CLIAMS:We Claim:
1. A method to prepare fermentable sugars from biomass resources, said method comprising the steps of:
(f) pretreating the biomass with dilute acid;
(g) neutralizing the pretreated biomass of the step (a) with acid-neutralizing neutralizers;
(h) separating the pentose sugars from the pretreated biomass of steps (a) and (b) to obtain a pretreated biomass slurry;
(i) hydrolysing the biomass slurry of step (c); and
(j) obtaining fermentable sugars.

2. A method as claimed in claim 1, wherein the step (a) the dilute acid is selected from group comprising of citric acid, isocitric acid, aconitic acid, propane-1,2,3-tricarboxylic acid and trimesic acid alone or as a mixture.

3. A method as claimed in any one of claims 1-2, wherein the step (a) the dilute acid is in the concentration of 0.5-20% w/v.

4. A method as claimed in claim 1, wherein the step (b) the neutralizer is selected from the group comprising of sodium hydroxide (NaOH), calcium carbonate (CaCO3), sodium carbonate (Na2CO3), magnesium hydroxide (Mg(OH)2, slaked lime (Ca(OH)2, quicklime (CaO) or calcium carbonate (CaCO3 may be used. More specifically, sodium hydroxide (NaOH), sodium carbonates (Na2CO3) or calcium carbonate (CaCO3).

5. A method as claimed in any one of claims 1 and 4, wherein the step (b) the neutralizer is in the concentration of 1-10M.

6. A method as claimed in any one of claims 1 and 4-5, wherein the step (b) the neutralizer is 5M.

7. A method as claimed in claim 1, wherein the step (c), the penstose sugars and solid pretreated biomass slurry is separated using various known filtration methods selected from group comprising a filter press, a centrifuge, a membrane filter or a nanofilter.

8. A method as claimed in claim 1, wherein step (d) hydrolysis of pre-treated biomass slurry is carried out using cellulase at a pH 4-7 at a temperature of 30°C-70°C for 12-48 hours.

9. A method as claimed in any one of claims 1 or 8, wherein the step (d) hydrolysis of pre-treated biomass slurry is carried out using cellulase at a pH 4.8-5.5 at a temperature of 48°C-52°C for 24-36 hours.

10. A method as claimed in claim 1, wherein step (a) is carried out under high pressure of at 130°C-220°C.

11. A method as claimed in claim 1, wherein the step (a) the biomass concentration is in the range of 5-50% (w/w).

12. A method as claimed in claim 10, wherein the step (a) the pressure is in the range of 5-10 bars.

13. A method as claimed in claim 12, wherein the step (a) the pressure is 5.5 bar.
,TagSPECI:TITLE OF THE INVENTION: A METHOD OF PREPARING FERMENTABLE SUGARS FROM BIOMASS RESOURCES

FIELD OF INVENTION:
The present invention relates to a method of preparing fermentable sugars from biomass resources.
BACKGROUND OF THE INVENTION
Ethanol is one of the most promising alternative fuels to replace or supplement gasoline being used across the world. Mostly, current production of ethanol is based on sugarcane, corn and other starch rich grains. For the ethanol industry to realize its goal of more than 10 billion gallons production per year it needs to rely on a more sustainable and inexpensive feed stocks. Moreover, the use of these feed stocks raised the issue of food vis-à-vis fuel and, therefore, prompts to look for the production of ethanol from the non-food materials like liginocellulosic biomass. All these biomass contains a large amount of liginocellulose, which could be a potential feedstock for commercial ethanol production.
Plant cell walls are composed of lignocellulosic materials, which are represented by cellulose (linear glucose polymers), hemicellulose (highly branched heteropolymers) and lignin (crosslinked aromatic macromolecules with large molecular weight). The bonding between the polysaccharide components (cellulose and hemicellulose) and non-polysaccharide components (lignin) is the main cause of mechanical and biological resistance. Cellulose, the most abundant polysaccharide on earth, is a polymer accounting about 50% of the wood weight. The cellulose chain which forms fibrils consists of about 10,000 glucose units. The cellulosic material has a crystal domain separated from the less-ordered, amorphous domain, which allows chemical and biochemical attack. Cellulases can hydrolyze the cellulose polymer to monomers, and the resulting glucose is fermented into ethanol by the yeast Saccharomyces cerevisiae.
Hemicellulose is a short (100-200 sugar units), highly-branched heteropolymer consisting of the predominant xylose as well as glucose, mannose, galactose, arabinose and other uronic acids. C5 and C6 sugars are linked by 1,3-, 1,6- or 1,4-glucosidic linkages, which differentiate cellulose from lignin, and are often acetylated.
Lignin is a 3-dimensional polyphenolic network of dimethoxylated, monomethoxylated and non-methoxylated phenylpropanoid units, derived from p-hydroxycinnamyl alcohol. Lignin is hydrophobic and highly resistant to chemical and biological degradation. Cellulosic fibrils are embedded in an amorphous matrix network of hemicellulose and lignin, and they serve as glues between the plant cells, providing resistance to biodegradation. Other non-structural components (phenols, tannins, fats, sterols, sugars, starches, proteins and ashes) of the plant tissue generally accounts for 5% or less of the dry weight of biomass.
In order to hydrolyze the biomass polysaccharides into fermentable sugars, for example by depolymerization, pretreatment processes such as steam explosion, mild acid treatment, strong acid treatment, ammonia treatment, alkali treatment, etc. are employed. No matter what it is, the pretreatment process should be environment-friendly and economically feasible. The pretreatment method will be selected considering process dependency and cost, as well as process yield and production parameters.
U.S. Pat. No. 5,628,830 (Brink) discloses the use of calcium carbonate to adjust the pH of an aqueous sugar solution containing xylose, glucose, mannose and galactose arising from acid hydrolysis of lignocellulosic feedstock. After pH adjustment of the aqueous sugar solution, the solution is submitted to fermentation. However, Brink's process employs full acid hydrolysis, which suffers from the disadvantage discussed above.
Shortcoming of processing lignocellulosic feedstocks to produce glucose is the large amounts of alkali that are required to adjust the pH of the acid pretreated feedstock prior to enzymatic hydrolysis with cellulase enzymes and special metallurgy of the vessel like hastalloy, which is very expensive. The addition of alkali adds significant cost to the process. In addition, the alkali reacts with the acid to produce salt, which must be processed or disposed of.
U.S. Pat. No. 4,425,433 (Neves) discloses the use of sodium carbonate or sodium bicarbonate to neutralize an acidic feedstock slurry containing glucose, which slurry is produced by acid hydrolysis of the cellulose and hemicellulose components of the feedstock. After the neutralization, the acidic slurry or “wort”, as referred to therein, is submitted to fermentation. However, a disadvantage of this process is that the amount of sodium carbonate and sodium bicarbonate required for the pH adjustment would add significant cost to the process and produce a large amount of salt to be disposed of. Moreover, special metallurgy of the vessel like Hastelloy is required, which is very expensive.
U.S. Pat. No. 6,927,048 (Verser et al.) discloses a process in which calcium carbonate and an amine or an alcohol are added during the fermentation of glucose to acetic acid. The calcium carbonate controls the pH while the amine or alcohol complexes with the acetic acid. After the fermentation, the calcium carbonate is precipitated by the addition of carbon dioxide and then recovered from the fermentation broth. The recovered calcium carbonate is then reused in the subsequent fermentation. Thus, Verser et al. does not address the reduction of alkali use during the pretreatment and neutralization of a lignocellulosic feedstock. Moreover, special metallurgy of the vessel like Hastelloy is required, which is very expensive.
U.S. Pat. No. 6,043,392 (Holtzapple et al.) also does not address reducing alkali usage during a neutralization conducted after acid pretreatment of a lignocellulosic feedstock. Rather, Holtzapple discloses a process that involves lime (alkali) treatment of lignocellulosic feedstocks with a subsequent fermentation step to produce volatile fatty acids (VFAs), followed by a thermal conversion of the VFAs to produce ketones. Calcium carbonate may be produced during an evaporation step involving carbon dioxide addition prior to thermal conversion of the VFAs. The calcium carbonate is recycled to the fermentor to neutralize acids that are produced by the fermentation or is burned in a lime kiln to produce lime which may be used in the lime treatment. Moreover, special metallurgy of the vessel like Hastelloy is required, which is very expensive.
Similarly, U.S. Pat. No. 5,693,296, also to Holtzapple, discloses a process involving treating biomass with calcium oxide or hydroxide, followed by carbonating the pretreated material to form calcium carbonate or bicarbonate. The calcium carbonate may be heated in a lime kiln to form calcium oxide, which can be hydrated to form calcium hydroxide, which, in turn, can be used to treat the biomass. Thus, this process also does not address reducing chemical usage during a neutralization of an acid pretreated feedstock in the production of glucose. Moreover, special metallurgy of the vessel like hastalloy is required, which is very expensive. A similar process is disclosed by Chang et al., 1998, Applied Biochemistry and Biotechnology, 74:135-159.
US 2006/0188965 (Wyman and Lloyd) discloses a process involving acid pretreatment of cellulosic biomass. The acid-pretreated feedstock slurry is then mixed with a lime solution to impart a pH of 10 to 11, followed by the addition of sulfuric acid to adjust the pH into a range of 5-7 prior to cellulose hydrolysis by cellulase. Following the enzymatic hydrolysis, a fermentation of the hydrolyzed material is carried out to produce alcohol, which is then concentrated by distillation. Remaining liquids and/or solids from the distillation are subjected to a recycle processing step to filter fine particulates. The resulting material is then sent back to the acid pretreatment, along with lignocellulosic material fed to the process. However, the recycling of this material back to pretreatment does not reduce the amount of alkali used to neutralize the pretreated cellulose. Moreover, special metallurgy of the vessel like Hastelloy is required, which is very expensive.
EP 2336195 relates a process of obtaining sugars and lignin from lignocellulosic biomass material. The process uses formic acid for the purpose of treating lignocellulose biomass.
US20110144359 relates to a method of producing furfural from lignocellulosic biomass material.
WO2011002832 relates to a biomass process comprising removal and/or inactivation of an enzyme inhibitors from recycled washing solution.
WO 2010059825 relates to a process of obtaining sugar solutions from polysaccharide enriched biomass by contracting biomass with water and at least one nucelophilic base to produce a polysaccharide enriched biomass comprising a solid fraction and a liquid fraction. The solid fraction is separated from the lignin-containing liquid fraction with an acid solution, the acid solution comprising about 70 weight percent to about 100 weight percent sulphuric acid or an acid mixture comprising phosphoric acid and sulphuric acid.
Thus there is need in the art for methods for producing renewable fuel allowing coping with the global climate change by removing carbon dioxide from the atmosphere by photosynthesis, without affecting the global grain prices. And, the by-product produced during bioethanol production may be used as livestock feed additives, fuels for steam and power generation, raw materials of gypsum board, cement additives, fertilizers etc. There is also need in the art to produce ethanol from lingo-cellulosic biomass (LCB) involving the steps of pretreatment, enzymatic hydrolysis followed by fermentation. Particularly there is also need in the art for a process of pretreatment by organic acids which has beneficial effects in the subsequent enzymatic hydrolysis as there is no need to add additional buffers, which are needed in conventional methods disclosed in the prior art.

SUMMARY OF THE INVENTION
Accordingly the present invention provides a method to prepare fermentable sugars from biomass resources, said method comprising the steps of:
(a) pretreating the biomass with dilute acid;
(b) neutralizing the pretreated biomass of the step (a) with acid-neutralizing neutralizers;
(c) separating the pentose sugars from the pretreated biomass of steps (a) and (b) to obtain a pretreated biomass slurry;
(d) hydrolysing the biomass slurry of step (c); and
(e) obtaining fermentable sugars.

DETAILED DESCRIPTION
While the invention is susceptible to various modifications and/or alternative processes and/or compositions, specific embodiment thereof has been shown by way of example in the tables will be described in detail below. It should be understood, however that it is not intended to limit the invention to the particular processes and/or compositions disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternative falling within the spirit and the scope of the invention as defined by the appended claims.

The tables and protocols have been represented where appropriate by conventional representations in the drawings, showing only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.

The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the elements described without departing from the scope of the invention.

The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that one or more processes or composition/s or systems or methods proceeded by "comprises... a" does not, without more constraints, preclude the existence of other processes, sub-processes, composition, sub-compositions, minor or major compositions or other elements or other structures or additional processes or compositions or additional elements or additional features or additional characteristics or additional attributes.

The efforts have been made to prepare fermentable sugars from biological resources using an environment-friendly and less expensive process. As a result, we have found that fermentable sugars can be produced with high yield via using an environment-friendly chemical e.g. tricarboxylic organic acid which includes citric acid, isocitric acid, aconitic acid, propane-1,2,3-tricarboxylic acid and trimesic acid. These carboxylic organic acids require no special metallurgy like Hastalloy, which is very expensive, for reaction vessel, which is required for acid pretreatment. This process can be done in the steel vessel.
In one general aspect, the present disclosure provides a method for preparing fermentable sugars from biomass, comprising: (a) pretreatment of biomass with dilute citric acid, isocitric acid, aconitic acid, propane-1,2,3-tricarboxylic acid and trimesic acid as an individual or in mixture from 0.5-20% w/v; (b) neutralizing the pretreated biomass mixture (c) separating the pentose sugars and solid pretreated biomass; (d) hydrolyzing the pretreated biomass slurry and (e) Integration of pretreatment process with enzymatic hydrolysis to convert them into fermentable sugars.
The individual steps of the method for preparing fermentable sugars from biomass according to the present disclosure are described here:
(a) Pretreatment of biomass with dilute citric acid, isocitric acid, aconitic acid, propane-1,2,3-tricarboxylic acid and trimesic acid as an individual or in mixture from 0.5-20% w/v;
The biomass used as the source material in the present disclosure may include various known biological resources containing cellulose or lignocellulosic materials. Specifically, it may be rice straw, Jatropha pruning, wheat straw, corn cob, corn stover, rice husk, paper, wood, sawdust, agricultural waste, grass, sugar cane bagasse, cotton, flax, bamboo, abaca, algae, fruit skin or seaweed. More specifically, it may be wheat straw, corn stover, rice straw, sorghum stalk, rice husk, wood, sawdust, sugar cane bagasse or fruit skin.
The biomass can be pretreated with dilute solution from 0.5-20% of citric acid, isocitric acid, aconitic acid, propane-1,2,3-tricarboxylic acid and trimesic acid as an individual or in mixture thereof. The experiment was carried out in high pressure reactor at 130-2200C with 5-50% (w/w) biomass loading for 5-60 min resident time.
(b) Neutralizing the pretreated biomass mixture
The neutralizer used may include various acid-neutralizing neutralizers known in the art. Specifically sodium hydroxide (NaOH), calcium carbonate (CaCO3), sodium carbonate (Na2CO3), magnesium hydroxide (Mg(OH)2, slaked lime (Ca(OH)2, quicklime (CaO) or calcium carbonate (CaCO3 may be used. More specifically, sodium hydroxide (NaOH), sodium carbonate (Na2CO3) or calcium carbonate (CaCO3) may be used.
(c) Separating the pentose sugars and solid pretreated biomass
The resultant of the step (b), which is a mixture of liquid hydrolysate rich in pentose sugars and solid biomass rich in cellulose, may be separated using various known filtration apparatuses. For example, a filter press, a centrifuge, a membrane filter or a nanofilter may be used for the separation.
(d) Hydrolyzing the pretreated biomass slurry
In the present disclosure, the cellulase treatment may be performed at pH 4-7 and 30-700C for 12-48 hours. More specifically, the cellulase treatment may be performed at pH 4.8-5.5 and 48-520C for 24-36 hours.
(e) Integration of pretreatment process with enzymatic hydrolysis to convert them into fermentable sugars
Enzymatic hydrolysis of the pretreated slurry has to be done in citrate or phosphate buffer. In the reported, literature the citrate buffer is commonly used. All the effective pretreatment process reported like sulfuric acid and alkali pretreatment. After pretreatment these chemicals need to be neutralized either in the separable salts like calcium sulphate if neutralized with calcium carbonate or soluble salts like sodium sulphate if neutralized with alkali and vice-versa in case of alkali pretreatment. Hence, poses problems in the downstream process of hydrolysis and fermentation and make the process expensive.
Therefore, in the present invention pretreatment is carried out in citric acid and pretreated slurry was neutralized with alkali, which provide in-situ citrate buffer and the solid stream received from the pretreatment can be directly incubated for enzymatic hydrolysis without any wash and without any separation process.
In almost all prior work, the LCB is treated with sulfuric, nitric or hydrochloric acids at higher temperatures to open up the LCB structure for subsequent enzymatic hydrolysis. Enzymatic hydrolysis by cellulase enzymes works best at a pH of 5 to 5.5, however the LCB after pre-treatment yields a solution with much lower pH, in range of 1-1.5. Therefore this solution needs to be neutralized with bases like calcium carbonate, sodium hydroxide etc to get a pH of about 5-5.5. This basic treatment generally produces insoluble alkali salts which need to be removed by filtration before these can be taken for enzymatic hydrolysis. Furthermore, in order to keep the pH stable in range of 5.0-5.5, appreciable amounts of buffer- generally citrate or phosphate buffers need to be added.
In our invention, the LCB pretreatment is effected by dilute organic acids like citric acid and after completion of pre-treatment, the acidic solution is treated with sodium hydroxide to get a pH of 5.0-5.5. This treatment with base generates sodium citrate and the need to add buffer is avoided. It has been observed that the final concentration of citric acid buffer, after neutralization with strong base, falls between 0.5-0.1 molar which is an apt condition for enzymatic hydrolysis. Also the need to separate insoluble inorganic salts by filtration is done away with. Additional benefits of pretreatment catalysed with organic acids include avoidance of use of special metallurgy reactors, Hastelloy type, which are essentially used when inorganic acids are employed in pretreatment.
Accordingly, the main embodiment of the present invention relates to a method to prepare fermentable sugars from biomass resources, said method comprising the steps of:
(a) pretreating the biomass with dilute acid;
(b) neutralizing the pretreated biomass of the step (a) with acid-neutralizing neutralizers;
(c) separating the pentose sugars from the pretreated biomass of steps (a) and (b) to obtain a pretreated biomass slurry;
(d) hydrolysing the biomass slurry of step (c); and
(e) obtaining fermentable sugars.

Another embodiment of the present invention relates to a method, wherein the step (a) the dilute acid is selected from group comprising of citric acid, isocitric acid, aconitic acid, propane-1,2,3-tricarboxylic acid and trimesic acid alone or as a mixture.
Another embodiment of the present invention relates to a method, wherein the step (a) the dilute acid is in the concentration of 0.5-20% w/v.
Yet another embodiment of the present invention relates to a method, wherein the step (b) the neutralizer is selected from the group comprising of sodium hydroxide (NaOH), calcium carbonate (CaCO3), sodium carbonate (Na2CO3), magnesium hydroxide (Mg(OH)2, slaked lime (Ca(OH)2, quicklime (CaO) or calcium carbonate (CaCO3 may be used. More specifically, sodium hydroxide (NaOH), sodium carbonates (Na2CO3) or calcium carbonate (CaCO3).
Yet another embodiment of the present invention relates to a method, wherein the step (b) the neutralizer is in the concentration of 1-10M.
Yet another embodiment of the present invention relates to a method, wherein the step (b) the neutralizer is 5M.

Yet another embodiment of the present invention relates to a method, wherein the step (c), the penstose sugars and solid pretreated biomass slurry is separated using various known filtration methods selected from group comprising a filter press, a centrifuge, a membrane filter or a nanofilter.
Another embodiment of the present invention relates to a method, wherein step (d) hydrolysis of pre-treated biomass slurry is carried out using cellulase at a pH 4-7 at a temperature of 30°C-70°C for 12-48 hours.
Another embodiment of the present invention relates to a method, wherein the step (d) hydrolysis of pre-treated biomass slurry is carried out using cellulase at a pH 4.8-5.5 at a temperature of 48°C-52°C for 24-36 hours.
Yet another embodiment of the present invention relates to method, wherein step (a) is carried out under high pressure of at 130°C-220°C.
Yet another embodiment of the present invention relates to a method, wherein the step (a) the biomass concentration is in the range of 5-50% (w/w).
Yet another embodiment of the present invention relates to a method, wherein the step (a) the pressure is in the range of 5-10 bars.
Yet another embodiment of the present invention relates to a method, wherein the step (a) the pressure is 5.5 bar.

Thus with the application of present invention the energy intensive filtration step and need to add extra buffer are eliminated. This in addition to use of a normal SS metallurgy reactor makes the total process of cellulosic ethanol preparation from LCB cost effective.
EXAMPLES
Example-1: A mixture of biomass (150 g) in de-mineralized water (1200ml) was taken in the high pressure reactor equipped with agitator, temperature and pressure probe and temperature control, sampling port and re-circulating coil. The reactor was also has a port to add catalyst at high pressure and temperature. The stirred mixture was heated to 160°C (pressure 5.5bar). After the reaction temperature reached to 1600C a solution of citric acid (30g) and de-mineralized water (150g) was pumped in the reactor using pressure vessel at 8 bar. The stirring was continued for 30 min and the reaction mixture was cooled to room temperature in 15 min using chilling water circulation. The whole mixture was neutralized by 5M Sodium hydroxide solution till pH 5. Solid and liquid materials were separated using centrifuge. The liquid hydrolysate was tested for inhibitors and sugars. Solid pretreated slurry (300g on dry weight basis) having pH 5 was taken in the hydrolysis reactor equipped with agitator and a temperature probe. Reactor temperature was maintained at 500C and cellulase preparation from cellulases was added at 15 FPU/g of pretreated biomass and total reaction volume was making up to 1000g using de-mineralized water at 5 pH. 20g of sodium azide was added to the reactor to prevent the growth of any micro-organisms during the hydrolysis process. The continuous agitation of reaction mixture was carried out for 24 hours to release maximum monomeric sugars. The liquid sample after complete hydrolysis was subjected to DNS for sugar analysis. The data received is summarized in Table-1.
Example-2: A mixture of biomass (100 g) in de-mineralized water (900ml) was taken in the high pressure reactor equipped with agitator, temperature and pressure probe and temperature control, sampling port and re-circulating coil. The reactor was also has a port to add catalyst at high pressure and temperature. The stirred mixture was heated to 170°C (pressure 5.5bar). After the reaction temperature reached to 1700C a solution of citric acid (20g) and de-mineralized water (100g) was pumped in the reactor using pressure vessel at 8 bar. The stirring was continued for 30 min and the reaction mixture was cooled to room temperature in 15 min using chilling water circulation. The whole mixture was neutralized by 5M Sodium hydroxide solution till pH 5. Solid and liquid materials were separated using centrifuge. The liquid hydrolysate was tested for inhibitors and sugars.
Solid pretreated slurry (300g on dry weight basis) having pH 5 was taken in the hydrolysis reactor equipped with agitator and a temperature probe. Reactor temperature was maintained at 500C and cellulase preparation from cellulases was added at 15 FPU/g of pretreated biomass and total reaction volume was making up to 1000g using de-mineralized water at 5 pH. 20g of sodium azide was added to the reactor to prevent the growth of any micro-organisms during the hydrolysis process. The continuous agitation of reaction mixture was carried out for 24 hours to release maximum monomeric sugars. The liquid sample after complete hydrolysis was subjected to DNS for sugar analysis. The data received is summarized in Table-1.
Example-3: A mixture of biomass (100 g) in de-mineralized water (900ml) was taken in the high pressure reactor equipped with agitator, temperature and pressure probe and temperature control, sampling port and re-circulating coil. The reactor was also has a port to add catalyst at high pressure and temperature. The stirred mixture was heated to 170°C (pressure 5.5bar). After the reaction temperature reached to 1700C a solution of citric acid (30g) and de-mineralized water (100g) was pumped in the reactor using pressure vessel at 8 bar. The stirring was continued for 60 min and the reaction mixture was cooled to room temperature in 15 min using chilling water circulation. The whole mixture was neutralized by 5M Sodium hydroxide solution till pH 5. Solid and liquid materials were separated using centrifuge. The liquid hydrolysate was tested for inhibitors and sugars. Solid pretreated slurry (300g on dry weight basis) having pH 5 was taken in the hydrolysis reactor equipped with agitator and a temperature probe. Reactor temperature was maintained at 500C and cellulase preparation from cellulases was added at 15 FPU/g of pretreated biomass and total reaction volume was making up to 1000g using de-mineralized water at 5 pH. 20g of sodium azide was added to the reactor to prevent the growth of any micro-organisms during the hydrolysis process. The continuous agitation of reaction mixture was carried out for 24 hours to release maximum monomeric sugars. The liquid sample after complete hydrolysis was subjected to DNS for sugar analysis. The data received is summarized in Table-1.
Example-4: A mixture of biomass (100 g) in de-mineralized water (900ml) was taken in the high pressure reactor equipped with agitator, temperature and pressure probe and temperature control, sampling port and re-circulating coil. The reactor was also has a port to add catalyst at high pressure and temperature. The stirred mixture was heated to 170°C (pressure 5.5bar). After the reaction temperature reached to 1700C a solution of citric acid (10g) and isocitric acid (20g) and de-mineralized water (100g) was pumped in the reactor using pressure vessel at 8 bar. The stirring was continued for 60 min and the reaction mixture was cooled to room temperature in 15 min using chilling water circulation. The whole mixture was neutralized by 5M Sodium hydroxide solution till pH 5. Solid and liquid materials were separated using centrifuge. The liquid hydrolysate was tested for inhibitors and sugars. Solid pretreated slurry (300g on dry weight basis) having Ph 5 was taken in the hydrolysis reactor equipped with agitator and a temperature probe. Reactor temperature was maintained at 500C and ellulose preparation from cellulases was added at 15 FPU/g of pretreated biomass and total reaction volume was making up to 1000g using de-mineralized water at 5 Ph. 20g of sodium azide was added to the reactor to prevent the growth of any micro-organisms during the hydrolysis process. The continuous agitation of reaction mixture was carried out for 24 hours to release maximum monomeric sugars.
Example 5: (Control): Same as example 4 except that the organic acid was not used in pre-treatment. Data received was summarised in Table-1.
Sugar content in the pre-treatment hydrolysate was 4.72 mg/ml when the experiments were carried out without any acid, whereas, in presence of organic acid it was 10.32, 12.36, 27.49, 24.70 mg/ml respectively in experiment 1-4. Similarly, sugar content in enzymatic hydrolysate was 56mg/ml with the pre-treated biomass in absence of organic acid where as it was 123, 179, 172, 163 mg/ml respectively in the experiments 1-4. Low level of sugar in control experiments shows lower pre-treatment and high level of sugar in experiment No. 3 shows high degree of pre-treatment, showing the effectiveness of the organic acid catalyst.
Table-1: Inhibitors and sugars in pretreatment and enzymatic hydrolysate
Example No. Inhibitors (ppm) Pretreated hydrolysate (Sugars, mg/ml) Enzymatic hydrolysate (sugar, mg/ml)
Acetic
Acid HMF Furfural
1 843 276 674 10.32 123
2 1314 563 2187 12.36 179
3 1760 607 2334 27.49 172
4 1555 504 2312 24.70 163
5 355 42 243 4.72 56

We Claim:
1. A method to prepare fermentable sugars from biomass resources, said method comprising the steps of:
(f) pretreating the biomass with dilute acid;
(g) neutralizing the pretreated biomass of the step (a) with acid-neutralizing neutralizers;
(h) separating the pentose sugars from the pretreated biomass of steps (a) and (b) to obtain a pretreated biomass slurry;
(i) hydrolysing the biomass slurry of step (c); and
(j) obtaining fermentable sugars.

2. A method as claimed in claim 1, wherein the step (a) the dilute acid is selected from group comprising of citric acid, isocitric acid, aconitic acid, propane-1,2,3-tricarboxylic acid and trimesic acid alone or as a mixture.

3. A method as claimed in any one of claims 1-2, wherein the step (a) the dilute acid is in the concentration of 0.5-20% w/v.

4. A method as claimed in claim 1, wherein the step (b) the neutralizer is selected from the group comprising of sodium hydroxide (NaOH), calcium carbonate (CaCO3), sodium carbonate (Na2CO3), magnesium hydroxide (Mg(OH)2, slaked lime (Ca(OH)2, quicklime (CaO) or calcium carbonate (CaCO3 may be used. More specifically, sodium hydroxide (NaOH), sodium carbonates (Na2CO3) or calcium carbonate (CaCO3).

5. A method as claimed in any one of claims 1 and 4, wherein the step (b) the neutralizer is in the concentration of 1-10M.

6. A method as claimed in any one of claims 1 and 4-5, wherein the step (b) the neutralizer is 5M.

7. A method as claimed in claim 1, wherein the step (c), the penstose sugars and solid pretreated biomass slurry is separated using various known filtration methods selected from group comprising a filter press, a centrifuge, a membrane filter or a nanofilter.

8. A method as claimed in claim 1, wherein step (d) hydrolysis of pre-treated biomass slurry is carried out using cellulase at a pH 4-7 at a temperature of 30°C-70°C for 12-48 hours.

9. A method as claimed in any one of claims 1 or 8, wherein the step (d) hydrolysis of pre-treated biomass slurry is carried out using cellulase at a pH 4.8-5.5 at a temperature of 48°C-52°C for 24-36 hours.

10. A method as claimed in claim 1, wherein step (a) is carried out under high pressure of at 130°C-220°C.

11. A method as claimed in claim 1, wherein the step (a) the biomass concentration is in the range of 5-50% (w/w).

12. A method as claimed in claim 10, wherein the step (a) the pressure is in the range of 5-10 bars.

13. A method as claimed in claim 12, wherein the step (a) the pressure is 5.5 bar.