Title of Invention	A PROCESS FOR ALCOHOL PRODUCTION FROM PANICUM SUMATRENSE (LITTLE MILLET) GRAINS
Abstract	The present invention provides the process for the production of ethanol from organic solid feedstock. Little millet grains were hydrolyzed using dilute acid which released maximal level of fermentable aqueous material. The present invention utilizes the hydrolysate for ethanol production using Saccharomyces cerevisiae (MTCC 170) and Issatchenkia orientalis (MTCC 10641) strains. The process results in maximum theoretical yield of ethanol. Ethanol fermentation efficiencies were between 79.15 percentage and 97.21 percentage. This is the two step process on production of ethanol from little millet grains using the separate hydrolysis and fermentation process. The present invention provides a simple and cost-effective way to produce ethanol by selectively ethanol tolerant yeast utilizing fermentable sugar based solutions; it results in maximum ethanol yield with a reduced amount of energy utilization.

Title of Invention

A PROCESS FOR ALCOHOL PRODUCTION FROM PANICUM SUMATRENSE (LITTLE MILLET) GRAINS

Abstract

The present invention provides the process for the production of ethanol from organic solid feedstock. Little millet grains were hydrolyzed using dilute acid which released maximal level of fermentable aqueous material. The present invention utilizes the hydrolysate for ethanol production using Saccharomyces cerevisiae (MTCC 170) and Issatchenkia orientalis (MTCC 10641) strains. The process results in maximum theoretical yield of ethanol. Ethanol fermentation efficiencies were between 79.15 percentage and 97.21 percentage. This is the two step process on production of ethanol from little millet grains using the separate hydrolysis and fermentation process. The present invention provides a simple and cost-effective way to produce ethanol by selectively ethanol tolerant yeast utilizing fermentable sugar based solutions; it results in maximum ethanol yield with a reduced amount of energy utilization.

Full Text	TITLE: A PROCESS FOR ALCOHOL PRODUCTION FROM PANICUM SUMATRENSE (LITTLE MILLET) GRAINS CROSS REFERENCE TO RELATED APPLICATIONS The present application claims priority to Indian Provisional Patent Application No. 389/CHE/2010, filed Feb. 15, 2010, which is incorporated by reference herein. FIELD OF INVENTION The invention relates ethanol production, more particularly method for the production of ethanol from little millet which is rich in carbohydrate and fiber using yeast under anaerobic condition. The plant is drought tolerant and adapted to acidic soils. BACKROUND OF THE INVENTION Ethanol, unlike the fossil fuels, is a safe and environment-friendly energy carrier and therefore the importance of ethanol as the fuel for internal combustion engines has, of late, increased considerably. At present, commercial production of ethanol relies on the fermentation of sugars by microorganisms. There is growing need for the industry to improve the technology and expand production due to the ruling high oil prices, environmental pressures and the expected fossil oil crisis as well as the relatively high cost of fuel alcohol. Sugars can be fermented directly to ethanol, while complex carbohydrates like starch and cellulose must be broken down first to simple sugars before the fermentation. There are two important process involved in the break down technology. They are hydrolysis and pretreatment. Hydrolysis is generally carried out by two different treatments like chemical and enzymatic. A number of chemical are used/tried by wide group of people. But the optimum concentration and conditions are substrate specific. The main interest of hydrolysis process is to obtain maximum fermentable sugars. In case of enzymatic hydrolysis the substrate needs pretreatment because of the complexity of substrate. Unlike hydrolysis the main concept of pretreatment is to remove /reduce the rigidity of complex substrate here the sugar production is not in concern. Dilute acid hydrolysis is probably the most commonly applied method among the chemical hydrolysis methods. Starch is water-insoluble polymer and a major component of agricultural crops. It is widely distributed in the vegetable kingdom and is stored in all grains and tubers. A typical starch molecule is constructed from a-1,4 and a -1,6 glucosidic bonds, each of which links two dehydrated glucose molecules. These molecules form either unbranched or somewhat branched polymer chains with up to 360 or 1000 glucose units, respectively. Very few studies have reported ethanol production from millet. Wu and co-worker, Cereal chemistry, 83, (2006), 127-131 have disclosed that millet grains might be a useful feedstock for ethanol production. Four pearl millet genotypes were tested for their potential as raw material for fuel ethanol production in this study. Ethanol fermentation was performed by using Saccharomyces cerevisiae (ATCC 24860). The final ethanol yields ranged from 8.7% to 16.8% (v/v) at dry mass concentrations of 20 to 35%, and the ethanol fermentation efficiencies were between 90.0% and 95.6%. Results showed that the fermentation efficiencies of pearl millets, on a starch basis, were comparable to those of corn and grain sorghum. Because pearl millets have greater protein and lipid contents, distillers' dried grains with solubles (DDGS) from pearl millets also had greater protein content and energy levels than did DDGS from corn and grain sorghum. He also disclosed that pearl millets could be a potential feedstock for fuel ethanol production in areas too dry to grow corn and grain sorghum. The economic feasibility of using peart millet as a feedstock was compared to maize. The author disclosed the comparison on the following aspects, (i) they are composition of raw material (protein, fat, crude fiber, ash and moisture), (ii) cost of electricity to grind. Value of DDGS of pearl millet was high ($0.11/Kg) when compared with DDGS of maize ($0.07/Kg) due to its high protein content. Total profit from pearl millet as the sole feedstock was $25,175,000 per year compared to $23,758,000 for maize feed stocks. Wilson et al., Issues in new crops and new uses, (2007), 56-59 disclosed the maize as a feedstock for ethanol production. Panicum sumatrense, one of the minor millets belongs to the family Poaceae, is commonly known as little millet in English and in other local names as follows: Tamil-samai, Kannada-shame, Malayalam-shama, Telugu-samalu, save, Hindi-shavan, kungu, kutki, Bengali-gundli, gondola, Marathi-sava, Gujarathi-gadro, Oriya-suniva. It is a coarse perennial grass, with a thick creeping rootstock. It grows usually in clumps, in bushes and grazed by cattle only when quite young. When it grows old it gets hard and woody and acquires a bitter or salty taste. It is distributed mainly in the tropical and warm temperate parts of the world. Little millet grains are used as a food in India but in other countries, it is used as a fodder crop. In India it is cultivated as a sole crop or subsidiary crop mixed with other millets like Sorghum bicolor, Paspalum sp. and Eleusine sp. or pulses like horse gram, or with oil seeds like gingelli, castor and mustard. Till Green Revolution more people used little millet as staple food. Nowadays its usage as food has decreased. Obviously the cultivation of the millet has also simultaneously decreased. But it is a good source of starch and the plant is drought tolerant and adapted to acidic soils. Whole grains from this plant contains 11.0% moisture, 7.2 % protein, 4.9 % fat, 63.8% carbohydrates, 9.6 % fiber, 3.5% mineral matter, 0.024% calcium, 0.32 % phosphorous, 0.007% iron, and 0.00034 % thiamine. Husked grains contain 11.0% moisture, 7.1% protein, 2.1% fat, 77.4% carbohydrates, 0.7% fiber, 1.7% mineral matter, 0.019 calcium, 0.16 % phosphorous, 0.003% iron, and 0.00034% thiamine. In addition, millet contains essential amino acids like arginine 4.66%, histidine 1.87%, isoleucine 6.66%, leucine 10.80%, lysine 1.83%, methionine 2.27%, phenylalanine 4.76%, threonine 3.40%, tryptophane 0.56%, and valine 6.06 % of total nitrogen. Fresh leaves of the plant contain 18.50mg/100g of carotene. The grains of P. sumatrense, with 77% carbohydrate have not been tested for alcohol production till now. Hence this can be used as a substrate for ethanol production. Therefore it is an embodiment of the present invention is to provide a process for producing ethanol from organic feedstock, particularly little millet. Another embodiment of the present invention is to provide a process for producing ethanol from little millet using dilute sulphuric acid to convert starch into fermentable sugars, which is then fermented with reference strain and ethanol tolerant yeast to produce ethanol. Another embodiment of the present invention is to find the effect of nitrogen source like urea in the fermentation process and ethanol production. Yet another embodiment of the present invention is to provide an industrially feasible, less energy utilizing and economical process for producing ethanol from little millet. Still another embodiment of the present invention is to provide a method for producing ethanol from little millet wherein the said method consumes very less energy during conversion of sugars. SUMMARY OF THE INVENTION Accordingly, the first embodiment of the present invention is to provide a technology for producing ethanol from organic feedstock. It is further embodiment of the invention to provide a process wherein the organic feedstock is a under cultivation crop grain. It is further embodiment of the invention to provide a process wherein the organic feedstock undergone dry form. Yet another embodiment of the present invention, the little millet containing about 70 % starch is converted into monosaccharides when treated with dilute sulphuric acid at 80 degree C to 130 degree C for 1 - 50 minutes wherein the dilute sulphuric acid used between 1 % and 25 %. It is further embodiment of the invention to provide a process wherein the hydrolyzed product is a sugar containing aqueous material. Another further embodiment of the present invention; the hydrolyzed product was fermented by using reference and ethanol tolerant yeast under anaerobic condition to obtain ethanol. DETAILED DESCRIPTION OF THE INVENTION Acid hydrolysis of little millet Incubation at varying conditions The organic feedstock optionally milled was used for further studies. One part of organic feedstock treated with 5-10 parts of mineral acid at different concentrations (1 - 25) under pressurized (15 lb) at different temperature (80 - 130 degree C) for 1-50 minutes. The hydrolysate optionally separated by used centrifugation force at 10,000 rpm for 5-20 min. The hydrolysate was neutralized with alkali and determined for sugars. The most important results of the hydrolysis were summarized in Figure 1. The results indicate that the initiation of maximum sugar release occurred in 2 concentration mineral acid and it reduced dramatically in all other concentrations of H2S04. Since maximum initiation of sugar release has occurred in even diluted concentrations H2SO4, and it was also recommended that use of dilute acid for pretreatment would not have any post production problems in the form of acid disposal. The hydrolysate product was collected by centrifugation force, neutralized with alkali and estimated for reducing sugars. The resulting hydrolyzed fractions from dry grain are then subjected to fermentation as described below. The hydrolyzed product was found to be maximum 77.15 percentages. The hydrolyzed aqueous material was fermented by both reference strain and isolated yeast under anaerobic condition. BRIEF DESCRIPTION OF THE FIGUREURES Figure 1: Schematically illustrate the effect of dosage of acid, thermal conditions and the sugar yield of the process of the present invention on organic feedstocks of little millet, c - control without acid addition; Means do not differ significantly at 5% level of significance using Tukey HSD test Figure 2: Schematically illustrate the fermentation of acid hydrolysate of little millet grains by Issatchenkia orientalis (MTCC 10641): AB - little millet hydrolysate without urea: A- pH and cell number in the fermented broth, B-reducing sugars concentration and ethanol yield in fermented broth. CD- little millet hydrolysate with urea supplementation: C- pH and cell number in the fermented broth, D- reducing sugars concentration and ethanol yield in fermented broth. Means do not differ significantly at 5% level of significance using Tukey HSD test Figure 3: Schematically illustrate the ethanol production by the reference strain Saccharomyces cerevisiae MTCC 170: CD - little millet acid hydrolysate: C- pH and cell number of little millet hydrolysate fermented broth, D- reducing sugars consumption and ethanol yield in little millet hydrolysate fermented broth. Means do not differ significantly at 5% level of significance using Tukey HSD test EXAMPLE 1 Seed culture and inoculum preparation Yeast strains Saccharomyces cerevisiae (MTCC 170) and Issatchenkia orientalis (MTCC 10641) were used for the present invention. Seed cultures were prepared by inoculating a large single colony into liquid glucose yeast extract malt medium under agitation (125 rpm) at 30 ± 2 degree C for 18 h. Preparation of acid hydrolysates 100 parts of organic feedstock was used for the preparation of hydrolysates. 2 concentration of sulphuric acid and little millet were mixed and the substrate mixtures were hydrolysed under pressurized temperature at 80-130 degree C and 15 lb pressure with a residence time of 5-20 min. The hydrolysates were filtered using a nylon filter and neutralized to pH 5.0-5.5 with sodium hydroxide. The amount of fermentable sugars produced during hydrolyses of substrates were estimated. EXAMPLE 2 Ethanol Production from hydrolyzed organic feedstocks of little millet The resulted hydrolysate was taken in a pre-sterilized container. 10 parts of the mother culture was inoculated and fermented, at 25 - 32 degree C 240 hrs. The sample was taken to analyze the production of ethanol. In the process, the ethanol production was preceded by reacting the samples with optimized concentrations of mineral acid. Two different reference and isolate yeast strains were used as fermenting microorganisms for conversion of organic feedstock hydrolyzed product to ethanol. The final ethanol yield from separate hydrolysis and fermentation is 30.53g / 100g (w/w) of organic feedstock at solid basis and with urea supplementation 37.5g / 100g (w/w) of organic feedstock at solid basis. Ethanol fermentation efficiencies were between 79.15 percentage and 97.21 percentage. In this invention, grain based ethanol production was studied to decide the various parameters essential for maximum ethanol recovery. The time for maximum ethanol production was also reduced in urea supplementation from 96 hrs to 72 hrs in little millet hydrolysate in SHF. EXAMPLE 3 In the present invention, the pH of the urea supplement fermented aqueous material was found to regularly reduce from 5.0 to 3.5 and it was found reduced more to 2.95 in the lack of urea. The yeast growth also regularly increased initially and decreased in the later part of the process. The yeast cell number was high (25 log 5) in urea supplemented fermented broth (Figure. 2). From the results, it can be concluded that urea supplemented broth supports the growth of yeast cells and their fermentation ability. In the present study the dry milled little millet samples were used for ethanol production. The first step was the hydrolysis of the little millet to release maximum amount of sugars. This was done using physical, chemical and thermal reactions in the occurrence of dilute sulphuric acid. EXAMPLE 4 To evaluate the competence of the isolate used in this invention, the aqueous material after hydrolysis were concurrently fermented using Saccharomyces cerevisiae (MTCC 170) a reference strain procured from Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India. The ethanol yield from little millet grains hydrolysate was 25.26g / 100g (Figure. 3). Maximum ethanol production was noticed on day 2 in little millet hydrolysate. The ethanol yield was comparably higher in the native isolate, /. orientalis (MTCC 10641) based fermentation than S. cerevisiae (MTCC 170) based fermentation. The pH was found to regularly reduce from 5.0 to 4.75 in the fermented broth. Though there was no drastic reduction in the pH of the fermented aqueous material, a gradual drop in the same was observed over a period of incubation. The yeast cell number was maximum (14 log 5) in the same (Figure. 3C). Selected citations and bibliography Wu, X., Wang, D., Bean, S., and Wilson, J.P. (2006). Ethanol production from pearl millet using saccharomyces cerevisiae. Cereal chem. 83:127-131. Wilson, J.P., McAloon, A.J., Yee, W., McKinney, J., Wang, D., Bean, S.R. (2007). Biological and Economic Feasibility of Pearl Millet as a Feedstock for Ethanol Production. Reprinted from: Issues in new crops and new uses. J. Janick and A. Whipkey (eds.). ASHS Press, Alexandria, VA. pp 56-59. Sivasankari, S., Senthilkumar, M., Seshadri, S. (2008). Bioethanol Production from Panicum sumatrense (little millet). 49th Annual Conference International symposium on Microbial Biotechnology. Diversity, Genomics and Metagenomics. pp-242. What is claimed is: 1. A method of making an ethanol product comprising: hydrolyzing carbohydrates under acidic condition with dilute acid solution to form a hydrolyzed product; and fermenting the hydrolyzed product to form the ethanol product. 2. The method of claim 1, wherein the acidic solution include sulphuric acid with the concentrations of 1.0 to 25.0. 3. The method of claim 1, wherein the method is carried out in a glass vessel comprising 1 part to 5 parts of dry solids. 4. The method of claim 1, wherein the acid solution included in an amount of from 5.0 to 10.0 volumes per gram of dry solids. 5. The method of claim 1, wherein hydrolyzing the carbohydrates is carried out in the form of dry; wherein hydrolyzing the carbohydrate is carried out in without incubation; wherein hydrolyzing the carbohydrate is carried out under pressurized temperature (151b) at 80 degree C to 130 degree C; wherein hydrolyzing the carbohydrate is carried out for 5 or 10 or 15 min. 6. A method of making hydrolyzed product comprising: hydrolyzing little millet grain carbohydrate under acidic conditions with dilute acid solution to form a hydrolyzed product; and fermenting the hydrolyzed product by two yeasts (Issatchenkia orientalis (MTCC 10641) & Saccharomyces cerevisiae (MTCC 170)) to form the ethanol product. 7. The method of claim 6, where in the organic feedstock Panicum sumatrens; wherein hydrolyzing the carbohydrate is of little millet grains. 8. The method of claim 6, wherein the yeast fermentation included pH 5.0 to 5.5 and temperature 25 to 35 degree C. 9. The method of claim 8, wherein the yeast fermentation medium included potassium dihydrogen phosphate, magnesium sulphate and urea 10. The method of claim 8, wherein the solid material is separated from the hydrolysate or ethanol product and the solid material dried to form dried grain.

Full Text

TITLE: A PROCESS FOR ALCOHOL PRODUCTION FROM PANICUM
SUMATRENSE (LITTLE MILLET) GRAINS CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to Indian Provisional Patent Application
No. 389/CHE/2010, filed Feb. 15, 2010, which is incorporated by reference
herein.

FIELD OF INVENTION

The invention relates ethanol production, more particularly method for the
production of ethanol from little millet which is rich in carbohydrate and fiber
using yeast under anaerobic condition. The plant is drought tolerant and adapted
to acidic soils.

BACKROUND OF THE INVENTION

Ethanol, unlike the fossil fuels, is a safe and environment-friendly energy carrier and therefore the importance of ethanol as the fuel for internal combustion engines has, of late, increased considerably. At present, commercial production of ethanol relies on the fermentation of sugars by microorganisms. There is growing need for the industry to improve the technology and expand production due to the ruling high oil prices, environmental pressures and the expected fossil oil crisis as well as the relatively high cost of fuel alcohol. Sugars can be fermented directly to ethanol, while complex carbohydrates like starch and cellulose must be broken down first to simple sugars before the fermentation. There are two important process involved in the break down technology. They are hydrolysis and pretreatment. Hydrolysis is generally carried out by two different treatments like chemical and enzymatic. A number of chemical are used/tried by wide group of people. But the optimum concentration and conditions are substrate specific. The main interest of hydrolysis process is to obtain maximum fermentable sugars. In case of enzymatic hydrolysis the substrate needs pretreatment because of the complexity of substrate. Unlike hydrolysis the main concept of pretreatment is to remove /reduce the rigidity of complex substrate here the sugar production is not in concern. Dilute acid hydrolysis is probably the most commonly applied method among the chemical hydrolysis methods.

Starch is water-insoluble polymer and a major component of agricultural crops. It is widely distributed in the vegetable kingdom and is stored in all grains and tubers. A typical starch molecule is constructed from a-1,4 and a -1,6 glucosidic bonds, each of which links two dehydrated glucose molecules. These molecules form either unbranched or somewhat branched polymer chains with up to 360 or 1000 glucose units, respectively.

Very few studies have reported ethanol production from millet. Wu and co-worker, Cereal chemistry, 83, (2006), 127-131 have disclosed that millet grains might be a useful feedstock for ethanol production. Four pearl millet genotypes were tested for their potential as raw material for fuel ethanol production in this study. Ethanol fermentation was performed by using Saccharomyces cerevisiae (ATCC 24860). The final ethanol yields ranged from 8.7% to 16.8% (v/v) at dry mass concentrations of 20 to 35%, and the ethanol fermentation efficiencies were between 90.0% and 95.6%. Results showed that the fermentation efficiencies of pearl millets, on a starch basis, were comparable to those of corn and grain sorghum. Because pearl millets have greater protein and lipid contents, distillers' dried grains with solubles (DDGS) from pearl millets also had greater protein content and energy levels than did DDGS from corn and grain sorghum. He also disclosed that pearl millets could be a potential feedstock for fuel ethanol production in areas too dry to grow corn and grain sorghum. The economic feasibility of using peart millet as a feedstock was compared to maize. The author disclosed the comparison on the following aspects, (i) they are composition of raw material (protein, fat, crude fiber, ash and moisture), (ii) cost of electricity to grind. Value of DDGS of pearl millet was high ($0.11/Kg) when compared with DDGS of maize ($0.07/Kg) due to its high protein content. Total profit from pearl millet as the sole feedstock was $25,175,000 per year compared to $23,758,000 for maize feed stocks. Wilson et al., Issues in new crops and new uses, (2007), 56-59 disclosed the maize as a feedstock for ethanol production.

Panicum sumatrense, one of the minor millets belongs to the family Poaceae, is commonly known as little millet in English and in other local names as follows: Tamil-samai, Kannada-shame, Malayalam-shama, Telugu-samalu, save, Hindi-shavan, kungu, kutki, Bengali-gundli, gondola, Marathi-sava, Gujarathi-gadro, Oriya-suniva. It is a coarse perennial grass, with a thick creeping rootstock. It grows usually in clumps, in bushes and grazed by cattle only when quite young. When it grows old it gets hard and woody and acquires a bitter or salty taste. It is distributed mainly in the tropical and warm temperate parts of the world. Little millet grains are used as a food in India but in other countries, it is used as a fodder crop. In India it is cultivated as a sole crop or subsidiary crop mixed with other millets like Sorghum bicolor, Paspalum sp. and Eleusine sp. or pulses like horse gram, or with oil seeds like gingelli, castor and mustard. Till Green Revolution more people used little millet as staple food. Nowadays its usage as food has decreased. Obviously the cultivation of the millet has also simultaneously decreased. But it is a good source of starch and the plant is drought tolerant and adapted to acidic soils.

Whole grains from this plant contains 11.0% moisture, 7.2 % protein, 4.9 % fat, 63.8% carbohydrates, 9.6 % fiber, 3.5% mineral matter, 0.024% calcium, 0.32 % phosphorous, 0.007% iron, and 0.00034 % thiamine. Husked grains contain 11.0% moisture, 7.1% protein, 2.1% fat, 77.4% carbohydrates, 0.7% fiber, 1.7% mineral matter, 0.019 calcium, 0.16 % phosphorous, 0.003% iron, and 0.00034% thiamine. In addition, millet contains essential amino acids like arginine 4.66%, histidine 1.87%, isoleucine 6.66%, leucine 10.80%, lysine 1.83%, methionine 2.27%, phenylalanine 4.76%, threonine 3.40%, tryptophane 0.56%, and valine 6.06 % of total nitrogen. Fresh leaves of the plant contain 18.50mg/100g of carotene.

The grains of P. sumatrense, with 77% carbohydrate have not been tested for alcohol production till now. Hence this can be used as a substrate for ethanol production.

Therefore it is an embodiment of the present invention is to provide a process for producing ethanol from organic feedstock, particularly little millet.

Another embodiment of the present invention is to provide a process for
producing ethanol from little millet using dilute sulphuric acid to convert starch
into fermentable sugars, which is then fermented with reference strain and
ethanol tolerant yeast to produce ethanol.

Another embodiment of the present invention is to find the effect of nitrogen
source like urea in the fermentation process and ethanol production.

Yet another embodiment of the present invention is to provide an industrially
feasible, less energy utilizing and economical process for producing ethanol from
little millet.

Still another embodiment of the present invention is to provide a method for
producing ethanol from little millet wherein the said method consumes very less
energy during conversion of sugars.

SUMMARY OF THE INVENTION

Accordingly, the first embodiment of the present invention is to provide a
technology for producing ethanol from organic feedstock.

It is further embodiment of the invention to provide a process wherein the organic
feedstock is a under cultivation crop grain.

It is further embodiment of the invention to provide a process wherein the organic
feedstock undergone dry form.

Yet another embodiment of the present invention, the little millet containing about
70 % starch is converted into monosaccharides when treated with dilute
sulphuric acid at 80 degree C to 130 degree C for 1 - 50 minutes wherein the
dilute sulphuric acid used between 1 % and 25 %.

It is further embodiment of the invention to provide a process wherein the
hydrolyzed product is a sugar containing aqueous material.

Another further embodiment of the present invention; the hydrolyzed product was
fermented by using reference and ethanol tolerant yeast under anaerobic
condition to obtain ethanol.

DETAILED DESCRIPTION OF THE INVENTION

Acid hydrolysis of little millet

Incubation at varying conditions

The organic feedstock optionally milled was used for further studies. One part of
organic feedstock treated with 5-10 parts of mineral acid at different
concentrations (1 - 25) under pressurized (15 lb) at different temperature (80 -
130 degree C) for 1-50 minutes. The hydrolysate optionally separated by used
centrifugation force at 10,000 rpm for 5-20 min. The hydrolysate was
neutralized with alkali and determined for sugars. The most important results of
the hydrolysis were summarized in Figure 1.

The results indicate that the initiation of maximum sugar release occurred in 2
concentration mineral acid and it reduced dramatically in all other concentrations
of H2S04. Since maximum initiation of sugar release has occurred in even diluted
concentrations H2SO4, and it was also recommended that use of dilute acid for
pretreatment would not have any post production problems in the form of acid
disposal.

The hydrolysate product was collected by centrifugation force, neutralized with
alkali and estimated for reducing sugars. The resulting hydrolyzed fractions from dry grain are then subjected to fermentation as described below. The hydrolyzed product was found to be maximum 77.15 percentages. The hydrolyzed aqueous material was fermented by both reference strain and isolated yeast under anaerobic condition.

BRIEF DESCRIPTION OF THE FIGUREURES

Figure 1: Schematically illustrate the effect of dosage of acid, thermal conditions and the sugar yield of the process of the present invention on organic feedstocks of little millet, c - control without acid addition; Means do not differ significantly at 5% level of significance using Tukey HSD test

Figure 2: Schematically illustrate the fermentation of acid hydrolysate of little millet grains by Issatchenkia orientalis (MTCC 10641): AB - little millet hydrolysate without urea: A- pH and cell number in the fermented broth, B-reducing sugars concentration and ethanol yield in fermented broth. CD- little millet hydrolysate with urea supplementation: C- pH and cell number in the fermented broth, D- reducing sugars concentration and ethanol yield in fermented broth. Means do not differ significantly at 5% level of significance using Tukey HSD test

Figure 3: Schematically illustrate the ethanol production by the reference strain Saccharomyces cerevisiae MTCC 170: CD - little millet acid hydrolysate: C- pH and cell number of little millet hydrolysate fermented broth, D- reducing sugars consumption and ethanol yield in little millet hydrolysate fermented broth. Means do not differ significantly at 5% level of significance using Tukey HSD test

EXAMPLE 1

Seed culture and inoculum preparation

Yeast strains Saccharomyces cerevisiae (MTCC 170) and Issatchenkia orientalis (MTCC 10641) were used for the present invention. Seed cultures were prepared by inoculating a large single colony into liquid glucose yeast extract malt medium under agitation (125 rpm) at 30 ± 2 degree C for 18 h.

Preparation of acid hydrolysates 100 parts of organic feedstock was used for the preparation of hydrolysates. 2 concentration of sulphuric acid and little millet were mixed and the substrate mixtures were hydrolysed under pressurized temperature at 80-130 degree C and 15 lb pressure with a residence time of 5-20 min. The hydrolysates were filtered using a nylon filter and neutralized to pH 5.0-5.5 with sodium hydroxide. The amount of fermentable sugars produced during hydrolyses of substrates were estimated.

EXAMPLE 2

Ethanol Production from hydrolyzed organic feedstocks of little millet The resulted hydrolysate was taken in a pre-sterilized container. 10 parts of the mother culture was inoculated and fermented, at 25 - 32 degree C 240 hrs. The sample was taken to analyze the production of ethanol.

In the process, the ethanol production was preceded by reacting the samples with optimized concentrations of mineral acid. Two different reference and isolate yeast strains were used as fermenting microorganisms for conversion of organic feedstock hydrolyzed product to ethanol. The final ethanol yield from separate hydrolysis and fermentation is 30.53g / 100g (w/w) of organic feedstock at solid basis and with urea supplementation 37.5g / 100g (w/w) of organic feedstock at solid basis. Ethanol fermentation efficiencies were between 79.15 percentage and 97.21 percentage.

In this invention, grain based ethanol production was studied to decide the various parameters essential for maximum ethanol recovery. The time for maximum ethanol production was also reduced in urea supplementation from 96 hrs to 72 hrs in little millet hydrolysate in SHF. EXAMPLE 3

In the present invention, the pH of the urea supplement fermented aqueous material was found to regularly reduce from 5.0 to 3.5 and it was found reduced more to 2.95 in the lack of urea. The yeast growth also regularly increased initially and decreased in the later part of the process. The yeast cell number was high (25 log 5) in urea supplemented fermented broth (Figure. 2). From the results, it can be concluded that urea supplemented broth supports the growth of yeast cells and their fermentation ability.

In the present study the dry milled little millet samples were used for ethanol production. The first step was the hydrolysis of the little millet to release maximum amount of sugars. This was done using physical, chemical and thermal reactions in the occurrence of dilute sulphuric acid.

EXAMPLE 4

To evaluate the competence of the isolate used in this invention, the aqueous material after hydrolysis were concurrently fermented using Saccharomyces cerevisiae (MTCC 170) a reference strain procured from Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India. The ethanol yield from little millet grains hydrolysate was 25.26g / 100g (Figure. 3). Maximum ethanol production was noticed on day 2 in little millet hydrolysate. The ethanol yield was comparably higher in the native isolate, /. orientalis (MTCC 10641) based fermentation than S. cerevisiae (MTCC 170) based fermentation.

The pH was found to regularly reduce from 5.0 to 4.75 in the fermented broth.

Though there was no drastic reduction in the pH of the fermented aqueous
material, a gradual drop in the same was observed over a period of incubation.

The yeast cell number was maximum (14 log 5) in the same (Figure. 3C).
Selected citations and bibliography

Wu, X., Wang, D., Bean, S., and Wilson, J.P. (2006). Ethanol production from pearl millet using saccharomyces cerevisiae. Cereal chem. 83:127-131.

Wilson, J.P., McAloon, A.J., Yee, W., McKinney, J., Wang, D., Bean, S.R. (2007). Biological and Economic Feasibility of Pearl Millet as a Feedstock for Ethanol Production. Reprinted from: Issues in new crops and new uses. J. Janick and A. Whipkey (eds.). ASHS Press, Alexandria, VA. pp 56-59.

Sivasankari, S., Senthilkumar, M., Seshadri, S. (2008). Bioethanol Production from Panicum sumatrense (little millet). 49th Annual Conference International symposium on Microbial Biotechnology. Diversity, Genomics and Metagenomics. pp-242.

What is claimed is:

1. A method of making an ethanol product comprising: hydrolyzing carbohydrates under acidic condition with dilute acid solution to form a hydrolyzed product; and fermenting the hydrolyzed product to form the ethanol product.

2. The method of claim 1, wherein the acidic solution include sulphuric acid with the concentrations of 1.0 to 25.0.

3. The method of claim 1, wherein the method is carried out in a glass vessel comprising 1 part to 5 parts of dry solids.

4. The method of claim 1, wherein the acid solution included in an amount of from 5.0 to 10.0 volumes per gram of dry solids.

5. The method of claim 1, wherein hydrolyzing the carbohydrates is carried out in the form of dry; wherein hydrolyzing the carbohydrate is carried out in without incubation; wherein hydrolyzing the carbohydrate is carried out under pressurized temperature (151b) at 80 degree C to 130 degree C; wherein hydrolyzing the carbohydrate is carried out for 5 or 10 or 15 min.

6. A method of making hydrolyzed product comprising: hydrolyzing little millet grain carbohydrate under acidic conditions with dilute acid solution to form a hydrolyzed product; and fermenting the hydrolyzed product by two yeasts (Issatchenkia orientalis (MTCC 10641) & Saccharomyces cerevisiae (MTCC 170)) to form the ethanol product.

7. The method of claim 6, where in the organic feedstock Panicum sumatrens; wherein hydrolyzing the carbohydrate is of little millet grains.

8. The method of claim 6, wherein the yeast fermentation included pH 5.0 to 5.5 and temperature 25 to 35 degree C.

9. The method of claim 8, wherein the yeast fermentation medium included potassium dihydrogen phosphate, magnesium sulphate and urea

10. The method of claim 8, wherein the solid material is separated from the hydrolysate or ethanol product and the solid material dried to form dried grain.

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=OSl7E5Ab6nw3sMVDMY26SA==&loc=egcICQiyoj82NGgGrC5ChA==

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Patent Number

278962

Indian Patent Application Number

389/CHE/2010

PG Journal Number

01/2017

Publication Date

06-Jan-2017

Grant Date

05-Jan-2017

Date of Filing

15-Feb-2010

Name of Patentee

SIVAGURUNATHAN.P

Applicant Address

49A/6, MAIN ROAD, KATCHIPERUMAL (PO) UDAYARPALAYAM(TK) ARIYALUR DT. PIN-621804

Inventors:

#	Inventor's Name	Inventor's Address
1	SIVASANKARI.S	49A/6, MAIN ROAD, KATCHIPERUMAL (PO) UDAYARPALAYAM(TK) ARIYALUR DT. PIN-621804
2	SIVAGURUNATHAN.P	49A/6, MAIN ROAD, KATCHIPERUMAL (PO) UDAYARPALAYAM(TK) ARIYALUR DT. PIN-621804

PCT International Classification Number

C12P

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA