FROM MOLASSES FERMENTATION, BY ADDITION OF ENZYMES WHICH CONVERT UNFERMENT ABLE SUGARS

INTO FERMENTABLE SUGARS

Technical Field

[1] The present invention relates to a process to enhance ethanol yield from molasses fermentation by enzymatic reaction which convert unfermentable sugars into fermentable sugars. Background Art

[2] The term molasses (from Greek word 'meli' meaning honey) is referred specifically to the final effluent obtained as a thick byproduct in the preparation of sucrose by repeated evaporation, crystallization and centrifugation of sugarcane or sugar beet juice etc. There are various sources of molasses such as sugar cane, sugar beet, starch, maize, sweet sorghum and citrus juice etc that are rich source of carbohydrates. Such carbohydrates can be consumed by a large number of microorganisms to produce various products. Generally molasses obtained after sugar recovery contains about 40-55% total fermentable sugars. Molasses is a cheap source of fermentable sugar that in addition to sugar also contains a number of other important nutrients. Other advantages of molasses are that they are by-products, thereby prices are not politically controlled and sugars in molasses can be easily utilized by most of the microorganisms.

[3] Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a somewhat potent psychoactive drug, best known as the type of alcohol found in alcoholic beverages and in modern thermometers. Ethanol is one of the oldest recreational drugs. In common usage, it is often referred to simply as alcohol or spirits. The fermentation of sugar into ethanol is one of the earliest organic reactions employed by humanity. The intoxicating effects of ethanol consumption have been known since ancient times. In modern times, ethanol intended for industrial use is also produced from by-products of petroleum refining. Ethanol has widespread use as a solvent of substances intended for human contact or consumption, including scents, flavorings, colorings, and medicines. In chemistry, it is both an essential solvent and a feedstock for the synthesis of other products. It has a long history as a fuel for heat and light, and more recently as a fuel for internal combustion engines. Disclosure of Invention Technical Problem

[4] With the dramatic increase in demand of ethanol across the globe and the increase in the cost of molasses, there is an immediate need to improve the efficiency of ethanol production from molasses. By converting unfermented sugars and oligosaccharides into fermentable sugars during molasses fermentation, ethanol yield can be enhanced by about 5% or by 5000 million liters worldwide. The value of this increased ethanol in India alone will be approximately 402 crores rupees annually.

[5] Production of ethanol from molasses by industrial microorganisms such as yeast (

Saccharomyces cerevisiae) is of great commercial importance. Most of the readily available sugars (sucrose, glucose and fructose) in molasses can be consumed by the yeast during fermentation or opportunistic contaminants during the molasses storage prior to fermentation. In addition to these sugars, there are some oligosaccharides and other pentose sugars in molasses, such as xylose, which are not readily fermented by yeasts or other contaminating microorganisms.

[6] US Pat. No 5372939 (Lastick et al.) discloses a process for producing ethanol from mixed sugar streams from pretreated biomass comprising xylose and cellulose using enzymes to convert these substrates to fermentable sugars; selecting and isolating a yeast Schizosaccharomyces pombe ATCC No. 2476, having the ability to ferment these sugars as they are being formed to produce ethanol by fermentation. The process further provides for obtaining ethanol by xylose being isomerized to xylulose, the cellulose being converted to glucose, and these sugars being concurrently converted to ethanol by yeast by anaerobic fermentation. However, this process does not describe the use of Sacchromyces cerevisiae or the use of molasses as starting material for fermentation.

[7] US Pat. No. 5254468 (Fournier et al) describes a bilayered immobilized enzyme pellet and a process to manufacture this pellet for use in a process involving the simultaneous isomerization of xylose to xylulose and fermentation of xylulose to ethanol. This process also describes the use of both xylose and glucose sugars effectively as a feedstock for ethanol production by isomerizing the xylose to xylulose and then making the xylulose immediately available for the fermentation process. This patent is however, restricted to use of glucose and xylose as the substrate and two enzymes xylose isomerase and urease immobilized on a bilayered pellet.

[8] US Pat. No. 4490468 (Gong et al) discloses a process for ethanol production by the fermentation of xylulose using yeast under fermentative conditions. This process is in no way combined with the fermentation of complex sugar mixture such as molasses.

[9] Another US Pat. No. 4368268 (Gong et al) describes a process for obtaining ethanol directly from D-xylose through fermentation of D-xylose by xylose-fermenting yeast mutants. The process provides for obtaining ethanol from hemicellulose hydrolyzates through yeast fermentation of D-xylose to ethanol and is not combined in any way with the use of molasses for fermentation.

[10] US Pat. No.5789210 (Ho et al) describes recombinant yeasts containing genes encoding xylose reductase, xylitol dehydrogenase and xylulokinase, and DNA molecules, vectors and methods useful for producing such yeasts. The recombinant yeasts effectively ferment xylose to ethanol, and preferred yeasts are capable of simultaneously fermenting glucose and xylose to ethanol thereby taking full advantage of these two sugar sources as they are found in agricultural biomass. However, this invention does not describe the use of wild type yeast strain, molasses and does not combine the use of enzymes for fermentation in any way. Technical Solution

[11] Efficiency of the ethanol production from molasses by Saccharomyces cerevisiae can be enhanced to a significant level by enzymatic processes. The present invention relates to a process to enhance ethanol yield from molasses fermentation by using a mix of specific enzymes and an antibiotic during molasses fermentation process. Using the present process un-fermented sugars and oligosaccharides are converted into fermentable sugars resulting in better availability of fermentable sugar for ethanol fermentation. The invention has great commercial application specifically in sugar and molasses industries, breweries, distilleries, alcohol-making industries, animal and poultry feed manufacturing industries and any other unit using molasses as raw material.

[12] The object of the present invention is to enhance ethanol yield from molasses fermentation by addition of enzymes which converts unfermentable sugars into fermentable sugars.

[13] The present invention provides a process to enhance ethanol yield from molasses fermentation, by addition of enzymes which convert unfermentable sugars into fermentable sugars and the process comprises of following steps:

1. Molasses sample were obtained from various distilleries across India and other molasses producing countries.

2. A mixture of enzymes such as xylanase, alpha-amylase, glucoamylase, beta glucanase, cellulase, alpha galactosidase and xylose isomerase in various combinations was added to molasses along with the antibiotic virginiamycin during anaerobic fermentation carried out using Saccharomyces cerevisiae.

3. Total reducing sugar was estimated by Fehling's titrimetric method. Advantageous Effects [14] Ethanol is an important chemical with numerous applications in various industries.

One of the important starting materials to manufacture ethanol on commercial scale is molasses. With the dramatic increase in demand of ethanol across the globe and the increase in the cost of molasses, there is an immediate need to improve the efficiency of ethanol production from molasses. By converting unfermented sugars and oligosaccharides into fermentable sugars during molasses fermentation, ethanol yield can be enhanced by about 5% or by 5000 million liters worldwide. The value of this increased ethanol in India alone will be approximately 402 crores rupees annually.

[15] The present invention relates to a process to enhance ethanol yield from molasses fermentation by using a mix of specific enzymes and an antibiotic during molasses fermentation process. Using the present process, the un-fermented sugars and oligosaccharides are converted into fermentable sugars resulting in better availability of fermentable sugar for ethanol fermentation. Description of Drawings

[16] Figure 1: Alcohol concentration during fermentation process with and without enzymes

[17] Figure 2: Xylose concentration during fermentation process with and without enzymes

[18] Figure 3: Sugar profile, and biomass of fermentation process without addition of enzyme

[19] Figure 4: Sugar profile, and biomass of fermentation process with carbohydrate de- polymerising enzymes

[20] Figure 5: Sugar profile, and biomass of fermentation process with carbohydrate de- polymerising enzymes and xylose isomerase Best Mode

[21] More especially, the present invention provides a process to enhance ethanol yield from molasses fermentation, by addition of enzymes which convert unfermentable sugars into fermentable sugars.

[22] Specific enzymes such as alpha amylase, glucoamylase, alpha galactosidase, xylanase, amylase, cellulase are added to molasses during the process of fermentation by Saccharomyces cerevisiae. These enzymes in specific amount and combination break down the oligosaccharides to easily fermentable monosaccharides. Since xylose and other pentose sugars remain unutilized by Saccharomyces cerevisiae, xylose isomerase along with the other carbohydrate depolymerizing enzymes has been used to isomerize the unfermented xylose to a readily fermentable xylulose. However, the iso- merization step is a reversible process. Therefore, addition of the same enzymes (particularly xylose isomerase) is necessary during the fermentation process so that more xylose is isomerized as xylulose is fermented by the yeast. Addition of an antibiotic such as virginiamycin reduces contamination by opportunistic bacteria during molasses storage and fermentation.

[23] Addition of enzymes resulted in increased alcohol yield (79 g/L) as compared to the control (74 g/L) within 36 h of fermentation (Table 1). Fermentation process was carried out at 30 oC under anaerobic condition with pH maintained at 4.5 by continuous addition of IM NaOH. The dynamics of alcohol concentration for three different fermentation process has been shown in Figure 1. The fermentation reaction was carried out in the presence of carbohydrate depolymersing enzymes. Another reaction was carried out in the presence of depolymersing enzymes as well as xylose isomerase. Maximum increase in the levels of alcohol was observed in presence of depolymersing enzymes & xylose isomerase which can be attributed to combined effect of the conversion of xylose to xylulose by the enzyme xylose isomerase and increase in reducing sugars such as glucose and fructose due to the action of carbohydrate depoly- merising enzymes. Xylose is otherwise not fermentable by Saccharomyces cerevisiae and once it is isomerised to xylulose by xylose isomerase, it becomes a readily available substrate for alcohol production as xylulose is readily fermented by Saccharomyces cerevisiae. This is evident from the decrease in xylose concentration observed in the fermentation set up with enzymes where the xylose concentration dropped from 2.4 g/L to 0.06 g/L within the 52 h fermentation period whereas no apparent decrease was noticed in the control set (Fig. X). Therefore, as a result of this enzymatic process, additional substrate in form of xylulose is available or yeast fermentation thereby utilizing non-fermentable sugar in molasses resulting in the enhanced alcohol production without any additional capital investment.

[24] Table 1: Alcohol content during fermentation process in the presence of enzymes

[25]

[Table 1] [Table ]

[26] Increase in other reducing sugars such as glucose and fructose was observed upon addition of enzymes to molasses (Figure 4) as compared to the control set (Figure 3) due to the action of enzymes such as alpha amylase, glucoamylase and cellulase on complex carbohydrates such as starch and cellulose in the molasses. This increase also contributed to the increase in alcohol content. In case of the control set, the alcohol yield was found to be lower compared to the enzyme treated set. Addition of enzymes also resulted in higher biomass as compared to the control and this was due to availability of surplus carbon source in form of released reducing sugar due to enzymatic action (Figure 4 & 5). In the fermentation processes, the presence of reducing sugars was observed even after 52 h of fermentation as compared to control set where the sugars were completely used up. This happened due to the continuing action of carbohydrate depolymerising enzymes even after the cessation of yeast growth.

[27] Fermentation of molasses was carried out with the addition of carbohydrate depolymerising enzymes but without the addition of xylose isomerase to study the individual effect of carbohydrate depolymerising enzymes on increasing alcohol levels. Increase in alcohol was observed in this case as compared to the control without the addition of enzymes. However, the alcohol yields were lower (76 g/L) as compared to the process where xylose isomerase was added along with the other enzymes (79 g/L). This establishes that although increase in alcohol can be obtained by addition of enzymes that specifically break down complex polysaccharides to reducing sugars, this is further enhanced by addition of xylose isomerase that isomerizes unfermentable xylose to fermentable xylulose and subsequent formation of alcohol. This process can be extended to other fermentable substrates for fermentation particularly hemicellulosic plant matter that contain xylose. Mode for Invention

[28] In order that this invention be more fully understood, the following preparative and testing examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.

[29] Example 1

[30] Culture conditions

[31] Molasses sample were obtained from various distilleries across India. Enzymes such as alpha amylase (85000U/g), glucoamylase (1,50,000 U/g), cellulase (85000 U/g), xylanase (85000 U/g), alpha galactosidase (2,00,000 U/g) and xylose isomerase (3200 U/ml) were procured from commercially available sources and were of industrial grade. The qualities of individual enzymes were assayed by routine biochemistry before the start of the experiment. Sugar standards for High Performance Liquid Chromatography (HPLC) were procured from Sigma. All other chemicals were of reagent grade procured locally.

[32] The molasses fermentations was carried out under anaerobic conditions using Sac- charomyces cerevisiae (0.5% v/v) inoculum. The fermenters were continuously controlled to maintain pH at 4.5 by automatic addition of IM NaOH. The temperature was automatically maintained at 30 oC throughout the period of fermentation. Samples are drawn aseptically at regular intervals and analyzed for sugars, alcohol and biomass.

[33] Example 2

[34] Analytical determination

[35] Total reducing sugar was estimated by Fehling's titrimetric method. Complete profiling of soluble sugars was done by HPLC on Zorbax carbohydrate column using acetonitrile:

[36] water 75: 25 as mobile phase at flow rate of 1 ml/min at 28 oC. Detection by was Refractive Index detector at 28 oC. Alcohol content was estimated by specific gravity measurement and biomass was determined by weighing.

[37] Example 3

[38] Enhancement of alcohol yield by addition of enzymes [39] Molasses sample were fermented using 0.5 % v/v Saccharomyces cerevisiae culture

[40] anaerobically at 30oC and at pH 4.5, using combination of enzymes (α- amylase)

(85000U/g), glucoamylase (1, 50,000 U/g), cellulose (85000 U/g), xylanase (85000 U/ g), alpha galactosidase (2,00,000 U/g) along with the inoculums and xylose isomerase (3200 U/mL) after 4 hours of start of fermentation. Control for this process consisted of fermentation set up under similar conditions and inoculums without the addition of enzymes.