soda or crushed refined soda added to the lignocellulosic biomass in an amount ranging from 0.1 % to 20 % wt of the dry biomass, adding a predetermined amount of water to the mixture of lignocellulosic biomass to obtain a feedstock, holding the feedstock for a residence time and adding steam to the feedstock to heat the feedstock to a temperature in the range of 150 to 250 °C to obtain pretreated biomass, hydrolyzing the pretreated biomass and fermenting the hydrolyzed biomass using microorganisms to ethanol.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment described and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the system, and such further applications of the principles of the invention therein being contemplated as would normally occur to one skilled in the art to which the invention relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.


The invention provides for a process for increasing the chemical and biological reactivity of agricultural residues, stalks, stovers, leaves and other materials of plant origin commonly called as biomass. The invention relates to a process for increasing the enzymatic digestibility/hydrolysis of biomass by a pretreatment involving crushed refined soda, trona soda or sodium sesquicarbonate. The invention also relates to the process of fermenting the hydrolyzed biomass post pretreatment using suitable microorganisms to ethanol. Biomass is presoaked with known quantities of crushed refined soda and heated with steam. The pretreated biomass can be enzymatically hydrolyzed and fermented to ethanol.
Trona is a mineral ore that usually contains 70-95% of a complex salt of sodium carbonate (Na2CO3) and sodium bicarbonate (NaHCO3) in a hydrated crystal form and is also referred to as sodium sesquicarbonate, (Na2CO3. NaHCO3.2H2O). Trona also contains between 6-30% insolubles, usually shale oil, and a small amount of NaCl, usually less than 0.3%. Trona soda is used primarily to make soda ash though has numerous other applications including feed to dairy cows as a rumen buffer. Crushed Refined Soda [CRS] is obtained from trona soda. Trona soda is washed with water in two stages. It is then dewatered by cyclones and finally reduced down to about 3% moisture by centrifuging. The centrifuged material is put into a buffer stock and is known at this stage as Crushed Refined Soda [CRS].
Though the invention has been described for crushed refined soda, the teachings of this document can be equally applied to other sources of soda or sodium sesquicarbonate.
The teachings of this document may be applied for all biomass including sugar cane bagasse, sugar cane leaves, sweet sorghum bagasse, sweet sorghum leaves, rice straw, rice husk, wheat straw, corn stover, and other lignocellulosic materials.


In accordance with the teachings of this document, biomass is presoaked with a predetermined amount of water and crushed refined soda [CRS] for a predetermined period of residence time. The biomass is then fed to a reactor where steam is added to heat the biomass to a predetermined temperature. The biomass so pretreated is removed from the reactor and excess liquid is filtered out. The cake is neutralized to the desired pH and treated with cellulases to release fermentable glucose.
In accordance with the teachings of this document, biomass is presoaked with a predetermined amount of water and crushed refined soda [CRS] for a predetermined period of residence time. The biomass is then fed to a high pressure reactor where steam is added to heat the biomass to a predetermined temperature. The biomass so pretreated is removed from the reactor and excess liquid is filtered out. The cake is neutralized to the desired pH and treated with cellulases to release fermentable glucose.
The amount of crushed refined soda or trona soda that is added to the biomass is in the range of 0.1% to 20% wt of the dry biomass. The preferred amount of crushed trona soda to be added is under 10% wt of the dry biomass and the most preferred amount of crushed trona soda to be added is under 5% wt of the dry biomass. The crushed refined soda is preferably added to the biomass in the form of fine or coarse particles.
In accordance with an embodiment, a mixture of crushed refined soda and trona soda are added to the biomass in the range of 0.1% to 20% wt of the dry biomass.
To the mixture of biomass and crushed refined soda, water is added to obtain feedstock. The amount of water added is to make the moisture content of the feedstock preferably in the range of 50 to 75%.


The feedstock so obtained is maintained for a predetermined residence time. The residence time is to ensure a proper mixing of the crushed refined soda and the biomass and may range from 1 to 24 hours. The preferred residence time is an overnight soak or between 10 to 15 hours. The residence time does not require elevated temperature or pressure.
The feedstock after the maintenance of the residence time is transferred to a reactor into which steam is input. The steam is used to heat the feedstock to a temperature in the range of 150 to 250 degrees centigrade. The steam is input to the reactor for a reaction time ranging from one to sixty minutes. The pretreated biomass so obtained is decanted or centrifuged to separate solids from the liquid and the solids are harvested. The pH of the solids so harvested is then adjusted to a value in the range of 4.5 to 5. The pH adjustment is carried out using a mineral acid and water is added so as to obtain a solution for further hydrolysis.
In accordance with the preferred embodiment, the feedstock is transferred to a high pressure reactor into which steam is input. The steam is used to heat the feedstock to a temperature in the range of 150 to 250 degrees centigrade. The steam is input to the reactor for a reaction time ranging from one to sixty minutes. Post reaction period the pressure is released and the reactor is brought to atmospheric pressure, preferably immediately. The pretreated biomass so obtained is decanted or centrifuged to separate solids from the liquid and the solids are harvested. The pH of the solids so harvested is then adjusted to a value in the range of 4.5 to 5. The pH adjustment is carried out using a mineral acid and water is added so as to obtain a solution for further hydrolysis.
In accordance with the preferred embodiment the pH value of the reaction in the reactor is maintained in the range of 7 to 12.


It is also preferred that the biomass particle size be maintained in the range of 0.5 cm to 10 cm.
The pressure range in the reactor is preferably maintained at in the range 100 to 350 psig. The pressure is a dependent variable and depends on the temperature of steam.
The pretreated biomass is enzymatically hydrolyzed using cellulases, hemicellulases, and cellobiases in varying quantities to release fermentable sugars. The fermentable sugars are converted to ethanol using yeast, bacteria or any suitable microorganism.
In accordance with an aspect of the invention a process of preparing ethanol from lignocellulosic biomass is provided comprising pre-treating the lignocellulosic biomass with trona soda or crushed refined soda. The trona soda or crushed refined soda added to the lignocellulosic biomass in an amount ranging from 0.1% to 20 % wt of the dry biomass and to this mixture a predetermined amount of water is added to obtain a feedstock. The feedstock is maintained for a predetermined residence time and steam is subsequently added to heat the feedstock to a temperature in the range of 150 to 250 ° C, to obtain pretreated biomass. The pretreated biomass is hydrolyzed and fermented using microorganisms to ethanol.
As the crushed refined soda or trona soda are naturally available waste streams can be returned to the soda source without contaminating the environment. No complex chemical recovery systems or processes are required. Lake Magadi in Africa is one known source of trona soda. Waste streams from the pretreatment process may be returned to the lake without requiring any additional expenses or chemical recovery processes. Moreover, the use of crushed refined soda or trona soda for pretreatment is inexpensive and high energy requirements are avoided.


The use of crushed refined soda or trona soda also does not adversely affect the subsequent hydrolysis or fermentation process and thus the process does not raise concerns on toxicity. Experimental data suggests that the use of other minerals and compounds present in naturally available trona soda or crushed refined soda synergistically affect the pretreatment process.
Thus, the use of crushed refined soda or trona soda for the pretreatment of biomass is economically superior to the known process on account of both low input and processing cost.
The following examples are provided to explain and illustrate certain preferred embodiments of the process of the invention.
Example 1: 100g of rice straw with a moisture content of 5-8% and 1-2 inches in length was soaked with 2% by weight of CRS and tap water to make upto 50 to 75% moisture. The mixture was left to soak overnight and charged into a high pressure reactor. Steam was added from another reactor to heat the contents to 150-250 C. The reaction was maintained for 5 to 60 minutes following which the pressure was quickly released to atmospheric level. The solids were harvested. pH is then adjusted to around 4.5 to 5 using a mineral acid and make-up water was added to a solid loading of 2.5 wt%. Specific quantities of cellulases and beta glucosidases are added to mixture and placed in an orbital shaker-incubator for 3-4 days. The release of glucose during the course of the hydrolysis was measured using a Waters HPLC fitted with a Shodex column and RI detector. The glucose concentration was plotted vs. time. The results showed that 8 g/ml of glucose was produced in the first 24 hours and 10 g/1 was


produced by 48 hrs. This corresponds to a yield of greater than 70% based on the composition of the pretreated biomass fed to the hydrolysis.
Example 2: l00g of sugar cane bagasse with a moisture content of 8% and 0.5 to 1 inch in length was soaked with 4% by weight of CRS and tap water to make upto 50 to 75% moisture. The mixture was left to soak overnight and charged into a high pressure reactor. Steam was added from another reactor to heat the contents to 150-250 C. The reaction was maintained for 5 to 60 minutes following which the pressure was quickly released to atmospheric level. The solids were harvested, pH is then adjusted to around 4.5 to 5 using a mineral acid and make-up water was added to a solid loading of 4 wt%. Specific quantities of cellulases and beta glucosidases are added to mixture and placed in an orbital shaker-incubator for 3-4 days. The release of glucose during the course of the hydrolysis was measured using a Waters HPLC fitted with a Shodex column and RI detector. The glucose concentration was plotted vs. time. The results showed that 14 g/ml of glucose was produced in the first 24 hours and 15 g/1 was produced by 48 hrs. This corresponds to a yield of greater than 70% based on the composition of the pretreated biomass fed to the hydrolysis.

Example 3: 40g of sweet sorghum bagasse with a moisture content of 10% and 0.5 to 2 inches in length was soaked with 4% by weight of CRS and tap water to make upto 50 to 75% moisture. The mixture was left to soak overnight and charged into a high pressure reactor. Steam was added from another reactor to heat the contents to 150 to 250 C. The reaction was maintained for 5 to 60 minutes following which the pressure was quickly released to atmospheric level. The solids were harvested, pH is then adjusted to around 4.5 to 5 using a mineral acid and make-up water was added to a solid loading of 8 wt%. Specific quantities of cellulases and beta glucosidases are added to mixture and placed in an orbital shaker-incubator for 3-4 days. The release of glucose during the course of the hydrolysis was measured using a Waters HPLC fitted with a Shodex column and RI detector. The glucose concentration was plotted vs. time. The results showed that 22 g/ml of glucose was produced in the first 24 hours and 27 g/1 was produced by 48 hrs. This corresponds to a yield of greater than 70% based on the composition of the pretreated biomass fed to the hydrolysis.

Solid Loading Glucose Titer Yield
2% CRS 4 12.92 56.84
2% CRS 4 12.32 57.98
2% CRS 4 13.54 63.11
3% CRS 4 13.00 65.00
3% CRS 8 27.00 67.50
4% CRS 8 25.23 63.08
4% CRS 4 18.20 77.21
4% CRS 4 16.16 77.20


We Claim:
1. A process for the pretreatment of lignocellulosic biomass for the separation of cellulose from other constituents of cellulosic biomass characterized by an alkali pretreatment of the biomass comprising adding crushed refined soda or trona soda and water to the biomass for a predetermined residence period.
2. A process for the pretreatment of lignocellulosic biomass comprising adding trona soda or crushed refined soda to the lignocellulosic biomass in an amount ranging from 0.1% to 20 % wt of the dry biomass, adding a predetermined amount of water to the mixture of lignocellulosic biomass to obtain a feedstock, holding the feedstock for a residence time and adding steam to the feedstock to heat the feedstock to a temperature in the range of 150 to 250 ° C to obtain pretreated biomass.
3. A process for the pretreatment of lignocellulosic biomass as claimed in claim 2 wherein the feedstock is transferred to a high pressure reactor for the addition of steam.
4. A process for the pretreatment of lignocellulosic biomass as claimed in claim 2 or 3 wherein the steam reacts with the feedstock for a period in the range of one minute to sixty minutes.
5. A process for the pretreatment of lignocellulosic biomass as claimed in claim 4 wherein the pH of the reaction is in the range of 7 to 12.


6. A process for the pretreatment of lignocellulosic biomass as claimed in claim 1 or 2 wherein the crushed refined soda is added in the form of coarse or finely ground particles.
7. A process for the pretreatment of lignocellulosic biomass as claimed in claim 1 or 2 wherein the residence time for the feedstock is between 2 to 24 hours.
8. A process for the pretreatment of lignocellulosic biomass as claimed in claim 1 or 2 wherein a combination of trona soda and crushed refined soda is added to the biomass.

9. A process for the pretreatment of lignocellulosic biomass as claimed in claim 1 or 2 wherein the biomass particle size is in the range of 0.5 cm to 10 cm.
10. A process for the pretreatment of lignocellulosic biomass as claimed in claim 2 wherein the pretreated biomass is enzymatically hydrolyzed using cellulases, hemicellulases, and cellobiases in varying quantities to release fermentable sugars.
11. A process for the pretreatment of lignocellulosic biomass as claimed in claim 10 wherein the fermentable sugars are converted to ethanol using yeast, bacteria or any suitable microorganism.
12. A process of preparing ethanol from lignocellulosic biomass characterized by an alkali pretreatment of the biomass comprising adding crushed refined soda or trona soda and water to the biomass for a predetermined residence period.


13. A process of preparing ethanol from lignocellulosic biomass comprising pretreating the lignocellulosic biomass with trona soda or crushed refined soda, the trona soda or crushed refined soda added to the lignocellulosic biomass in an amount ranging from 0.1% to 20 % wt of the dry biomass, adding a predetermined amount of water to the mixture of lignocellulosic biomass to obtain a feedstock, holding the feedstock for a residence time and adding steam to the feedstock to heat the feedstock to a temperature in the range of 150 to 250 °C to obtain pretreated biomass, hydrolyzing the pretreated biomass and fermenting the hydrolyzed biomass using microorganisms to ethanol.
14. A process for the pretreatment of lignocellulosic biomass substantially as herein described.
15. A process of preparing ethanol substantially as herein described.

ABSTRACT
The invention provides for a process for the pretreatment of lignocellulosic biomass comprising adding trona soda or crushed refined soda to the Lignocellulosic biomass in an amount ranging from 0.1% to 20 % wt of the dry biomass, adding a predetermined amount of water to the mixture of Lignocellulosic biomass to obtain a feedstock, holding the feedstock for a residence time and adding steam to the feedstock to heat the feedstock to a temperature in the range of 150 to 250 ° C to obtain pretreated biomass.