FIELD OF INVENTION
The invention relates to a process and system for the preparation of ethanol from lignocellulosic materials and more particularly from lignoceliulosic materials like corncob, corn stover, sugarcane bagasse or any other lignocellulosic materials using and method of co-fermentation.
BACKGROUND
Due to the future limitations on the availability of fossil fuels particularly crude oil, many national governments are promoting the use of alternate fuels such as ethanol in motor vehicles. Ethanol is a major motor vehicle fuel in Brazil and is used at a large scale in other countries like the US and Europe; while in India it has been promoted significantly since past few years. However, preparation of fuel ethanol is mostly done from food crops like maize, sugarcane or beet, causing major social and economic issues of use of these food materials for . non-food applications. Therefore, the governments are promoting use of non-food feedstocks like lignocellulosic materials for the preparation of fuel ethanol at large scales to fulfil the growing demands for the renewable energy sources.
Lignocellulosic materials [LCM] from the agriculture industry are waste by-products. It is mostly used inefficiently as an energy source or fed to

animals; however, a large part is wasted as such without any use. LCM is a complex structure of cellulose, hemicelluiose and lignin forming a composite, which depending on its source is differentially resistant to hydrolysis compared with other carbohydrate-based materials like starch. LCM form structural components of plants and has varying composition based on its location in host or type of the host. In the art, there are present several methods of chemical and thermal hydrolysis of LCM using high temperature water, acid, alkali and other chemicals. These treatments are performed to achieve effective degradation of LCM to fermentable sugars like xylose and glucose. These sugars on fermentation by yeasts lead to ethanol that is used in various applications including as a fuel additive.
LCM biomass refers to plant biomass that is composed of cellulose, hemicelluiose, and lignin. This biomass comes in many different types such as wood, wood residues, municipal waste, agricultural residues and energy crops like fast growing tall and woody grasses. In all these categories, the carbohydrate polymers (cellulose and hemicelluloses) are tightly bound to the lignin by hydrogen and covalent bonds forming the strong structures of LCM. One barrier to the production of ethanol from LCM is that a large fraction of the sugars necessary for fermentation present in the form of lignocelluloses. LCM has evolved to resist degradation and to confer hydrolytic stability and structural robustness to the cell walls of the plants. This "recalcitrance" is attributable to the cross linking between the polysaccharides (cellulose

and hemicellulose) and the lignin via ester and ether linkages thus creating a material that is physically hard to access. This means that for an efficient use of these components, said LCM should be disintegrated, separated and/or decrystallized.
Although there are several methods of hydrolysis of LCM known; however, there exists a need to find more effective and economic methods of treating LCM in more benign and economic conditions as the ethanol is a commodity product and the prices are very competitive. Besides, during LCM treatment using chemical and thermal treatment several inhibitors of the fermentative process are produce that lead to marked reduction of efficiencies of fermenting organisms as these inhibitors are toxic to these microbes. These inhibitors are mainly phenolics, certain organic acids and other organics produced from the components of LCM during the treatment of hydrolysis.
Therefore, the control of LCM hydrolysis treatment [also called pre-treatment] is a major step in the processing of LCM for the preparation of ethanoi. In the art current methods of the pre-treatment use acids, alkalis or other chemicals like ammonia at high temperatures to . hydrolyze the hemicellulose part of LCM liberating xylose sugar in the first step. Next, the cellulose rich remaining part is subjected to enzymatic or chemical hydrolysis to liberate, glucose sugar and remaining lignin rich part is burned to generate energy. These two sugars are then fermented to make ethanol or used to prepare other

bio-based chemicals. However, two major problems occur with such a treatment: 1] the heating may only be short, because otherwise too many unwanted by-products are formed from the carbohydrates; and 2) it is difficult to produce a biomass slurry with more than 30% solids by weight, which is necessary for an economic use in further processing.
The invention disclosed herein in addresses several problems of previous methods like: 1] high concentration of inhibitors in hydrolysates, 2] low efficiency of the hydrolysis of LCM at benign conditions and 3] low efficiency of conversion of LCM derived sugars to ethanol in the fermentative process. To this end a novel method is disclosed having advantages like: 1] low level of inhibitors that do not limit the growth of microbes, 2] more benign and economic processing conditions of LCM, 3] higher efficiency of conversion of sugars to ethanol, and 4] use of an efficient recombinant yeast that is able to ferment both glucose and xylose sugars simultaneously in a novel fermentation process.
DESCRIPTION OF DRAWINGS
These and other features, aspects, and advantages of the present invention will become better understood when the following detailed

description is read with reference to the accompanying drawings, wherein:
FIGURE 1 depicts a block diagram of mass flow during the production of ethanol from a LCM using the separate hydrolysis and co-fermentation [SHCF] process. Different elements of the process are identified and directional movement of different additives and streams formed during the process are shown to describe the features of one embodiment of the present invention.
FIGURE 2 depicts a block diagram of mass flow during the production of ethanol from a LCM using the simultaneous hydrolysis and co-fermentation [SSCF] process. Different elements of the process are identified and directional movement of different additives and streams formed during the process are shown to describe the features of one embodiment of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
In one embodiment of the disclosed invention as illustrated in FIGURE 1 and FIGURE 2 to produce ethanol from-LCM biomass, said biomass like corncob, bagasse, stover or other similar agricultural material is size reduced to a particulate matter. This particulate matter of about 40 mm size is then soaked and washed in water to remove any soil or other contaminating agents. Next, said soaked biomass is pre-treated

in the presence of'one or more acids [inorganic or organic] at certain high pressure and temperature provided by high-pressure steam as energy and water source. On performing said pre-treantment process for desired time period at desired conditions, a hydrolyzed stream of LCM is obtained [the first stream], which is subsequently subjected to a - neutralization and enzymatic hydrolysis process. The pre-treatment on said LCM releases xylose from the hemicellulosic part, while crystalline cellulose fibres are loosen so that it may be further treated with cellulolytic enzymes. The said hydrolyzed stream is first neutralized with an alkali like NaOH or MgO to increase the pH to about 5.5 [forming the neutralized stream] and then one or more celllulases and are added, and reaction is allowed for several hours at desired conditions. This step releases glucose from the cellulose fibres leading to substantial increase of glucose in formed stream [the second stream]. The said stream is then subjected to a pentose and hexose fermenting recombinant yeast of Saccharomyces sp. leading to conversion of said sugars to ethanol forming a fermented stream [the final stream]. Then said fermented stream is subjected to a distillation to separate aqueous ethanol. The aqueous ethanol so afforded is further processed to obtain anhydrous ethanol or other ethanol products. The spent wash stream obtained at the end of this process is then subjected to solid/ liquid separation using a device like a filter press to remove the solids [which is mostly lignin] from waste water. These solids are burnt in a boiler to generate energythat is further-

used to run said system described herein. The wastewater is subjected to evaporation and further treatment to obtain process water for recycle/ reuse in said system. Again, solids obtained in said evaporation step are burnt in said boiler.
In another embodiment,, the cellulose enzyme treatment of the pre-treated LCM is done in two different modes. In the first mode, called separate hydrolysis and co-fermentation [SHCF] as shown in FIGURE 1, the cellulase treatment is carried out separated at optimum conditions to get maximum conversion of cellulose fibres to glucose before the fermentation of the sugars. Once the separate hydrolysis is completed, the co-fermentation of xylose and glucose present in said stream is performed using recombinant yeast of Saccharomyces sp. that is capable of fermenting pentose and hexose sugars simultaneously. In the second mode, called simultaneous saccharification and co-fermentation [SSCF] as shown in FIGURE 2, the cellulase treatment is carried out partially at optimum conditions to get about 20 % to about 30 % conversion of cellulose fibres to glucose before the fermentation of the sugars. Once this partial hydrolysis is completed, the co-fermentation of xylose and glucose present in said, stream is performed using recombinant yeast of Saccharomyces sp. that is capable of fermenting pentose and hexose sugars simultaneously. Herein during the fermentation process, saccharification process is also carried out by addition of cellulases and the glucose so liberated is immediately consumed for ethanol

production. The SSCF has advantage that any substrate inhibition of cellulases is avoided increasing the enzymatic hydrolysis efficiency.
In another embodiment of the invention, the recombinant yeast of Saccharomyces sp. is created by genetic engineering of xylose metabolism pathway in said yeast. This include addition of genes related to xylose isomerise, xylose-1-epimerase, PPP pathway genes or other genes that are essential for conversation of xylose to ethanol by said yeast.
Examples provided below give wider utility of the invention without any limitations as to the variations that may be appreciated by a person skilled in the art. A non-limiting summary of various experimental results is given in the examples, which demonstrate the advantageous and novel aspects of the process of using LCM for the preparation Sf ethanol.
EXAMPLE 1: SEPARATE HYDROLYSIS AND CO-FERMENTATlON OF CORNCOB BIOMASS
In first step, a batch of about 130 kg of corncobs having total solids of about 92% by weight, cellulose of about 33% by weight, hemicelluloses of about 26% by weight and lignin of about 13% by weight was used as a feedstock. It was subjected to mechanical shearing for size reduction to less than 40 mm particles affording about 120 Kg of the particulate

material. This particulate material was soaked in water for about 30 min. Then about 400 kg slurry [also called mixture] containing about 30% by weight total solids was prepared and continuously introduced into a plug screw type hydrolyser. Here the slurry was mixed with about 160 L of the admixture of oxalic and sulphuric acids. This admixture of mixed acids contained about 1% by weight oxalic acid and about 2% by weight sulphuric acid on dry biomass weight basis [total 3% acid on dry biomass weight basis]. The resultant reaction mixture was then subjected to hydrolysis in said hydrolyser at a temperature of about 160 °C and pressure of about 6 bar [absolute] for a period of about 24 minutes at pH of about 1.3. At the end of this pre-treatment the final slurry of about 563 kg contained about 18 % of total solids; and about 0.54 % of glucose, about 5.5 % of xylose, about 0.01% of furfural, about 0.03% of HMF and about 3800 PPM of phenolic components along with undigested cellulose and lignin as detected by the HPLC methods. In this pre-treatment, the efficiency of xylan to xylose conversion was about 86%. In second step, this pretreated hydrolysate rich with C5 sugars along with C6 solids [cellulose + lignin] was subjected to enzymatic hydrolysis by cellulases. Before enzymatic hydrolysis, it was neutralized with MgO to increase pH to about 5 and diluted to total solids of 15 %. Then a commercially available cellulase cocktail [about 20 mg/g of cellulose] was added to the pretreated material and allowed to digest said solids at desired conditions for about 120 h at 52 °C. After said enzymatic hydrolysis, glucose of about

5 % by weight and xylose of about 4.5 % by weight of was afforded in the stream with cellulose hydrolysis efficiency of about 77 %. At the end of these treatments, the stream contained xylose of about 4.34% by weight. In third step,, said sugar rich slurry was subjected to fermentation using a recombinant strain of S. cerevisiae, which fermented both glucose and xylose present in said slurry to ethanol leading to ethanol concentration in the stream at about 4 % by weight. Here glucose to ethanol conversion efficiency was about 90 % and xylose to ethano! was about 80 % of theoretical maximum after about 72 h of fermentation time [overall efficiency was about 85.%]. Here corncob to ethanol conversion efficiency [enzyme treatment [for 120 h] + fermentation [for 72 h]] was about 70 % at the end. After fermentation, this stream was subjected to distillation to obtain ethanol. About 35 L of ethanol was afforded from about 120 kg of dry corncob biomass. This process afforded ethanol from both xylose and glucose sugars in a more efficient and economic way than any process previously disclosed.

EXAMPLE 2: SIMULTANEOUS HYDROLYSIS AND CO-FERMENTATION OF CORNCOB BIOMASS
In first step, a batch of about 124 kg of corncobs having total solids of about 92% by weight, cellulose of about 33% by weight, hemicelluloses of about 27% by weight and lignin of about 13% by weight was used as

a feedstock. It was subjected to mechanical shearing for size reduction to less than 40 mm particles affording about 115 Kg of the particulate material. This particulate material was soaked in water for about 30 min. Then about 383 kg slurry [also called mixture] containing about 30% by weight total solids was prepared and continuously introduced Into a plug screw type hydrolyser. Here the slurry was mixed with about 160 L of the admixture of oxalic and sulphuric acids. This admixture of mixed acids contained about 1% by weight oxalic acid and about 2% by weight sulphuric acid on dry biomass weight basis [total 3% acid on dry biomass weight basis]. The resultant reaction mixture was then subjected to hydrolysis in said hydrolyser at a temperature of about 160 °C and pressure of about 6 bar [absolute] for a period of about 24 minutes at pH of about 1.3. At the end of this pre-treatment the final slurry of about 566 kg contained about 20 % of total solids; and about 0.68% of glucose, about 5 % of xylose, about 0.01% of furfural, about 0.03% of HMF and about 4500 PPM of phenolic components along with undigested cellulose and lignin as detected by the HPLC methods. In this pre-treatment, the efficiency of xylan to xylose conversion was about 83 %. In second step, said pre-treated hydrolysate rich with C5 sugars along with C6 solids [cellulose + lignin] was subjected to enzymatic hydrolysis by cellulases. Before enzymatic hydrolysis, it was neutralized with MgO to increase pH to about 5 and diluted with water to total solids of about 15 %. Then a commercially available cellulase cocktail [about 20 mg/g of cellulose] was added to the pre-treated

material and allowed digest said solids at desired conditions for about 18 h at 52 °C. After said enzymatic hydrolysis, glucose of about 2.5 % by weight was afforded in the stream with cellulose hydrolysis efficiency of about 40 %. At the end of these treatments, the stream contained xylose of about 4.20 % by weight and glucose of about 3 % by weight. In third step, said partially enzyme-hydrolysed slurry was subjected to fermentation by using a recombinant strain of S. cerevisiae, which fermented both glucose and xylose present in said slurry to ethanol leading to ethanol concentration in the stream at about 3.3 % by weight. Here corncob to ethanol conversion efficiency [enzyme treatment [for 18 h] + fermentation [for 90 h]] was about 66 % at the end. After fermentation, the obtained slurry was subjected to distillation and about 33 L of ethanol was afforded from about 115 kg of dry corncob biomass.
EXAMPLE 3: SEPARATE HYDROLYSIS AND CO-FERMENTATION OF BAGASSE BIOMASS
In first step, a batch of about 135 kg of sugarcane bagasse having total solids of about 90 % by weight, cellulose of about 35 % by weight, hemicelluloses of about 21 % by weight and lignin of about 21 % by weight was used as a feedstock. It was subjected to mechanical shearing for size reduction to less than 40 mm particles affording about 122 kg of the particulate material. This particulate material was soaked

in water for about 30 min. Then about 451 kg of slurry containing about 30% by weight total solids was prepared and continuously introduced into a plug-screw type hydrolyser. Here the slurry was mixed with about 180 L of the admixture of oxalic and sulphuric acids. This admixture of mixed acids contained about 1% by weight oxalic acid and about 1.5% by weight sulphuric acid on dry biomass weight basis [total 2.5% acid on dry biomass weight basis]. The resultant reaction mixture was then subjected to hydrolysis in said hydrolyser at a temperature of about 150 °C and pressure of about 5 bar[a] for a period of about 15 minutes at pH of about 1.2. At the end of this pre-treatment the final slurry of about 658 kg contained about 18 % of total solids; and about 0.4 % of glucose, about 4.0 % of xylose, and about 4000 PPM of phenolic components along with undigested cellulose and lignin as detected by the HPLC methods. In this pre-treatment, the efficiency of xylan to xylose conversion was about 90 %. In second step, this pre-treated hyd.rolysate rich with C5 sugars along with C6 solids [cellulose + lignin] was subjected to enzymatic hydrolysis by cellulases. Before enzymatic hydrolysis it was neutralized with MgO to increase pH to about 5. Then a commercially available cellulase cocktail [about 30 mg/g of cellulose] was added to the pre-treated material and allowed digest said solids at desired conditions for about 120 h at 52 °C. After said enzymatic . hydrolysis, glucose of about 3.5 % by weight was afforded in the stream with cellulose hydrolysis efficiency of about 58 %. At the end of these treatments, the slurry contained xylose of about 3.5 % by weight

and glucose of about 3.7 % by weight. In third step, said sugar rich slurry was subjected to co-fermentation by using a recombinant strain of S. cerevisiae, which fermented glucose and xylose present in said slurry to ethanol leading to ethanol concentration in the stream at about 3 % by weight. Here glucose to ethanol conversion efficiency was about 90% of theoretical maximum and xylose to ethanol conversion efficiency was about 88%, after about 72 h of fermentation time [overall efficiency was about 89 %]. Here bagasse to ethanol conversion efficiency [enzyme treatment [for 120 h] + fermentation [for 72 h]] was about 52 % at the end. After fermentation, this stream was subjected to distillation to obtain ethanol. After fermentation, the obtained slurry was subjected to distillation to obtain ethanol. About 22 L of ethanol was afforded from about 122 kg of dry sugarcane bagasse biomass.
EXAMPLE 4: SIMULTANEOUS HYDROLYSIS AND CO-FERMENTATION OF BAGASSE BIOMASS
In first step, a batch of about 250 kg of sugarcane bagasse having total solids of about 70 %. by weight, cellulose of about 35 % by weight; hemicelluloses of about 21 % by weight and lignin of about 21 % by weight was used as a feedstocks It was subjected to mechanical shearing for size reduction to less than 40 mm particles affording about 175 kg of the particulate material. This particulate material was soaked in water for about 30 min. Then about 582 kg of slurry containing about

30% by weight total solids was prepared and continuously introduced into a plug-screw type hydrolyser. Here the slurry was mixed with about 195.L of the admixture of oxalic and sulphuric acids. This admixture of mixed acids contained about 1% by weight oxalic acid and about 1.5% by weight sulphuric acid on dry biomass weight basis [total 2.5% acid on dry biomass weight basis]. The resultant reaction mixture was then subjected to hydrolysis in said hydrolyser at a temperature of about 150 °C and pressure of about 5 bar[a] for a period of about 15 minutes at pH of about 1.2. At the end of this pre-treatment the final slurry of about 717 kg contained about 22 % of total solids; and about 0.7 % of glucose, about 4.7 % of xylose, and about 4500 PPM of phenolic components along with undigested cellulose and lignin as detected by the HPLC methods. In this pre-treatment, the efficiency of xylan to xylose conversion was about 81 %. In second step, this pre-treated hydrolysate rich with C5 sugars along with C6 solids [cellulose + lignin] was subjected to enzymatic hydrolysis by cellulases. Before enzymatic hydrolysis, it was neutralized with MgO to increase pH to about 5, and diluted with water to total solids of about 17 %. Then a commercially available cellulase cocktail [about 30 mg/g of cellulose] was added to the pre-treated materia! and allowed digest said solids at desired conditions for about 18 h at 55 °C. After said enzymatic hydrolysis, glucose of about 2 % by weight was afforded in the stream with partially cellulose hydrolysis efficiency of about 28%. At the end of these treatments, the slurry contained xylose of about 3.64 % by

weight. In third step, said partial hydrolyzed sugar slurry was subjected to fermentation at 37 °C by using a recombinant strain of S. cerevisiae, which fermented glucose and xylose present in said slurry to ethanol leading to ethanol concentration in the stream at about 3.7 % by weight. Here corncob to ethanol conversion efficiency [enzyme treatment [for 18 h] + fermentation [for 96 h]] was about 53 % at the end. After fermentation, the obtained slurry was subjected to distillation to obtain ethanol. About 46 L of ethanol was afforded from about 175 kg of dry sugarcane bagasse biomass.
While the invention.has been particularly shown and described with reference to embodiments listed in examples, it will be appreciated that several of the above disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. The fermentation processes : disclosed herein afforded ethanol from both xylose and glucose sugars in a more efficient and economic way than previously disclosed. Also that various presently unforeseen and unanticipated alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims. Although the invention has been described with reference to specific preferred embodiments, it is not intended to be limited thereto, rather those having ordinary skill in the art will recognize that variations and modifications may be made

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

5. CLAIMS
WE CLAIM:
1. A process for the preparation of ethanol from a dry
lignocellulosic biomass comprising:
(a) converting said biomass to a particulate matter;
(b) preparing a mixture of said matter in water;
(c) contacting said mixture with a blend of acids at a desired temperature for a desired time period to obtain a first stream;
(d) adjusting pH of said first stream with a base to obtain a neutralised stream;
(e) contacting said neutralised stream with cellulolytic enzymes at desired temperature for a desired time period to obtain a second stream;
(f) subjecting said second stream to a hexose and pentose fermenting yeast to obtain a final stream;
(g) separating ethanol from said final stream by distillation.
2. The process of claim 1, wherein:
(a) said blend of acids comprise oxalic acid and sulphuric acid;
(b) said amount of oxalic acid is between 0.1 to 5 percent and amount of sulphuric acid is between 0.1 to 5 percent of said biomass by weight;

(c) said cellulolytic enzymes comprises cellulases of different sources;
(d) said enzymes are used between 10 mg and 100 mg per gram of cellulose present in said neutralised stream;
(e) said desired temperature to obtain said first stream ranges from about 140° C to about 210° C;
(f) said desired time period to obtain said first stream ranges from about 5 minutes to about 120 minutes;
(g) said desired temperature to obtain said second stream ranges from about 40° C to about 80° C; and
(h) said desired time period to obtain said final stream ranges from about 50 hours to about 120 hours.
3. The process of claim 1, wherein pH of said first stream is adjusted to between about 4 and about 6.
4. The process of claim 1, wherein said first .stream comprises pentose sugars derived from thermo-chemical hydrolysis of hemi-cellulose part of said biomass.
5. The process of claim 1, wherein the efficiency of hydrolysis of hemi-cellulose is at least 60 percent of theoretical efficiency.

6. The process of claim 1, wherein said second stream comprises hexose sugars derived from enzymatic hydrolysis of cellulose part of said biomass.
7. The process of claim 1, wherein the efficiency of enzymatic hydrolysis of cellulose is at least 55 percent of theoretical efficiency.
8. The process of claim 1, wherein the efficiency of conversion of hexose sugars to ethanol is at least 80% of theoretical efficiency.
9. The process of claim 1, wherein the efficiency of conversion of pentose sugars to ethanol is at least 65% of theoretical efficiency.
10. The process of claim 1, wherein said hexose and pentose fermenting recombinant yeast is a Saccharomyces sp.