DESC:FIELD OF THE INVENTION
The present invention relates to a cost effective method for producing readily hydrolysable polysaccharides from biomass by extracting lignin from lignocellulosic biomass. The present invention discloses use of refinery waste stream from the petroleum refinery unit for fuel sweetening to delignify the biomass and further hydrolysis of the delignified biomass with a saccharification enzyme consortium to obtain readily hydrolysable polysaccharides.
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 molasses, 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 sustainable and inexpensive feed stocks. Moreover, the use of feed stocks like sugarcane and corn raised the issue of food vis-à-vis fuel and, therefore, prompted to look for the production of ethanol from the non-food materials like lignocellulosic biomass. Cellulosic and lignocellulosic feedstocks like agricultural residues, forestry wastes, municipal wastes etc provide a very large and sustainable/ renewable feedstock for production of ethanol, fuel and chemicals. Therefore, plant based material present a good choice as a feedstock for producing ethanol and chemicals.

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 for their degradation. Cellulose, the most abundant polysaccharide in all plants and is a polymer mainly of glucose, accounting about 50% of the plant 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 e.g. 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.

Lignin is a 3-dimensional polyphenolic network of monomethoxylated, dimethoxylated and non-methoxylated phenylpropanoid units, derived mainly from p-hydroxycinnamyl alcohol. Lignin is hydrophobic and is 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.

Lignocellulosic biomass feedstocks and wastes, such as agricultural residues, wood, forestry wastes, sludge from paper manufacture, and municipal and industrial solid wastes, provide a potentially large renewable feedstock for the production of chemicals, plastics, fuels and feeds. Cellulosic and lignocellulosic feedstocks and wastes, composed of cellulose, hemicellulose, pectins and of lignin are generally treated by a variety of chemical, mechanical and enzymatic means to release primarily hexose and pentose sugars, which can then be fermented to useful products.

The presence of lignin in the pretreated biomass, e.g. by adopting steam explosion or acid treatment, is one of the major obstacles for efficient enzymatic hydrolysis. Cellulolytic enzymes tend to adsorb irreversibly on the surface of lignin, therefore much higher quantities of enzymes, translating to higher cost of process, are required. Therefore, it is highly desirable to adopt a pretreatment process which leads to outright extraction of lignin. Ammonia and alkali based pretreatment processes which can disrupt the lignocellulosic structure and at the same time extract lignin, are the preferred choices. However, higher cost of alkali and its consumption during the pretreatment process is the main impediment for their application on commercial scale.

Cellulosic and lignocellulosic feedstocks and wastes, such as agricultural residues, wood, forestry wastes, waste from paper manufacture, and municipal and industrial solid wastes, provide a potentially large renewable feedstock for the production of chemicals and fuels. Different types of biomass, such as woody plants, herbaceous plants, grasses, aquatic plants, agricultural crops and residues, municipal solid waste and manures, contain different amounts of cellulose, hemicellulose, lignin, and extractives (Chandra et al., Biofuels. Adv. Biochem. Eng. Biotechnol., 108: 67-93, 2007). Generally plant biomass contains 40–50% cellulose (with exception to a few plants, such as cotton and hemp bast-fibre that are made up of ˜80% cellulose), 20–40% hemicellulose, 20-30% lignin by weight (Chandra et al., Biofuels. Adv. Biochem. Eng. Biotechnol., 108:67-93, 2007; Mckendry, P., Bioresour. Technol. 83:37-46, 2002). Biomass recalcitrance to bioprocessing is directly related to the inherent properties of the biomass source. Properties such as lignin content, cellulose accessibility to cellulase (CAC), and cellulose crystallinity (CC) determine the overall digestibility of the biomass. The complexity of a given biomass type is reflected in the relationship between its structural and carbohydrate components. The factors that contribute to biomass recalcitrance include: crystallinity and degree of polymerization of cellulose; accessible surface area (or porosity); protection of cellulose by lignin; cellulose sheathing by hemicellulose; and fibre strength (Mosier et al., Appl. Biochem. Biotechnol., 125:77-97, 2005a; Mosier et al., Bioresour. Technol., 96:673-686, 2005b). It is this variability that accounts for differences in the digestibility/hydrolysis of a given biomass feedstock. Removal of lignin enhances biomass digestibility up to the point where the effect of lignin present is no longer sufficient to limit enzymatic hydrolysis or microbial digestibility (Chang and Holtzapple, Appl. Biochem. Biotechnol., 84-86,5-37, 2000; Draude et al., Bioresour. Technol., 79, 113-120, 2001; Jeoh et al., Biotechnol. Bioeng., 98:112-122, 2007). It has also been shown that that highly crystalline cellulose is less accessible to cellulase attack than amorphous cellulose (Chang and Holtzapple, Appl. Biochem. Biotechnol., 84-86, 5-37, 2000), and that cellulose accessibility to cellulase is one of the most important (rate limiting) factors in enzymatic hydrolysis when the affect of lignin is minimized (Jeoh et al., Biotechnol. Bioeng., 98:112–122, 2007). Pointing out that the difficulty is not in achieving good sugar, but in obtaining good yields at low energy input, Zhu and Pan suggested that bio-based research efforts targeting woody biomass should be focused on upstream activity such as size reduction and physico-chemical pretreatment to improve direct microbial utilization of polysaccharides and sugar yields following enzymatic saccharification (Zhu et al., Appl. Microbiol. Biotechnol., 87:847-857, 2010; Zhu and Pan., Bioresour. Technol., 100:4992–5002, 2010).

EP 0654096 B1 discloses methods for the pretreatment of a lignocellulose-containing biomass. Pretreatment comprises the addition of calcium hydroxide and water to the biomass to form a mixture, and subjecting the mixture to relatively high temperatures for a period of time sufficient to render the biomass amenable to digestion. The pretreated biomass is digested to produce useful products such as feedstocks, fuels, and compounds including fatty acids, sugars, ketones and alcohols. Alternatively, the pretreatment process includes the addition of an oxidizing agent, selected from the group consisting of oxygen and oxygen-containing gasses, to the mixture under pressure. The invention is also directed to a method for the recovery of calcium from the pretreated biomass.

U.S. Patent No. 4,048,341 is directed to a process for increasing the feed value of lignocellulosic material by contacting the material with an alkaline liquid, specifically, sodium hydroxide. The alkaline liquid, supplied in excess, is allowed to run off the material before any essential alkalization effect has been reached. After the liquid absorbed in the material has provided its effect, an acid solution is added to the material to neutralize the excess alkali. Large amount of alkali is consumed during the process.

WO/2008/095098 relates a new method of treating biomass with alkali, e.g. lime. In this method, lime and lignin were removed from the treated biomass by squeezing with a high pressure device to remove alkali and other potential inhibitors of the cellulose enzymes added for saccharification. The resulting fibrous material was rapidly solubilised by cellulases, without inhibitory effects on the cellulose activity.

WO 2010059825 relates to a process of obtaining sugar solutions from polysaccharide enriched biomass by contacting biomass with water and at least one nucleophilic 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.

U.S. Pat. No. 4,644,060 relates to the process for improving the bioavailability of polysaccharides in lignocellulosic materials. This method involves contacting the lignocellulosic materials with ammonia at a temperature from about 100 to about 35 MPa, and at an ammonia density from about 0.10 g per mL to about 0.45 g per mL. Lignocellulosic materials treated by the process of the invention can be nearly completely hydrolyzed by cellulases, employed directly as carbohydrate sources for microbial fermentation, or fed to livestock.

U.S. Pat. No. 4,356,196 describes a process for treating alfalfa and other cellulosic agricultural crops. The materials to be treated are contacted with ammonia at pressures from about 203 kPa (30 psi) to about 4.05 MPa (588 psi), and at temperatures from about 10° C to about 85° C, in a closed reactor, for about 30 minutes. Ammonia is then released from the reactor explosively, leaving a product having enhanced value as a foodstuff for livestock.

U.S. Pat. No. 5,693,296 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.

WO2004/081185 discusses methods for hydrolyzing lignocellulose, comprising contacting the lignocellulose with a chemical; the chemical may be a base, such as sodium carbonate or potassium hydroxide, at a pH of about 9 to about 14, under moderate conditions of temperature, pressure and pH.

US 2007/0031918 A1 discloses a process for treating biomass to produce fermentable sugars. The process involves a pretreatment step wherein biomass at relatively high concentration is treated with a low concentration of ammonia relative to the dry weight of biomass. The biomass is further treated with a saccharification enzyme consortium to produce fermentable sugars.

The prior art available does not specify the recovery of the base catalyst like alkali. The amount required for pretreatment of these bases is very high and therefore makes the process very expensive. The cost of these catalysts is another area of concern. Moreover, recovery of lignin from the residue by neutralising the base catalyst cost heavily and become environmental hazard leading to contamination of soil and water.

Additionally, in the prior art, it was noticed that the addition of some amount of other nucleophile compound like sulphur, amine or alcohol improved the enzymatic hydrolysis by delignifying the biomass, which reduce the cellulase amount significantly. Therefore search was made to look in the prior art which uses alkali along with other nucleophiles like sulphur, amines and alcoholic components. The prior art related to this is given below.

French Patent No. 2,518,573 discloses a process for saccharification of lignocellulosic materials involving pretreatment with an amine, for example, diethanolamine, for about 1 to 3 hours at a temperature from about 80° C to about 170° C., followed by enzymatic hydrolysis.

US 8304213 B2 discloses a process in which biomass is pretreated using an organic solvent solution under alkaline conditions in the presence of one of more sulfide (hydrosulfide) salt and optionally one or more additional nucleophile to fragment and extract lignin. Pretreated biomass is further hydrolyzed with a saccharification enzyme consortium. Fermentable sugars released by saccharification may be utilized for the production of target chemicals by fermentation.

EP 2358889 A2 describes the pretreatment of biomass using an organic solvent solution under alkaline conditions in the presence of elemental sulfur and optionally one or more alkylamine and/or one or more additional nucleophile to fragment and extract lignin. Pretreated biomass is further hydrolyzed with a saccharification enzyme consortium. Fermentable sugars released by saccharification may be utilized for the production of target chemicals by fermentation.

WO/2010/080434 relates to the process in which biomass is pretreated using an organic solvent solution under alkaline conditions in the presence of one or more alkylamine and optionally one or more additional nucleophile to fragment and extract lignin. Pretreated biomass is further hydrolyzed with a saccharification enzyme consortium. Fermentable sugars released by saccharification may be utilized for the production of target chemicals by fermentation.

US 20100143974 A1 discloses a method to treat biomass with alkali, for example lime. The lime and lignin was sufficiently removed from the treated biomass by squeezing with a high pressure device to remove alkali and other potential inhibitors of the cellulase enzymes added for saccharification. The resulting fibrous material was rapidly solubilized by cellulases, even at solid loads ranging from 10 to 30% (w/w) without inhibitory effects on the cellulase activity. The lime pretreatment removed lignin effectively and left the cellulose and hemicellulose almost intact. The method yielded a biomass with structure capable of being enzyme solubilized and fermented readily at a solids loading of 10-30% for a production of ethanol.

Hence, 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. Pretreatment is primarily used to make the polysaccharides of lignocellulosic biomass more readily accessible to cellulolytic enzymes. However, ideal 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.

The basic pretreatment methods could result in separation of lignin prior to enzymatic saccharification and thus save on costly enzymes. However, the separation of enzymes in most of the cases was not complete and to enhance lignin separation a host of additional chemicals were used which increased the cost, due to cost involved of the chemicals and in some cases due to the separation and recovery of these chemicals post pretreatment. There is therefore a need to develop a simple process, using substantially higher solid content of biomass, in a manner that the cost of the pretreatment step can be very substantially reduced and the subsequent saccharification step yields higher amount of monomeric sugars.

SUMMARY OF THE INVENTION
Accordingly, the present invention provides a method of producing fermentable sugars from lignocellulosic biomass comprising the steps of:
(a) treating a biomass mixture comprising lignocellulosic biomass and a waste refinery stream produced by petroleum refineries during production of low sulphur fuels to obtain a treated biomass mixture;
(b) filtering the treated biomass mixture and washing with water and pressing to remove excess refinery waste, water and lignin to obtain a de-lignified biomass; and
(c) reacting the delignified biomass with one or more saccharification enzymes to obtain fermentable sugars.

According to one embodiment of the present invention, the lignocellulosic biomass is selected from a group consisting of rice straw, wheat straw, cotton stalk, corn cobs, corn stover, sugarcane bagasse, wood chips, Jatropha cuttings, mustard stalk and combination thereof. These lignocellulosic biomasses as used in present invention are commercial waste obtained from India after a commercial process.

According to one embodiment of the present invention, the lignocellulosic biomass is of size from 2 mm to 10 cm.

According to one embodiment of the present invention, the waste refinery stream is selected from LPG treating unit.

According to another embodiment of the present invention, the waste refinery stream is selected from merox unit for producing low sulphur kerosene and ATF.

According to another embodiment of the present invention, the waste refinery stream is selected from refinery unit for producing low sulphur gasoline.

According to one embodiment of the present invention, the ratio of lignocellulosic biomass to waste refinery stream is from 0.2-1.

According to one embodiment of the present invention, the lignocellulosic biomass is treated at a temperature of between 80 to 200 °C in step (a).

According to one embodiment of the present invention, the step (a) is conducted at a time between 20 minutes to 2 hours.

According to one embodiment of the present invention, the pressing step (b) is conducted at a pressure in the range of 600 psi to 2500 psi.

According to one embodiment of the present invention, the waste refinery stream comprises alkali concentration in the range of 1% to 10%, more preferably in the range of 2% to 8%, the percentage being expressed in terms of the weight of the waste stream.

According to another embodiment of the present invention, the waste refinery stream comprises about 4-10% of alkali and about 1-5% nucleophile, the percentage being expressed in terms of the weight of the waste stream.

According to one embodiment of the present invention, the biomass mixture comprises alkali concentration in the range of 1% to 10%, preferably in the range of 1% to 5%, and more preferably in the range of 1% to 3%, the percentage being expressed in terms of the weight of the biomass mixture.

According to another embodiment of the present invention, optionally the biomass mixture is diluted to make the overall percentage of alkali equal to 5% to 1%, the percentage being expressed in terms of the weight of the biomass mixture.

According to one embodiment of the present invention, the waste refinery stream comprises: water from 1-99%, alkali 1-10%, alkali metal sulphide and disulphide from about 0.1 to 5%, thiols from about 0.01 to 4% having C-2 to C-10, alkali mercaptides having C-2 to C-10 from about 0.01 to 3%, alkyl disulphide from C 2-10 mercaptans, sodium salt of naphthenic acid from 0.01 to 1% by weight, alkali carbonate from 0.01 to 1%, the percentage being expressed in terms of the weight of the waste stream.

In yet another embodiment of the present invention, the step (c) comprises adjusting of pH 4 to 7 with buffer selected from citrate, acetate and phosphate so that pH and temperature are compatible with saccharification enzymatic hydrolysis. The temperature used in step (c) is between 35 to 60°C. The saccharification enzyme used in step (c) comprises one or more cellulases. The total sugars release in step (c) is from 75 to 95% of the original hollocellulose present in the biomass.

The present invention also provides a method of producing de-lignified biomass from lignocellulosic biomass comprising the steps of:
(a) treating a biomass mixture comprising lignocellulosic biomass and a waste refinery stream produced by petroleum refineries during production of low sulphur fuels to obtain a treated biomass mixture; and
(b) filtering the treated biomass mixture and washing with water and pressing to remove excess refinery waste, water and lignin to obtain a de-lignified biomass.

According to one embodiment of the present invention, the waste alkali refinery stream is selected from LPG treating unit, merox unit for producing low sulphur kerosene and ATF and refinery unit for producing low sulphur gasoline.

According to one embodiment of the present invention, the biomass mixture comprises alkali concentration in the range of 1% to 10%, preferably in the range of 1% to 5%, and more preferably in the range of 1% to 3%, the percentage being expressed in terms of the weight of the biomass mixture.

BRIEF DESCRIPTION OF DRAWINGS
Fig 1 shows the effect of sodium hydroxide concentration on delignification (%) and enzymatic hydrolysis (%) of rice straw.
Fig 2 shows the effect of refinery waste stream concentration on delignification (%) and enzymatic hydrolysis (%) of rice straw.
Fig 3 shows the effect of sodium hydroxide concentration on delignification (%) and enzymatic hydrolysis (%) of cotton stalk.
Fig 4 shows the effect of refinery waste stream concentration on delignification (%) and enzymatic hydrolysis (%) of cotton stalk.

DETAILED DESCRIPTION OF THE INVENTION
While the invention is susceptible to various modifications and alternative forms, specific embodiment thereof will be described in detail below. It should be understood, however that it is not intended to limit the invention to the particular forms disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternative falling within the scope of the invention as defined by the appended claims.

The present invention discloses a method of producing de-lignified biomass from lignocellulosic biomass using refinery alkali waste stream. The present invention provides a method for producing readily available and hydrolysable polysaccharide-enriched biomass and for extracting lignin from lignocellulosic biomass. In one of the embodiment the present invention provides a method for producing readily available and hydrolysable polysaccharide-enriched biomass and for extracting lignin from lignocellulosic biomass while quantitatively retaining carbohydrate.

The methods include treating lignocellulosic biomass with an aqueous solution at elevated temperatures in a simple process using the caustic process waste streams. The biomass may be further treated with a saccharification enzyme consortium to produce fermentable sugars. These sugars may be subjected to further processing for the production of ethanol.

An aspect of the present invention discloses a method of producing hydrolysable polysaccharides-enriched lignocellulosic biomass.

The hydrolysable polysaccharides-enriched lignocellulosic biomass is produced by suspending the lignocellulosic biomass in an aqueous solution of refinery waste stream from 1% to 100%, heating the biomass suspension to a temperature of about 60 °C to about 220°C or about 100 °C to about 220°C for about 5 minutes to about 4 hours whereby lignin is fragmented and is dissolved in the suspension and filtering the free liquid; whereby the dissolved lignin is removed and hydrolysable polysaccharide-enriched biomass is produced.

Refinery waste stream used in accordance with present invention comprises a mixture of water from 1-99%, alkali 1-10%, alkali metal sulphide and disulphide from about 0.1 to 5%, thiols from about 0.01 to 4% having C-2 to C-10, alkali mercaptides having C-2 to C-10 from about 0.01 to 3%, alkyl disulphide from C 2-10 mercaptans, sodium salt of naphthenic acid from 0.01 to 1% by weight, alkali carbonate from 0.01 to 1%. Refinery waste stream contains about 4-10% of sodium hydroxide and some sulphur and other compounds includes organic and inorganic in the form of divalent sulphur.

The lignocellulosic biomass used in accordance to the present invention includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste and sludge from paper manufacture, wood and forestry waste. Examples of biomass include, but are not limited to corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, cotton stalk, mustard stalk, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sugar cane straw, Jatropha cuttings, sorghum, soy, lentil stalks, trees, branches, roots, leaves, wood chips, sawdust, Jatropha and Karanja cake and other like materials.

In one embodiment of the present invention, the lignocellulosic biomass includes agricultural residues such as corn stover, wheat straw, cotton stalk, mustard stalk, rice straw, canola straw, and soybean stover; grasses such as switchgrass, miscanthus, Jatropha cuttings, fiber process residues such as corn fiber, beet pulp, pulp mill fines and rejects and sugar cane bagasse; sugar cane straw and sorghum; forestry wastes, other hardwoods, softwood and sawdust as well as other crops or sufficiently abundant lignocellulosic material.

In another embodiment, biomass that is useful for the invention has a relatively high carbohydrate content, is relatively dense, and/or is relatively easy to collect, transport, store and/or handle.

In another embodiment of the invention, biomass that is useful includes corn cobs, corn stover, sugar cane bagasse, sugar cane straw, rice straw, cotton stalk, mustard stalk, Jatropha cuttings and switchgrass. The lignocellulosic biomass may be derived from a single source, or can comprise a mixture derived from more than one source; for example, biomass could comprise a mixture of corn cobs and corn stover, or a mixture of stems or stalks and leaves.

Another aspect of the invention discloses a method of simultaneous fragmentation and selective extraction of lignin from lignocellulosic biomass to produce a substantially lignin-free biomass.

In accordance with the present invention lignin-free biomass is produced from lignocellulosic biomass by
a) contacting said biomass with the caustic refinery waste water to form a waste water-biomass mixture;
b) placing the waste water-biomass mixture in a sealed pressure vessel whereby the mixture obtained in step (a) is heated at a temperature of about 100°C to about 180°C for about 10 to about 80 minutes whereby lignin is fragmented and dissolved in the solvent;
c) removing the dissolved lignin obtained in step (b) by filtration and
d) washing the residual biomass with water, whereby substantially lignin-free biomass is produced.

Definitions
The following definitions are used in this disclosure:
"Room temperature" and "ambient" when used in reference to temperature refer to any temperature from about 20°C to about 40°C.
"Fermentable sugars" refers to a sugar content primarily comprising monosaccharides and some disaccharides that can be used as a carbon source by a microorganism (some polysaccharides may be present) in a fermentation process to produce a target product.
"Lignocellulosic" refers to material comprising both lignin and cellulose. Lignocellulosic material may also comprise hemicellulose. In the processes described herein, lignin is dissolved and substantially removed from the lignocellulosic biomass to produce a carbohydrate-enriched biomass.
"Soluble lignin" as referred to herein means the lignin that is dissolved in an in the pretreatment media.
“AI lignin” refers to acid insoluble lignin
"Cellulosic" refers to a composition comprising cellulose.
"Target product" refers to a chemical, fuel, or chemical building block produced by fermentation. Product is used in a broad sense and includes are ethanol and butanol.
"Dry weight of biomass" refers to the weight of the biomass essentially all water removed. Dry weight is typically measured according to American Society for Testing and Materials (ASTM) Standard E1756-01 (Standard Test Method for Determination of Total Solids in Biomass) or Technical Association of the Pulp and Paper Industry, Inc. (TAPPI) Standard T-412 om-02 (Moisture in Pulp, Paper and Paperboard).
"Selective extraction" means removal of lignin while substantially retaining carbohydrates.
"Biomass" and "lignocellulosic biomass" as used herein refer to any lignocellulosic material, including cellulosic and hemi-cellulosic material along with lignin, for example, bioenergy crops, agricultural residues, municipal solid waste, wood, forestry waste and combinations thereof, and as further described below. Biomass has a carbohydrate content that comprises polysaccharides and oligosaccharides and may also comprise additional components, such as protein and/or lipid and lignin.
"Preprocessing" as used herein refers to processing of lignocellulosic biomass prior to pretreatment. Preprocessing is any treatment of biomass that prepares the biomass for pretreatment, such as mechanically milling and/or drying to the appropriate moisture contact.
“Solvent" as used herein refers to the mixture of water and refinery waste stream from 1-99% and vice versa.
Biomass-solvent suspension" refers to a mixture of biomass and solvent. The biomass-solvent solution may comprise additional components such as sulfides, sodium salt of mercaptans having C-2 to C-15, disulphide of mercaptans having C-2 to C10.
"Saccharification" refers to the production of fermentable sugars from primarily polysaccharides by the action of hydrolytic enzymes. Production of fermentable sugars from pretreated biomass occurs by enzymatic saccharification by the action of cellulolytic and hemicellulolytic enzymes.
"Pretreating biomass" or "biomass pretreatment" as used herein refers to subjecting native or preprocessed biomass to chemical or physical action, or any combination thereof, rendering the biomass more susceptible to enzymatic saccharification or other means of hydrolysis prior to saccharification. For example, the methods claimed herein may be referred to as pretreatment processes that contribute to rendering biomass more accessible to hydrolytic enzymes for saccharification.
"Pretreatment filtrate/hydrolysate" means the free liquid that is in contact with the biomass following pretreatment and which is separated by filtration.
"Air-drying the filtered biomass" can be performed by allowing the biomass to dry through equilibration with the air of the ambient atmosphere.
"Carbohydrate-enriched" as used herein refers to the biomass produced by the process treatments described herein. In one embodiment the readily saccharifiable carbohydrate-enriched biomass produced by the processes described herein has a carbohydrate concentration of greater than or equal to 85% of the dried biomass by weight, while having removed 80% or greater of the starting biomass lignin content based on dry weight.
"Heating the biomass suspension" means subjecting the biomass suspended in a solvent to a temperature greater than ambient or room temperature. Temperatures relevant to the present pretreatments are from about 60 to about 220°C, or from about 100 to about 220°C, or from about 140 to about 180° C, or any temperature within or approximately these ranges.
"Filtering free liquid under pressure" means removal of unbound liquid through filtration, with some pressure difference on opposite faces of the filter.
"Alkaline" or "under alkaline conditions" means a pH of greater than 7.0. In the present invention, "under alkaline conditions", also means a pH of the biomass-solvent suspension equal to or greater than the pKa of the nucleophiles present such that these are substantially deprotonated and more highly reactive than in their protonated states.
"Substantially lignin-free biomass" means a pretreated sample in which about = 70% of the lignin is removed.
"Dry biomass" means biomass with a dry matter content of 90%. Methods for drying the biomass include exposure at ambient temperature to vacuum or flowing air at atmospheric pressure and or heating in an oven or a vacuum oven.
"Pressure vessel" is a sealed vessel that may be equipped or not with a mechanism for agitation of a biomass/solvent suspension, in which a positive pressure is developed upon heating the lignocellulosic biomass.
"Hydrolysate" refers to the liquid in contact with the lignocellulose biomass which contains the products of hydrolytic reactions acting upon the biomass (either enzymatic or not), in this case monomeric and oligomeric sugars.
"Enzyme consortium" or "saccharification enzyme consortium" is a collection of enzymes, usually secreted by a microorganism, which in the present case will typically contain one or more cellulases, xylanases, glycosidases, ligninases and esterases, pectinase.
"Monomeric sugars" or "simple sugars" consist of a single pentose or hexose unit, e.g., glucose, xylose, arabinose, galactose and mannose.
"Fragmentation" is a process in which lignocellulosic biomass is treated with refinery waste in alkaline conditions breaking the lignin down into smaller subunits.
"Simultaneous fragmentation and selective extraction" as used herein refers to a fragmentation reaction performed in refinery waste stream such that the lignin fragments go into solution as soon as they are released from the bulk biomass.

Methods for pretreating lignocellulosic biomass to produce readily saccharifiable biomass as disclosed herein provide economical processes for rendering components of the lignocellulosic biomass more accessible or more amenable to enzymatic saccharification. The pretreatment can be chemical, physicochemical and biological, or any combination thereof. In this disclosure the pretreatment is performed in the presence of nucleophiles may also be present, such as thiol, sulfide reagents, or combinations thereof. The presence of refinery waste stream and alkaline conditions assists lignin fragmentation and removal and carbohydrate recovery.

In addition, the methods described in the present disclosure minimize the loss of carbohydrate during the pretreatment process and maximize the yield of solubilized (monomeric+oligomeric) sugars in saccharification.

As disclosed above the methods described herein include pretreating lignocellulosic material, with a refinery waste stream solution comprising the components described below, to produce a readily saccharifiable carbohydrate-enriched biomass.

During pretreatment of biomass as described herein, the sulphur components promote fragmentation of the lignin, the mechanism of which might include nucleophilic attack on the lignin aryl ether linkages, nucleophilic attack at the alpha-position of the quinine methides, formed under alkaline conditions, promoting the rupture of the beta-aryl ether bond, or reduction of the quinine methide with elimination of the beta-aryl ether. This fragmentation of the lignin into lower molecular weight components and the dissolution of these fragments in the refinery waste stream leading to increases the exposure of the polysaccharide chains to cellulolytic and hemicellulolytic enzymes (such as cellulases and hemicellulases) for hydrolytic release of oligomeric and monomeric sugars.

Lignocellulosic Biomass

In the present method, the biomass dry weight is at an initial concentration of at least about 9% up to about 80% of the weight of the biomass-solvent suspension during pretreatment. More suitably, the dry weight of biomass is at a concentration of from about 10% to about 60%, 8% to about 50%, or about 12% to about 40% of the weight of the biomass-solvent suspension. The percent of biomass in the biomass-solvent suspension is kept high to reduce the total volume of pretreatment material, decreasing the amount of solvent and reagents required and making the process more economical.

The biomass may be used directly as obtained from the source, or may be subjected to some preprocessing, for example, energy may be applied to the biomass to reduce the size, increase the exposed surface area, and/or increase the accessibility of lignin and of cellulose, hemicellulose, and/or oligosaccharides present in the biomass to base pretreatment and to saccharification enzymes used, respectively, in the second and third steps of the method. Energy means useful for reducing the size, increasing the exposed surface area, and/or increasing the accessibility of the lignin, and the cellulose, hemicellulose, and/or oligosaccharides present in the biomass to the base pretreatment and to saccharification enzymes include, but are not limited to, milling, crushing, grinding, shredding and chopping. This application of energy may occur before or during pretreatment, before or during saccharification, or any combination thereof.

Drying prior to pretreatment may occur as well by conventional means, such as exposure at ambient temperature to vacuum or flowing air at atmospheric pressure and or heating in an oven at atmospheric pressure or a vacuum oven.

Pretreatment Conditions
Pretreatment of biomass with the solvent solution comprising of refinery waste stream and water is carried out in any suitable vessel. Typically the vessel is one that can withstand pressure, has a mechanism for heating, and has a mechanism for mixing the contents.

The pretreatment reaction may be performed in any suitable vessel, such as a batch reactor or a continuous reactor. One skilled in the art will recognize that at higher temperatures (above 100° C), a pressure vessel is required. The suitable vessel may be equipped with a means, such as impellers, for agitating the biomass, water and refinery waste stream mixture.

Prior to contacting the biomass with waste stream, vacuum may be applied to the vessel containing the biomass. By evacuating air from the pores of the biomass, better penetration of the media into the biomass may be achieved. The time period for applying vacuum and the amount of negative pressure that is applied to the biomass will depend on the type of biomass and can be determined empirically so as to achieve optimal pretreatment of the biomass (as measured by the production of fermentable sugars following saccharification).

The heating of the biomass in waste stream is carried out at a temperature of from about 60°C to about 220 °C, about 100 °C to about 220 °C, about 80 to about 200 °C, about 110 °C to 170°C, or about 140 °C to about 195 °C. The heated solution may be cooled rapidly to room temperature. In still another embodiment, the heating of the biomass is carried out at a temperature of about 180 °C. Heating of the biomass-solvent suspension may occur for about 10 minutes to about 3 hours or for about 20 minutes to about 2 hours or for about 30 minutes to about 2 hours or more preferably from about 20 minutes to 1.5 hours.

The pretreatment of biomass with the waste stream solution occurs under alkaline conditions. For the pretreatment methods described herein, the temperature, pH, time of pretreatment and concentration of reactants such as the waste stream solutions.

Following pretreatment at elevated temperature, the biomass is filtered under pressure. The filtration may either be preceded or not by cooling. Following filtration, the biomass may be washed one or more times with water at elevated or at ambient temperature. It may then either be washed with water to remove the water soluble material and excess moisture was removed by air drying and then saccharified. Methods for drying the biomass include exposure at ambient temperature to vacuum or flowing air at atmospheric pressure and or heating in an oven at atmospheric pressure or in a vacuum oven as described more fully herein.
To assess performance of the pretreatment, i.e., the production of readily saccharifiable carbohydrate-enriched biomass and subsequent saccharification, separately, the theoretical yield of sugars derivable from the starting biomass can be determined and compared to measured yields.

Further Processing
Saccharification
Following pretreatment and subsequent filtration/washing, the readily saccharifiable carbohydrate-enriched biomass comprises a solid containing mostly polysaccharides and a minor amount of residual lignin. This readily saccharifiable biomass may then be further hydrolyzed in the presence of a saccharification enzyme consortium to release oligosaccharides and/or monosaccharides in a hydrolysate.

Surfactants such as Tween 20 or Tween 80 or polyoxyethylenes such as PEG 2000, 4000 or 8000 may be added to improve the saccharification process (U.S. Pat. No. 7,354,743 B2, incorporated herein by reference).

The saccharification enzyme consortium may comprise one or more glycosidases; the glycosidases may be selected from the group consisting of cellulose-hydrolyzing glycosidases, hemicellulose-hydrolyzing glycosidases, and starch-hydrolyzing glycosidases. Other enzymes in the saccharification enzyme consortium may include, but not limited to, peptidases, lipases, ligninases and esterases.

The saccharification enzyme consortium comprises one or more enzymes selected primarily, but not exclusively, from the group "glycosidases" which hydrolyze the ether linkages of di-, oligo-, and polysaccharides and are found in the enzyme classification EC 3.2.1.x (Enzyme Nomenclature 1992, Academic Press, San Diego, Calif. with Supplement 1 (1993), Supplement 2 (1994), Supplement 3 (1995, Supplement 4 (1997) and Supplement 5 [in Eur. J. Biochem., 223:1-5, 1994; Eur. J. Biochem., 232:1-6, 1995; Eur. J. Biochem., 237:1-5, 1996; Eur. J. Biochem., 250:1-6, 1997; and Eur. J. Biochem., 264:610-650, 1999, respectively]) of the general group "hydrolases" (EC 3). Glycosidases useful in the present method can be categorized by the biomass component that they hydrolyze.

Glycosidases useful for the present method include, but not limited to, cellulose-hydrolyzing glycosidases such as cellulases, endoglucanases, exoglucanases, cellobiohydrolases and beta-glucosidases; hemicellulose-hydrolyzing glycosidases such as, xylanases, endoxylanases, exoxylanases, beta-xylosidases, arabino-xylanases, mannases, galactases, pectinases, glucuronidases, and starch-hydrolyzing glycosidases such as, amylases, alpha-amylases, beta-amylases, glucoamylases, alpha-glucosidases and isoamylases. It is well known in the art that microorganisms that produce polysaccharide-hydrolyzing enzymes often exhibit an activity, such as cellulose degradation, that is catalyzed by several enzymes or a group of enzymes having different substrate specificities. Thus, a "cellulase" from a microorganism may comprise a group of enzymes, all of which may contribute to the cellulose-degrading activity. Commercial or non-commercial enzyme preparations, such as cellulase, may comprise numerous enzymes depending on the purification scheme utilized to obtain the enzyme. Thus, the saccharification enzyme consortium of the present method may comprise enzyme activity, such as "cellulase", however it is recognized that this activity may be catalyzed by more than one enzyme.

Saccharification enzymes may be obtained commercially, in isolated form. In addition, saccharification enzymes may be expressed in host microorganisms at the biofuels plant, including using recombinant microorganisms.

One skilled in the art would know how to determine the effective amount of enzymes to use in the consortium and adjust conditions for optimal enzyme activity. One skilled in the art would also know how to optimize the classes of enzyme activities required within the consortium to obtain optimal saccharification of a given pretreatment product under the selected conditions.

The saccharification reaction is performed at or near the temperature and pH optima for the saccharification enzymes. The temperature optimum used with the saccharification enzyme consortium in the present method ranges from about 15°C to about 100°C. In another embodiment, the temperature optimum ranges from about 20°C to about 80°C, 35 to 60°C and most typically 45-50°C. The pH can range from about 2 to about 11. In another embodiment, the pH optimum used with the saccharification enzyme consortium in the present method ranges from about pH 4 to 7 or about 4 to about 5.5.

The saccharification can be performed for a time of about 60 minutes to about 120 hours and preferably from about 4 hours to about 24 hours. The time for the reaction will depend on enzyme concentration and specific activity, as well as the substrate used, its concentration (i.e., solids loading) and the environmental conditions, such as temperature and pH. One skilled in the art can readily determine optimal conditions of temperature, pH and time to be used with a particular substrate and saccharification enzyme(s) consortium.

The saccharification can be performed batch-wise or as a continuous process. The saccharification can also be performed in one step, or in a number of steps. For example, different enzymes required for saccharification may exhibit different pH or temperature optima. A primary treatment can be performed with enzyme(s) at one temperature and pH, followed by secondary or tertiary (or more) treatments with different enzyme(s) at different temperatures and/or pH. In addition, treatment with different enzymes in sequential steps may be at the same pH and/or temperature, or different pH and temperatures, such as using cellulases stable and more active at higher pHs and temperatures followed by hemicellulases that are active at lower pH and temperatures.

The degree of solubilization of sugars from biomass following saccharification can be monitored by measuring the release of monosaccharides and oligosaccharides. Methods to measure monosaccharides and oligosaccharides are well known in the art. For example, the concentration of reducing sugars can be determined using the 1,3-dinitrosalicylic (DNS) acid assay (Miller, G. L., Anal. Chem., 31: 426-428, 1959). Alternatively, sugars can be measured by HPLC using an appropriate column.

Having described the basic aspects of the present invention, the following non-limiting examples illustrate specific embodiment thereof. The following examples are illustrative of the invention but not to be construed to limit the scope of the present invention.

Example 1
The rice straw was milled through a 2-mm mesh screen using a knife mill and then dried in atmospheric air. Air-dried rice straw was stored in plastic bags at room temperature for further use. The chemical composition of this rice straw was; cellulose 38.08%, hemicellulose 19.98%, AI lignin 12.01 % and ash 16.40 %. The rice straw (20 gm) was taken in pressure tube of 500 ml and a solution of NaOH in different concentrations (1%, 2%, 3%, 4% and 5% w/v) in 200 ml of water was added to the pressure tube. The material was mixed properly so as to suspend the biomass in pressure tube. Pressure tube filled with above biomass and alkali solution was tightly closed and autoclaved at 121°C at 15 psi pressure for 1 h. The pretreated rice straw was then filtered through muslin cloth, pressed and washed with water until the pH of the filtrate reached nearly 7. Air died rice straw (300mg) was subjected to two stage complete acid hydrolysis. Acid insoluble lignin and total reducing sugars were measured by National Renewable Energy Laboratory (NREL) protocol and the data received from five set of experiments i.e. 1%, 2%, 3%, 4%, 5% NaOH solution is given in Table-1. Pretreated rice straw samples were then subjected to enzymatic hydrolysis. These experiments with NaOH solution of different concentrations were carried out to establish a base case and later compare with pretreatment using refinery waste caustic streams of similar caustic concentration.

Enzymatic Saccharification
The pretreated rice straw as obtained above (2.0g on oven dry basis) was suspended in 0.05 M sodium citrate buffer solution (20ml) pH 4.8containing sodium azide (0.01 % w/v) in a 100 ml Erlenmeyer flask. Before adding enzyme, the slurry was preheated in an incubator shaker at 50°C at 150 rpm for 15min. The hydrolysis was initiated by adding 20FPU (filter paper unit) of cellulase enzyme cocktail per gram of dry biomass. Sample was drawn from the reaction mixture after 24 h and centrifuged at 10,000 x g for 10min and analyzed for reducing sugars by 3, 5 - dinitrosalicylic acid (DNS) method. Sugar yield was expressed as mg reducing sugars produced by enzymatic action per gm dry biomass. Table 1 outlines the delignification and enzymatic hydrolysis of 1%, 2%, 3%, 4%, 5% NaOH treated biomass.

Results shown in tables indicate that total sugar present per gm dry biomass increased with increase in percentage of NaOH. Data also indicate that lignin extraction increased with increasing NaOH content presumably because the solubility of lignin increased with increasing NaOH concentration. The delignification at 1, 2, 3, 4 and 5% NaOH was 47.2, 52.5, 60.3, 69.1and 72% respectively. From the Figure 1, it can be seen that as the concentration of NaOH increased from 1 to 3 % NaOH, the enzymatic hydrolysis efficiency also increased. From 3% to 5% NaOH concentration, there was a decrease in the efficiency. As the NaOH concentration increased, partial degradation of cellulose and hemicellulose occur which results in the reduced concentration of reducing sugars in the enzymatic reactions i.e., reduced enzymatic hydrolysis. The concentration of the reducing sugars was 780 mg per 1 gram of pretreated biomass with a saccharification rate of 85.7% at 24 h of enzymatic reaction when the NaOH concentration reached 3.0%.

Example 2
Refinery waste stream was analyzed for alkali concentration and found to be 10% NaOH. Therefore, in order to replicate the conditions and maintain the same alkali level, a set of five experiments was devised i.e. 10, 20, 30, 40, 50% refinery waste stream which was equivalent to the 1, 2, 3, 4, 5% alkali solution as taken in Example 1.
The rice straw (20 gm) was taken in pressure tubes of 500 ml and a solution of refinery waste stream (10%, w/v) in 200 ml of water was added to the pressure tube. This makes the concentration of NaOH about 1 %. The material was mixed properly so as to suspend the biomass in pressure tube. Pressure tube filled with above biomass and alkali solution was tightly closed and autoclaved at 121°C at 15 psi pressure for 1h. The pretreated rice straw was then filtered through muslin cloth, pressedand washed with water until the pH of the filtrate reached nearly 7. Air died rice straw (300mg) was subjected to two stage complete acid hydrolysis. Acid insoluble lignin and total reducing sugars were measured by NREL protocol and the data received from this experiment is given in Table 1. This pretreated rice straw sample was then subjected to enzymatic hydrolysis.

Enzymatic Saccharification
The pretreated rice straw as obtained above (2.0g on oven dry basis) was suspended in 0.05 M sodium citrate buffer solution (20ml) pH 4.8 containing sodium azide (0.01 % w/v) in a 100 ml Erlenmeyer flask. Before adding enzyme, the slurry was preheated in an incubator shaker at 50°C at 150 rpm for 30 min. The hydrolysis was initiated by adding 20 FPU of cellulase enzyme cocktail per gram of dry biomass. Sample was drawn from the reaction mixture after 24 h and centrifuged at 10,000 x g for 30 min and analyzed for reducing sugars by 3, 5 - dinitrosalicylic acid (DNS) method. Sugar yield was expressed as mg reducing sugars produced by enzymatic action per gm dry biomass. When NaOH concentration was 1%, concentration of the reducing sugars was 599 mg per 1 gram of pretreated biomass with a saccharification rate of 74.5% at 24 h of enzymatic reaction, whereas with 10% refinery waste stream, concentration of the reducing sugars was 671 mg per 1 gram of pretreated biomass with a saccharification rate of 85.7%. Delignification achieved with 1% NaOH was 47.20% whereas 62.25% delignification was achieved with 10% refinery waste stream.

Example 3
The rice straw (20 gm) was taken in pressure tubes of 500 ml and a solution of refinery waste stream (20%, w/v) in 200ml of water was added to the pressure tube to make overall concentration of NaOH as 2%. The material was mixed properly so as to suspend the biomass in pressure tube. Pressure tube filled with above biomass and alkali solution was tightly closed and autoclaved at 121°C at 15 psi pressure for 1h. The pretreated rice straw was then filtered through muslin cloth, pressed and washed with water until the pH of the filtrate reached nearly 7. Air dried rice straw (300mg) was subjected to two stage complete acid hydrolysis. Acid insoluble lignin and total reducing sugars were measured by NREL protocol and the data obtained from this experiment is given in Table 1. This pretreated rice straw sample was then subjected to enzymatic hydrolysis.

Enzymatic Saccharification
The pretreated rice straw as obtained above (2.0g on oven dry basis) was suspended in 0.05 M sodium citrate buffer solution (20ml) pH 4.8 containing sodium azide (0.01 % w/v) in a 100 ml Erlenmeyer flask. Before adding enzyme, the slurry was preheated in an incubator shaker at 50°C at 150 rpm for 30 min. The hydrolysis was initiated by adding 20 FPU of cellulase enzyme cocktail per gram of dry biomass. Sample was drawn from the reaction mixture after 24 h and centrifuged at 10,000 x g for 35 min and analyzed for reducing sugars by 3, 5 - dinitrosalicylic acid (DNS) method. Sugar yield was expressed as mg reducing sugars produced by enzymatic action per gm dry biomass. When NaOH concentration was 2%, concentration of the reducing sugars was 700 mg per 1 gram of pretreated biomass with a saccharification rate of 77.9% at 24 h of enzymatic reaction, whereas with 20% refinery waste stream, concentration of the reducing sugars was 810 mg per 1 gram of pretreated biomass with a saccharification rate of 90.1%. Delignification achieved with 2% NaOH and 20% refinery waste stream was 52.5 and 64.5% respectively.

Example 4
The rice straw (20 gm) was taken in pressure tubes of 500 ml and a solution of refinery waste stream (30%, w/v) in 200ml of water was added to the pressure tube to make overall concentration of NaOH as 3%. The material was mixed properly so as to suspend the biomass in pressure tube. Pressure tube filled with above biomass and alkali solution was tightly closed and autoclaved at 121°C at 15 psi pressure for 1h. The pretreated rice straw was then filtered through muslin cloth, pressed and washed with water until the pH of the filtrate reached nearly 7. Air dried rice straw (300mg) was subjected to two stage complete acid hydrolysis. Acid insoluble lignin and total reducing sugars were measured by NREL protocol and the data received from this experiment is given in Table 1. This pretreated rice straw sample was then subjected to enzymatic hydrolysis.

Enzymatic Saccharification
The pretreated rice straw as obtained above (2.0g on oven dry basis) was suspended in 0.05 M sodium citrate buffer solution (20ml) pH 4.8 containing sodium azide (0.01 % w/v) in a 100 ml Erlenmeyer flask. Before adding enzyme, the slurry was preheated in an incubator shaker at 50°C at 150 rpm for 35 min. The hydrolysis was initiated by adding 25 FPU of cellulase enzyme cocktail per gram of dry biomass. Sample was drawn from the reaction mixture after 24 h and centrifuged at 10,000 x g for 30 and analyzed for reducing sugars by 3, 5 - dinitrosalicylic acid (DNS) method. Sugar yield was expressed as mg reducing sugars produced by enzymatic action per gm dry biomass. With 3% NaOH and 30% refinery waste stream, concentration of the reducing sugars was 780 and 862 mg per 1 gram of pretreated biomass respectively. Saccharification rate of 86.3% and 92% was achieved with 3% NaOH and 30% refinery waste respectively. Delignification achieved with 3% NaOH and 30% refinery waste stream was 60.3 and 68.92% respectively.

Example-5
The rice straw (20 gm) was taken in pressure tubes of 500 ml and a solution of refinery waste stream (40%, w/v) in 200ml of water was added to the pressure tube to make overall concentration of NaOH as 4%. The material was mixed properly so as to suspend the biomass in pressure tube. Pressure tube filled with above biomass and alkali solution was tightly closed and autoclaved at 121°C at 15 psi pressure for 1h. The pretreated rice straw was then filtered through muslin cloth, pressed and washed with water until the pH of the filtrate reached nearly 7. Air dried rice straw (300mg) was subjected to two stage complete acid hydrolysis. Acid insoluble lignin and total reducing sugars were measured by NREL protocol and the data received from this experiment is given in Table 1. This pretreated rice straw sample was then subjected to enzymatic hydrolysis.

Enzymatic Saccharification
The pretreated rice straw as obtained above (2.0g on oven dry basis) was suspended in 0.05 M sodium citrate buffer solution (20ml) pH 4.2-5.0 containing sodium azide (0.01 % w/v) in a 100 ml Erlenmeyer flask. Before adding enzyme, the slurry was preheated in an incubator shaker at 50°C at 150 rpm for 30 min. The hydrolysis was initiated by adding 20 FPU of cellulase enzyme cocktail per gram of dry biomass. Sample was drawn from the reaction mixture after 24 h and centrifuged at 10,000 x g for 20 and analyzed for reducing sugars by 3, 5 - dinitrosalicylic acid (DNS) method. Sugar yield was expressed as mg reducing sugars produced by enzymatic action per gm dry biomass. When NaOH concentration was 4%, concentration of the reducing sugars was 790 mg per 1 gram of pretreated biomass with a saccharification rate of 85.7% at 24 h of enzymatic reaction, whereas with 40% refinery waste stream, concentration of the reducing sugars was 845 mg per 1 gram of pretreated biomass with a saccharification rate of 90.4%. Delignification achieved with 4% NaOH and 40% refinery waste stream was 69.17 and 72% respectively.

Example-6
The rice straw (20 gm) was taken in pressure tubes of 500 ml and a solution of refinery waste stream (50%, w/v) in 200ml of water was added to the pressure tube to make overall concentration of NaOH as 5%. The material was mixed properly so as to suspend the biomass in pressure tube. Pressure tube filled with above biomass and alkali solution was tightly closed and autoclaved at 121°C at 15 psi pressure for 1h. The pretreated rice straw was then filtered through muslin cloth, pressed and washed with water until the pH of the filtrate reached nearly 7. Air dried rice straw (300mg) was subjected to two stage complete acid hydrolysis. Acid insoluble lignin and total reducing sugars were measured by NREL protocol and the data received from this experiment is given in Table 1. This pretreated rice straw sample was then subjected to enzymatic hydrolysis.

Enzymatic Saccharification
The pretreated rice straw as obtained above (2.0g on oven dry basis) was suspended in 0.05 M sodium citrate buffer solution (20ml) pH 4.8 containing sodium azide (0.01 % w/v) in a 100 ml Erlenmeyer flask. Before adding enzyme, the slurry was preheated in an incubator shaker at 50°C at 150 rpm for 40 min. The hydrolysis was initiated by adding 20 FPU of cellulase enzyme cocktail per gram of dry biomass. Sample was drawn from the reaction mixture after 24 h and centrifuged at 10,000 x g for 40 min and analyzed for reducing sugars by 3, 5 - dinitrosalicylic acid (DNS) method. Sugar yield was expressed as mg reducing sugars produced by enzymatic action per gm dry biomass. When NaOH concentration was 5%, concentration of the reducing sugars was 712 mg per 1 gram of pretreated biomass with a saccharification rate of 77.5% at 24 h of enzymatic reaction, whereas with 50% refinery waste stream, concentration of the reducing sugars was 801 mg per 1 gram of pretreated biomass with a saccharification rate of 85%. Delignification achieved with 5% NaOH was 72% whereas 72.5% delignification was achieved with 50% refinery waste stream. From the Figure 2, it can be seen that as the concentration of refinery waste stream increased from 10% to 30%, the enzymatic hydrolysis efficiency also increased. From 30 % to 50% concentration, there was a decrease in the efficiency. As the NaOH concentration increased, partial degradation of cellulose and hemicellulose occur which results in the reduced concentration of reducing sugars in the enzymatic reactions i.e., reduced enzymatic hydrolysis. The concentration of the reducing sugars was 862.5 mg per 1 gram of pretreated biomass with a saccharification rate of 93% at 24 h of enzymatic reaction when the concentration of refinery waste stream reached 30% (Table 3).

Example 7
In this example cotton stalk was taken in place of rice straw and similar experimental conditions as given in Example 1 were adopted. The chemical composition of cotton stalk was determined and was found as cellulose 39.36%; hemicellulose 19.16%; AI lignin 24.73 % and ash 3.22 %. The amount of acid insoluble lignin and total reducing sugars were measured by NREL protocol and the data obtained from these experiments is given in Table 2. The pretreated cotton stalk samples were subjected to enzymatic saccharification by adopting the similar conditions as in Example 1. Figure 3 outlines the effect of NaOH concentration on delignification (%) and enzymatic hydrolysis (%) of cotton stalk.

Example 8: In this example cotton stalk was taken in place of rice straw and similar experimental conditions as given in Example 2 were adopted. The amount of acid insoluble lignin and total reducing sugars were measured by NREL protocol and the data obtained from these experiments is given in Table 2. The pretreated cotton stalk sample was subjected to enzymatic saccharification by adopting the similar conditions as in Example 2 and data obtained from this experiment is given in Table 2.

Example 9: In this example cotton stalk was taken in place of rice straw and similar experimental conditions as given in Example 3 were adopted. The amount of acid insoluble lignin and total reducing sugars were measured by NREL protocol and the data obtained from these experiments is given in Table 2. The pretreated cotton stalk sample was subjected to enzymatic saccharification by adopting the similar conditions as in Example 3 and data obtained from this experiment is given in Table 2.

Example 10: In this example cotton stalk was taken in place of rice straw and similar experimental conditions as given in Example 4 were adopted. The amount of acid insoluble lignin and total reducing sugars were measured by NREL protocol and the data obtained from these experiments is given in Table 2. The pretreated cotton stalk sample was subjected to enzymatic saccharification by adopting the similar conditions as in Example 4 and data obtained from this experiment is given in Table 2.

Example 11: In this example cotton stalk was taken in place of rice straw and similar experimental conditions as given in Example 5 were adopted. The amount of acid insoluble lignin and total reducing sugars were measured by NREL protocol and the data obtained from these experiments is given in Table 2. The pretreated cotton stalk sample was subjected to enzymatic saccharification by adopting the similar conditions as in Example 5 and data obtained from this experiment is given in Table 2.

Example 12: In this example cotton stalk was taken in place of rice straw and similar experimental conditions as given in Example 6 were adopted. The amount of acid insoluble lignin and total reducing sugars were measured by NREL protocol and the data obtained from these experiments is given in Table 2. The pretreated cotton stalk sample was subjected to enzymatic saccharification by adopting the similar conditions as in Example 6 and data obtained from this experiment is given in Table 2. Figure 4 outlines the effect of refinery waste stream concentration on delignification (%) and enzymatic hydrolysis (%) of cotton stalk.

Example 13:
In this example mustard stalk was taken in place of rice straw and similar experimental conditions as given in Example 1 were adopted. The chemical composition of Mustard stalkwas determined and was found as Cellulose 39.94%, hemicellulose 22.30%, AI lignin 20.84%, ash 1.26%. The amount of acid insoluble lignin and total reducing sugars were measured by NREL protocol and the data obtained from these experiments is given in Table 3. The pretreated mustard stalk samples were subjected to enzymatic saccharification by adopting the similar conditions as in Example 1.

Example 14: In this example mustard stalk was taken in place of rice straw and similar experimental conditions as given in Example 2 were adopted. The amount of acid insoluble lignin and total reducing sugars were measured by NREL protocol and the data obtained from these experiments is given in Table 3. The pretreated mustard stalk sample was subjected to enzymatic saccharification by adopting the similar conditions as in Example 2 and data obtained from this experiment is given in Table 3.

Example 15: In this example mustard stalk was taken in place of rice straw and similar experimental conditions as given in Example 3 were adopted. The amount of acid insoluble lignin and total reducing sugars were measured by NREL protocol and the data obtained from these experiments is given in Table 3. The pretreated mustard stalk sample was subjected to enzymatic saccharification by adopting the similar conditions as in Example 3 and data obtained from this experiment is given in Table 3.

Example 16: In this example mustard stalk was taken in place of rice straw and similar experimental conditions as given in Example 4 were adopted. The amount of acid insoluble lignin and total reducing sugars were measured by NREL protocol and the data obtained from these experiments is given in Table 3. The pretreated mustard stalk sample was subjected to enzymatic saccharification by adopting the similar conditions as in Example 4 and data obtained from this experiment is given in Table 3.

Table 1: Rice straw. Comparison of pretreatment/ enzymatic saccharification with NaOH solutions and with equivalent caustic concentration of refinery wastes

Catalyst concentration Total sugar in pretreated biomass (mg/g) AI lignin (mg/g) Delignification (%) of the lignin present Enzymatic hydrolysis
Sugar released (mg/g) Sugar released (%) of present holocellulose
1% NaOH 803.64 63.30 47.20 599.00 74.50

10% Waste stream 782.55 45.30 62.25 671.00 85.70
2% NaOH 897.99 57.00 52.5 700.00 77.90

20% Waste stream 898.10 42.60 64.5 810.00 90.10
3% NaOH 904.65 47.60 60.33 780.00 86.20

30% Waste stream 926.85 37.30 68.92 862.50 93.00
4% NaOH 921.00 37.00 69.17 790.00 85.70

40% Waste stream 934.28 33.00 72.0 845.00 90.40
5% NaOH 919.08 33.60 72.0 712.50 77.50

50% Waste stream 941.39 33.00 72.5 801.00 85.00

Table 2: Cotton stalk. Comparison of pretreatment/ enzymatic saccharification with NaOH solutions and with equivalent caustic concentration of refinery wastes

Catalyst concentration Total sugar in pretreated biomass (mg/g) AI lignin (mg/g) Delignification (%) of the lignin present Enzymatic hydrolysis
Sugar released (mg/g) Sugar released (%) of present holocellulose
1% NaOH 722.22 123.65 50 505.50 70

10% Waste stream 753.33 111.28 55 565.00 75
2% NaOH 748.88 103.86 58 546.68 73

20% Waste stream 761.11 98.92 60 586.05 77
3% NaOH 765.55 93.97 62 597.12 78

30% Waste stream 783.33 86.5 65 626.66 80
4% NaOH 788.00 86.5 65 606.16 77

40% Waste stream 800.08 79.13 68 632.00 79
5% NaOH 815.20 81.6 67 603.45 74

50% Waste stream 825.55 74.19 70 618.25 75

Table 3: Mustard stalk: Comparison of pretreatment/ enzymatic saccharification with NaOH solutions and with equivalent caustic concentration of refinery wastes

Catalyst concentration Total sugar in pretreated biomass (mg/g) AI lignin (mg/g) Delignification (%) of the lignin present Enzymatic hydrolysis
Sugar released (mg/g) Sugar released (%) of present holocellulose
1% NaOH
795 200 30 580.00 72.9

10% Waste stream 810 190 32 651.00 80.30
2% NaOH 874 205 31 655.00 74.9

20% Waste stream 896 196 30 750.00 83.7
3% NaOH 878 205 31 660.00 75.1

30% Waste stream 900 195 30 760.00 84.4
,CLAIMS:We Claim:
1. A method of producing fermentable sugars from lignocellulosic biomass comprising the steps of:
(a) treating a biomass mixture comprising lignocellulosic biomass and a waste refinery stream produced by petroleum refineries during production of low sulphur fuels to obtain a treated biomass mixture;
(b) filtering the treated biomass mixture and washing with water and pressing to remove excess refinery waste, water and lignin to obtain a de-lignified biomass; and
(c) reacting the de-lignified biomass with one or more saccharification enzymes to obtain fermentable sugars.

2. The method of claim 1, wherein the lignocellulosic biomass is selected from a group consisting of rice straw, wheat straw, cotton stalk, corn cobs, corn stover, sugarcane bagasse, wood chips, Jatropha cuttings, mustard stalk and combination thereof.

3. The method of claim 1 wherein the lignocellulosic biomass is of size from 2 mm to 10 cm.

4. The method of claim 1 wherein the waste refinery stream is selected from LPG treating unit.

5. The method of claim 1 wherein the waste refinery stream is selected from merox unit for producing low sulphur kerosene and ATF.

6. The method of claim 1 wherein the waste refinery stream is selected from refinery unit for producing low sulphur gasoline.

7. The method of claim 1, wherein the ratio of lignocellulosic biomass to waste refinery stream is from 0.2-1.

8. The method of claim 1, wherein the lignocellulosic biomass is treated at a temperature of between 80 to 200 °C in step (a).

9. The method of claim 1, wherein the step (a) is conducted at a time between 20 minutes to 2 hours.

10. The method of claim 1, wherein the pressing step (b) is conducted at a pressure in the range of 600 psi to 2500 psi.

11. The method of claim 1, wherein the waste refinery stream comprises alkali concentration in the range of 1% to 10%, more preferably in the range of 2% to 8%, the percentage being expressed in terms of the weight of the waste stream.

12. The method of claim 1, wherein the waste refinery stream comprises about 4-10% of alkali and about 1-5% nucleophile, the percentage being expressed in terms of the weight of the waste stream.

13. The method of claim 1, wherein the biomass mixture comprises alkali concentration in the range of 1% to 10%, more preferably in the range of 1% to 5%, the percentage being expressed in terms of the weight of the biomass mixture.

14. The method of claim 1, wherein optionally the biomass mixture is diluted to make the overall percentage of alkali equal to 5% to 1%, the percentage being expressed in terms of the weight of the biomass mixture.

15. The method of claim 1, wherein the waste refinery stream comprises: water from 1-99%, alkali 1-10%, alkali metal sulphide and disulphide from about 0.1 to 5%, thiols from about 0.01 to 4% having C-2 to C-10, alkali mercaptides having C-2 to C-10 from about 0.01 to 3%, alkyl disulphide from C 2-10 mercaptans, sodium salt of naphthenic acid from 0.01 to 1% by weight, alkali carbonate from 0.01 to 1%, the percentage being expressed in terms of the weight of the waste stream.

16. The method of claim 1, wherein step (c) comprises adjusting of pH 4 to 7 with buffer selected from citrate, acetate and phosphate so that pH and temperature are compatible with saccharification enzymatic hydrolysis.

17. The method of claim 16, wherein the temperature is between 35 to 60°C.

18. The method of claim 1, wherein saccharification enzyme comprises one or more cellulases.

19. The method of claim 1, wherein the total sugars release is from 75 to 95% of the original hollocellulose present in the biomass.

20. A method of producing de-lignified biomass from lignocellulosic biomass comprising the steps of:
(a) treating a biomass mixture comprising lignocellulosic biomass and a waste refinery stream produced by petroleum refineries during production of low sulphur fuels to obtain a treated biomass mixture; and
(b) filtering the treated biomass mixture and washing with water and pressing to remove excess refinery waste, water and lignin to obtain a de-lignified biomass.

21. The method of claim 20, wherein the waste alkali refinery stream is selected from LPG treating unit, merox unit for producing low sulphur kerosene and ATF and refinery unit for producing low sulphur gasoline.

22. The method of claim 20, wherein the biomass mixture comprises alkali concentration in the range of 1% to 10%, more preferably in the range of 1% to 5%, the percentage being expressed in terms of the weight of the biomass mixture.