PREPARATION OF ETHANOL FROM BIOMASS AND SUGARCANE BASED FEED STOCKS.

FIELD OF INVENTION

The invention relates to the preparation of ethanol from biomass and molasses in general using a pentose and hexose fermenting yeast. It more particularly relates to used of lignocellulosic biomass like corn cob, corn stover, sugarcane bagasse or agricultural waste biomass that is simultaneously used as a partial feed in conventional ethanol fermentation system that uses molasses to achieve simultaneous high efficiency conversion of pentose and hexose sugars to ethanol.

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 feed stocks like lignocellulosic materials for the preparation of fuel ethanol at large scales to fulfil the growing demands for the renewable energy sources.

In India, ethanol production is mainly done using sugarcane as feedstock. A steady supply of sugarcane (or sugarcane juice) as feedstock is required for the continuous ethanol production. However, many times the sugarcane demand varies depending on the market condition. This leads to non availability of sufficiently molasses to operate the ethanol plants for whole year. Therefore, if an alternative feedstock like LCM is used with molasses [without major addition of capital expenditure] it should able to provide enough feedstock to operate the conventional ethanol plants. This invention addresses this need in the industry.

Like molasses, lignocellulosic materials [LCM] are other waste byproducts from the agriculture industry which is used for ethanol production. 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, hemicellulose 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.

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 de-crystallized. Recently, several efficient methods of LCM pre-treatment or hydrolysis are described in the art. The pre-treated LCM may then be used for fermentation of both pentose and hexose, present in it, for the production of ethanol by using yeasts that utilises both the sugar types and converts it to ethanol.

The invention disclosed herein provides a method of use of pre-treated LCM for fermentative production of ethanol more efficiently than single use of pre-treated biomass for making ethanol as the efficiencies of conversion of pentoses and hexoses present in pre-treated LCM materials as such do not reach the level of sucrose or starch based ethanol production processes.

Although there are effective method for molasses fermentation and several methods of hydrolysis of LCM known; however, there exists a need to find more effective and economic methods to use existing distillery infrastructure like fermentation, distillation and waste water treatment throughout the year to produce high value chemicals with reduced capital expenditure.

DETAILED DESCRIPTION OF THE INVENTION In one embodiment of the disclosed invention ethanol is produced from pre-treated LCM biomass and molasses. Wherein 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 materials. Next, said soaked biomass is contacted with an admixture of acids [inorganic or organic] at temperature of about 140 °C to about 210 °C for about 5 minutes to about 120 minutes time period using the high-pressure steam as energy and water source. The admixture of acids comprises the amount of oxalic acid is between 0.1 to 5 percent and the amount of sulphuric acid is between 0.1 to 5 percent of said biomass by weight. On performing said pre-treatment 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 r treated with cellulolytic enzymes. The said hydrolyzed stream is first neutralized with an alkali like NaOH or MgO to increase its pH to about 4 to about 6 [forming the neutralized stream] and then about 10 mg to about 100 mg of cocktail of cellulase enzymes is added, and reaction is allowed to digest said solid material for about 30 hours to about 120 hours at about 40 °C to about 80 °C temperature conditions. The said cellulolytic enzymes comprise one or more of a cellulase, a hemicellulase or a combination thereof. This step releases glucose from the cellulose fibres leading to substantial increase of glucose and xylose in the formed stream [the second stream]. The said second stream is further mixed with desired quantity of sugarcane molasses, which contains about 40% to about 60% fermentable sugars by weight. Mixing of molasses in second stream forms a third stream having about 12% to 16% total fermentable sugars by weight. Said sugar rich third stream is subjected to fermentation by using recombinant strain of Saccharomyces cerevisiae, which ferment both glucose and xylose present in third stream leading to the formation of ethanol in the end stream. Here nitrogen source like urea and di-ammonium phosphate is added in third stream to support in fermentation process.

In another embodiment, the cellulase treatment is carried out separately at optimum conditions to get maximum conversion of cellulose fibres to glucose before the fermentation of the sugars. Once the separate hydrolysis is completed, molasses is added and the co- fermentation of xylose and glucose present in said stream is performed using a recombinant yeast of Saccharomyces sp. that is capable of fermenting pentose and hexose sugars simultaneously.

In another embodiment of the invention, a 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.

In another embodiment of the invention, molasses based sugars and cellulosic based sugars are fermented with same conversion efficiency in combination to produce ethanol. That is about 90% conversion efficiency of hexose sugars and less than 88 % conversion efficiency of pentose sugars.

In yet another embodiment of the invention, the efficiency of enzymatic hydrolysis of cellulose is at least 60% of the theoretical efficiency of the reaction.

In another embodiment of the invention, the efficiency of conversion of hexose sugars to ethanol is at least 80% of theoretical efficiency of the reaction.

In another embodiment of the invention, the efficiency of conversion of pentose sugars to ethanol is at least 65% of theoretical efficiency of the reaction.

In yet another embodiment of the invention, molasses contributes up to 80% of total fermentable sugars that leads to formation of ethanol.

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 combination of molasses with LCM for the preparation of ethanol.

EXAMPLE 1

In first step, a batch of about 135 kg of sugarcane bagasse having total solids of about 90% by weight, comprising 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 405 kg of slurry containing about 30% total solids by weight was prepared and continuously introduced into a plug-screw type hydrolyser. Here the slurry was mixed with about 180 L of an admixture of acid catalysts. This admixture of mixed acids contained about 1 % of oxalic acid by weight and about 1 .5% of sulphuric acid by weight on dry biomass weight basis [total acid of about 2.5% by weight]. The resultant reaction mixture was then subjected to hydrolysis in said hydrolyser at a temperature of about 150 ^S C and pressure of about 5 bar[a] for a period of about 15 minutes at the 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 hydrolysate rich with C5 sugar along with C6 solids [primarily cellulose] was subjected to enzymatic hydrolysis by an admixture of cellulases. Before enzymatic hydrolysis it was neutralized with MgO to increase the pH to about 5. Then a cocktail of cellulases [about 30 mg/g of cellulose] was added to the pre-treated material and allowed to digest said solids at desired conditions for up to 72 h at 52 ^S C. This enzymatic hydrolysis afforded about 790 kg of hydrolysate having glucose of about 3.8% by weight and xylose of about 3.2% by weight with cellulose hydrolysis efficiency of about 62%. In third step, this hydrolysate was mixed with about 189 kg of sugarcane molasses containing about 45% by weight fermentable sugars forming a stream having about 13% total fermentable sugars. To this nitrogen source like urea and di-ammonium phosphate (about 500 PPM of each) was added to support the fermentation process. The said sugar-rich stream was subjected to fermentation by using a recombinant strain of Saccharomyces cerevisiae, which fermented both glucose and xylose present in said stream to ethanol leading to ethanol concentration in the end stream of about 6.4% by weight. Herein glucose to ethanol conversion efficiency was about 90% and xylose to ethanol was about 88% of theoretical maximum after about 72 h of fermentation time [overall efficiency was about 85%]. Herein the overall bagasse to ethanol conversion efficiency after 120 h [enzyme treatment 72 h and fermentation for 48 h] was about 58% at the end of process. After fermentation, this stream was subjected to distillation and afforded about 80 L of ethanol from about 122 kg of dry sugarcane bagasse and 189 kg of molasses containing about 45% total fermentable sugars by weight.

EXAMPLE 2:

In first step, a batch of about 135 kg of sugarcane bagasse having total solids of about 90% by weight, comprising 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 405 kg of slurry containing about 30% total solids by weight was prepared and continuously introduced into a plug-screw type hydrolyser. Here the slurry was mixed with about 180 L of an admixture of acid catalysts. This admixture of mixed acids contained about 1 % of oxalic acid by weight and about 1 .5% of sulphuric acid by weight on dry biomass weight basis [total acid of about 2.5% by weight]. The resultant reaction mixture was then subjected to hydrolysis in said hydrolyser at a temperature of about 150 ^S C and pressure of about 5 bar[a] for a period of about 15 minutes at the 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 hydrolysate rich with C5 sugar along with C6 solids [primarily cellulose] was subjected to enzymatic hydrolysis by an admixture of cellulases. Before enzymatic hydrolysis it was neutralized with MgO to increase the pH to about 5. Then a cocktail of cellulases [about 30 mg/g of cellulose] was added to the pre-treated material and allowed to digest said solids at desired conditions for about 72 h at 52 ^S C. This enzymatic hydrolysis afforded about 790 kg of hydrolysate having glucose of about 3.8% by weight and xylose of about 3.2% by weight with cellulose hydrolysis efficiency of about 62%. In third step, this hydrolysate is mixed with about 127 kg of cane syrup containing about 71 % fermentable sugars by weight forming a stream having about 14% total fermentable sugars by weight. To this nitrogen source like urea and di-ammonium phosphate (about 500 PPM of each) was added to support the fermentation process. The said sugar-rich stream was subjected to fermentation by using a recombinant strain of Saccharomyces cerevisiae, which fermented both glucose and xylose present in said stream to ethanol leading to ethanol concentration in the end stream of about 6.7% by weight. Herein glucose to ethanol conversion efficiency was about 90% and xylose to ethanol was about 88% of theoretical maximum after about 72 h of fermentation time [overall efficiency was about 85%]. Herein the overall bagasse to ethanol conversion efficiency after 120 h [enzyme treatment 72 h and fermentation for 48 h] was about 58% at the end of process. After fermentation, this stream was subjected to distillation and afforded about 80 L of ethanol from about 122 kg of dry sugarcane bagasse and 120 kg of concentrated cane syrup containing about 71 % total fermentable sugars by weight.

EXAMPLE 3

In first step, a batch of about 250 kg of bagasse pith having total solids of about 40 % by weight comprising cellulose of about 38 % by weight, hemicelluloses of about 21 % by weight and lignin of about 22% by weight was used as a feedstock. Then about 250 kg of bagasse pith material containing about 19% total solids by weight was prepared and continuously introduced into a plug-screw type hydrolyser. Here the slurry was mixed with about 135 L of an admixture of acid catalysts. This admixture of mixed acids contained about 1 % of oxalic acid by weight and about 1 .5% of sulphuric acid by weight on dry biomass weight basis [total acid of about 2.5% by weight]. The resultant reaction mixture was then subjected to hydrolysis in said hydrolyser at a temperature of about 165 ^S C and pressure of about 5 bar[a] for a period of about 15 minutes at the pH of about 1 .2. At the end of this pre- treatment the final slurry of about 530 kg contained about 19% of total solids; and about 0.5% of glucose, about 4.1 % of xylose, and about 3500 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 75%. In second step, this pre-treated hydrolysate rich with C5 sugar along with C6 solids [primarily cellulose] was subjected to enzymatic hydrolysis by an admixture of cellulases. Before enzymatic hydrolysis it was neutralized with MgO to increase the pH to about 5 and added water to reduce the total solid from about 19% to about 15% by weight of said hydrolysate. Then a cocktail of cellulases [about 30 mg/g of cellulose] was added to the pre-treated material and allowed to digest said solids at desired conditions for about 72 h at 52 ^S C. This enzymatic hydrolysis afforded about 635 kg of hydrolysate having glucose of about 4.4% by weight and xylose of about 2.5% by weight with cellulose hydrolysis efficiency of about 68%. In third step, this hydrolysate is mixed with about 134 kg of sugar cane molasses containing about 49% fermentable sugars by weight forming a stream having about 14.2% total fermentable sugars by weight. To this nitrogen source like urea and di-ammonium phosphate (about 500 PPM of each) was added to support the fermentation process. The said sugar-rich stream was subjected to fermentation by using a recombinant strain of Saccharomyces cerevisiae, which fermented both glucose and xylose present in said stream to ethanol leading to ethanol concentration in the end stream of about 6.2% by weight. Herein glucose to ethanol conversion efficiency was about 90% and xylose to ethanol was about 88% of theoretical maximum after about 72 h of fermentation time [overall efficiency was about 85%]. Herein the overall bagasse to ethanol conversion efficiency after 120 h [enzyme treatment 72 h and fermentation for 48 h] was about 58% at the end of process. After fermentation, this stream was subjected to distillation and afforded about 65 L of ethanol from about 100 kg of dry bagasse pith and 134 kg of concentrated molasses containing about 49% total fermentable sugars by weight.

While the invention has been particularly shown and described with reference to embodiments listed in examples, it will be appreciated that several of the above disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also that various presently unforeseen and unanticipated alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims. Although the invention has been described with reference to specific preferred embodiments, it is not intended to be limited thereto, rather those having ordinary skill in the art will recognize that variations and modifications may be made therein which are within the spirit of the invention and within the scope of the claims. The advantages of the disclosed invention are listed below:

1 . In the process the sugars from molasses and biomass feed stocks are fermented with the same efficiency with substantially improved sugar conversion efficiency compared with second generation cellulosic fermentation process.

2. The process has overall less energy requirements than second generation cellulosic ethanol processing plant.

3. The process also helps in operating the molasses based distillery units run to more than 300 days compared with the present seasonal operation the ethanol plants for about 160 days per year.

4. The capital investments required for implementing the process are substantially less compared with the erecting of green field cellulosic ethanol plants.