Title of Invention	A TECHNOLOGY FOR ETHANOL FROM BANANA PSEUDOSTEM WASTE
Abstract	The present invention provides the process for the production of ethanol from lignocellulosic feedstock. Banana pseudostem wastes were hydrolyzed using mild mineral 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. Ethanol fermentation efficiencies were between 69.72 percentage and 84.67 percentage. This is the two step process on production of ethanol from banana pseudostem waste 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 TECHNOLOGY FOR ETHANOL FROM BANANA PSEUDOSTEM WASTE

Abstract

The present invention provides the process for the production of ethanol from lignocellulosic feedstock. Banana pseudostem wastes were hydrolyzed using mild mineral 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. Ethanol fermentation efficiencies were between 69.72 percentage and 84.67 percentage. This is the two step process on production of ethanol from banana pseudostem waste 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 TECHNOLOGY FOR ETHANOL FROM BANANA PSEUDOSTEM WASTE CROSS REFERENCE TO RELATED APPLICATIONS The present application claims priority to Indian Provisional Patent Application No. 387/CHE/2010, filed Feb. 15, 2010, which is incorporated by reference herein. FIELD OF INVENTION The invention relates ethanol production, more specifically method for the production of ethanol from wasted banana pseudostem which is the finest source of cellulose and hemicellulose using yeast under anaerobic condition. BACKROUND OF THE INVENTION Ethanol has been known for a long time, perhaps the oldest product obtained through traditional biotechnology. Conventionally, ethanol containing beverages are used for human consumption. It is also used as the main ingredient in medical wipes as antibacterial and antiseptics, antifreeze agent, an antidote for methanol poisoning, disinfectants, extractant, hand sanitizer gels, in the manufacture of varnishes and perfumes, preservative for biological specimens, sketch art markers, flavorings and medicines. The demand for ethanol has been increasing day by day because of its multiple uses. Currently, commercial ethanol production relies on the fermentation of sucrose from cane sugar and molasses or glucose derived from starch based crops such as corn, wheat and cassava. There is a growing need for the industry to improve technology and enhance production due to high oil prices, environmental pressures and national security concerns. The global production of plant biomass, of which over 90% is lignocellulose, amounts to about 200X109 tonnes per year, whereas 8-20X109 tonnes of the primary biomass remains potentially inaccessible. Lignocellulose is the major structural component of woody plants and non-woody plants such as grass and represents a major source of the renewable organic matter. Plants consisting of lignocellulose can be divided into three categories according to plant taxonomy: softwood (gymnosperms), hardwood (woody angiosperms) and annual plants (herbaceous angiosperms). Lignocellulosic materials are the most appealing in this respect, due to their abundance and low cost. Banana is the most widely grown fruit crop in more than 132 countries including India, covering almost 10 million hectares of cropped area. Uma et al., Banana Fibre, (2003), Niseema printers, Cochin and Sharma et al., Ind. J. Microbiol, 47, (2007), 310-316 disclosed that India on an average produces 1.533107 MT banana / year with 24% of world's bananas production. Zuluaga and co-workers disclosed the cultivation of banana generates a considerable amount of residues, where the fruit constitutes 12 wt% of the plant, and the remaining parts become agricultural waste. Tamil Nadu Government disclosed that banana is cultivated in and around 94 thousand hectares in 14 districts of Tamil Nadu, India (2008) and the estimated production of banana is 4.488 106 tonnes in the year 2007-2008. Baig er al., African Journal of Biotechnology, 3, (2004), 447-450 and Khalil et al., Bioresources, 1, (2006), 220-232 disclosed that the remaining part of the plant includes leaves, stem and rhizome and after harvesting the fruit left in the field takes several months for degradation. Apart from its multifaceted uses like therapeutics, dye, starch extraction and other innumerable uses, it forms the finest source of cellulose. Very few reports have been accounted on alcohol production form banana pseudostem. Baig and oo workers, African Journal of Biotechnology, 3, (2004), 447-450 disclosed the sugar production from dried leaves and psuedostem using cellulase produced by Trichoderma lignorum. They disclosed 1.34 mg/ml of reducing sugars from steam treated the agro-waste after 24 h. They also disclosed the size of substrates, pH and temperature to impact the yield. Saeed and Qazi, Mycopath, 3, (2005), 73-78 disclosed the simultaneous saccharification and fermentation of banana by using Issatchenkia orientalis and reported 4.14 % ethanol yield in a single step process. In another report, Shafique and co-worker International Journal of Agriculture and Biology, 6, (2004), 488-491 disclosed the exoglucanase production using Bacillus subtilis on solid-state fermentation (SSF) from banana stalk. They also disclosed maximum exoglucanase activity (3.48 lU/mL/min) from the medium fermented with 70% moisture content, 5 mL inoculum, 0.1% peptone, 0.4% yeast extract and 0.2% Tween-80 at pH 7 and temperature 35 °C. A strong need exists for efficient ethanol production with cost effective raw material and energy consumption. Therefore it is an embodiment of the present invention is to provide a technology for producing ethanol from lignocellulosic waste, particularly banana pseudostem waste. Another embodiment of the present invention is to offer a technology for producing ethanol from banana pseudostem waste using dilute oxalic acid to convert lignocellulose 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 identify the effect of nitrogen source like urea in the fermentation process and ethanol production. Yet another embodiment of the present invention is to offer an industrially viable, less energy utilizing and inexpensive technology for producing ethanol from banana pseudostem waste. Still another embodiment of the present invention is to provide a method for producing ethanol from banana pseudostem 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 offer a technology for producing ethanol from lignocellulosic waste. It is further embodiment of the invention to provide a technology wherein the lignocellulosic waste is a waste product from agriculture sector. It is further embodiment of the invention to provide a process wherein the lignocellulosic waste undergone dry milled form. Yet another embodiment of the present invention, the banana pseudostem waste containing about 60-70 % cellulose and hemicellulose is converted into monosaccharides when treated with dilute oxalic acid under pressurized temperature at 80 degree C to 130 degree C for 5 - 150 minutes wherein the dilute oxalic acid used between 1 % and 25 %. It is further embodiment of the invention to provide a technology wherein the hydrolyzed product is a sugar containing aqueous material. Another embodiment of the present invention, the hydrolyzed product was fermented by using standard reference strain and ethanol tolerant yeast under anaerobic condition to obtain ethanol. DETAILED DESCRIPTION OF THE INVENTION Acid hydrolysis of banana pseudostem waste Incubation at varying conditions The lignocellulosic feedstock optionally milled was used for further studies. One part of lignocellulosic waste treated with 5-10 parts of mild mineral acid at different concentrations (1 - 25) under pressurized (15 lb) at different temperature (80 - 130 degree C) for 5-150 minutes of residence time. 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 sugar production was regularly increased from initial residence time and above but initiation of maximum sugar release occurred in 8 concentration mild mineral acid and it reduced dramatically in all other concentrations of C2H2O4. Since maximum initiation of sugar release has occurred in even mild mineral acid at diluted concentrations of C2H2O4, 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 total and reducing sugars. The resulting hydrolyzed fractions from dry milled lignocellulosic waste are then subjected to fermentation as described below. The hydrolyzed product was found to be maximum 53.5 percentages. The hydrolyzed aqueous material was fermented by both reference strain and isolated yeast under anaerobic condition. BRIEF DESCRIPTION OF THE FIGURES Figure 1: Schematically illustrate the effect of dosage of acid, thermal conditions and the sugar yield of the process of the present invention on lignocellulosic feedstocks of banana pseudostem waste, 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 banana pseudostem waste 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- banana pseudostem waste 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 cerew's/ae MTCC 170: AB - banana pseudostem acid hydrolysate: A- pH and cell number of little millet hydrolysate fermented broth, B-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 lignocellulosic feedstock was used for the preparation of hydrolysates. 8 concentrations of oxalic acid and banana pseudostem 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 5-150 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 lignocellulosic feedstocks of banana pseudostem waste 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 technology, the ethanol production was preceded by treating the samples with optimized concentrations of mild mineral acid. Two different reference and isolate yeast strains were used as fermenting microorganisms for conversion of lignocellulosic feedstock hydrolyzed product to ethanol. The final ethanol yield from separate hydrolysis and fermentation 18.65g / 100g (w/w) of lignocellulosic feedstock at solid basis and with urea supplementation 22.77g / 100g (w/w) of lignocellulosic feedstock at solid basis. Ethanol fermentation efficiencies were between 69.72 percentage and 84.67 percentage. In this present invention, lignocellulosic based ethanol production was invented to decide the various parameters essential for maximum ethanol recovery. The time for maximum ethanol production was also reduced in urea supplementation from 168 hrs to 72 hrs in banana pseudostem 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 4.5 in both the hydrolysates with and without urea supplementation. The yeast growth also regularly increased initially and decreased in the later part of the process. The yeast cell number was high (42 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 invention the dry milled banana pseudostem samples were used for ethanol production. The first step was the hydrolysis of the banana pseudostem to release maximum amount of sugars. This was done using physical, chemical and thermal reactions in the occurrence of dilute oxalic 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 banana pseudostem hydrolysate was 22.65g / 100g (Figure. 3). Maximum ethanol production was noticed on day 2 in banana pseudostem hydrolysate. The ethanol yield was comparably equal in the native isolate, /. orientalis (MTCC 10641) based fermentation of S. cerevisiae (MTCC 170) based fermentation. The pH was found to regularly reduce from 5.1 to 4.65 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 (14.9 log 5) in the same (Figure. 3A). Selected citations and bibliography Anonymous (2008). Agriculture Statistical Information of Tamil Nadu.Commissionerate of Agriculture, Tamil Nadu, India. Khali), H.P.S.A., Alwani, M.S., Omar, A.K.M. (2006). Chemical composition, anatomy, lignin distribution, and cell wall structure of Malaysian plant waste fibers. Bioresources. 1: 220-232. Saeed, A.A., Qazi, J.I. (2005) Simultaneous saccharification and ethanol fermentation of apples, bananas and potatoes by Issatchenkia orientalis. Mycopath. 3: 73-78. Shafique, S., Asgher, M., Sheikh, M.A., Asad, M.J. (2004). Solid State Fermentation of Banana Stalk for Exoglucanase Production, Int. J. Agri. S/0/.6: 488-491 Sharma, N., Kalra, K.L., Oberoi, H.S., Bansal, S. (2007). Optimization of fermentation parameters for production of ethanol from kinnow waste and banana peels by simultaneous saccharification and fermentation. Ind. J. Microbiol. 47:310-316. Uma, S., Kalpana, S., Sathiamoorthy .S. (2003). Banana Fibre, Niseema printers, Cochin. Zuluaga, R., Putaux , G.L., Restrepo, A., Mondragon, I., Ganan.P. (2007). Cellulose microfibrils from banana farming residues: isolation and characterization. Cellulose. 14: 585-592. What is claimed is: 1. A method of making an ethanol product comprising: hydrolyzing carbohydrates under acidic condition with mild diluted mineral 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 oxalic 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 10 or 100 min. 6. A method of making hydrolyzed product comprising: hydrolyzing banana pseudostem lignocellulose under acidic conditions with mild mineral diluted 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 lignocellulosic feedstock Poovan (var. Mysore AAB); wherein hydrolyzing the lignocellulose is of banana pseudostem. 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 manure.

Full Text

TITLE: A TECHNOLOGY FOR ETHANOL FROM BANANA PSEUDOSTEM
WASTE

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to Indian Provisional Patent Application
No. 387/CHE/2010, filed Feb. 15, 2010, which is incorporated by reference
herein.

FIELD OF INVENTION

The invention relates ethanol production, more specifically method for the
production of ethanol from wasted banana pseudostem which is the finest source
of cellulose and hemicellulose using yeast under anaerobic condition.

BACKROUND OF THE INVENTION

Ethanol has been known for a long time, perhaps the oldest product obtained
through traditional biotechnology. Conventionally, ethanol containing beverages
are used for human consumption. It is also used as the main ingredient in
medical wipes as antibacterial and antiseptics, antifreeze agent, an antidote for
methanol poisoning, disinfectants, extractant, hand sanitizer gels, in the
manufacture of varnishes and perfumes, preservative for biological specimens,
sketch art markers, flavorings and medicines. The demand for ethanol has been
increasing day by day because of its multiple uses. Currently, commercial
ethanol production relies on the fermentation of sucrose from cane sugar and
molasses or glucose derived from starch based crops such as corn, wheat and
cassava. There is a growing need for the industry to improve technology and
enhance production due to high oil prices, environmental pressures and national security concerns.

The global production of plant biomass, of which over 90% is lignocellulose, amounts to about 200X109 tonnes per year, whereas 8-20X109 tonnes of the primary biomass remains potentially inaccessible. Lignocellulose is the major structural component of woody plants and non-woody plants such as grass and represents a major source of the renewable organic matter. Plants consisting of lignocellulose can be divided into three categories according to plant taxonomy: softwood (gymnosperms), hardwood (woody angiosperms) and annual plants (herbaceous angiosperms). Lignocellulosic materials are the most appealing in this respect, due to their abundance and low cost.

Banana is the most widely grown fruit crop in more than 132 countries including India, covering almost 10 million hectares of cropped area. Uma et al., Banana Fibre, (2003), Niseema printers, Cochin and Sharma et al., Ind. J. Microbiol, 47, (2007), 310-316 disclosed that India on an average produces 1.533*107 MT banana / year with 24% of world's bananas production. Zuluaga and co-workers disclosed the cultivation of banana generates a considerable amount of residues, where the fruit constitutes 12 wt% of the plant, and the remaining parts become agricultural waste. Tamil Nadu Government disclosed that banana is cultivated in and around 94 thousand hectares in 14 districts of Tamil Nadu, India (2008) and the estimated production of banana is 4.488* 106 tonnes in the year 2007-2008. Baig er al., African Journal of Biotechnology, 3, (2004), 447-450 and Khalil et al., Bioresources, 1, (2006), 220-232 disclosed that the remaining part of the plant includes leaves, stem and rhizome and after harvesting the fruit left in the field takes several months for degradation. Apart from its multifaceted uses like therapeutics, dye, starch extraction and other innumerable uses, it forms the finest source of cellulose.

Very few reports have been accounted on alcohol production form banana pseudostem. Baig and oo workers, African Journal of Biotechnology, 3, (2004), 447-450 disclosed the sugar production from dried leaves and psuedostem using cellulase produced by Trichoderma lignorum. They disclosed 1.34 mg/ml of reducing sugars from steam treated the agro-waste after 24 h. They also disclosed the size of substrates, pH and temperature to impact the yield. Saeed and Qazi, Mycopath, 3, (2005), 73-78 disclosed the simultaneous saccharification and fermentation of banana by using Issatchenkia orientalis and reported 4.14 % ethanol yield in a single step process. In another report, Shafique and co-worker International Journal of Agriculture and Biology, 6, (2004), 488-491 disclosed the exoglucanase production using Bacillus subtilis on solid-state fermentation (SSF) from banana stalk. They also disclosed maximum exoglucanase activity (3.48 lU/mL/min) from the medium fermented with 70% moisture content, 5 mL inoculum, 0.1% peptone, 0.4% yeast extract and 0.2% Tween-80 at pH 7 and temperature 35 °C. A strong need exists for efficient ethanol production with cost effective raw material and energy consumption.

Therefore it is an embodiment of the present invention is to provide a technology
for producing ethanol from lignocellulosic waste, particularly banana pseudostem
waste.

Another embodiment of the present invention is to offer a technology for
producing ethanol from banana pseudostem waste using dilute oxalic acid to
convert lignocellulose 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 identify the effect of nitrogen
source like urea in the fermentation process and ethanol production.

Yet another embodiment of the present invention is to offer an industrially viable,
less energy utilizing and inexpensive technology for producing ethanol from
banana pseudostem waste.

Still another embodiment of the present invention is to provide a method for
producing ethanol from banana pseudostem 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 offer a technology
for producing ethanol from lignocellulosic waste.

It is further embodiment of the invention to provide a technology wherein the
lignocellulosic waste is a waste product from agriculture sector.

It is further embodiment of the invention to provide a process wherein the
lignocellulosic waste undergone dry milled form.

Yet another embodiment of the present invention, the banana pseudostem waste
containing about 60-70 % cellulose and hemicellulose is converted into
monosaccharides when treated with dilute oxalic acid under pressurized
temperature at 80 degree C to 130 degree C for 5 - 150 minutes wherein the
dilute oxalic acid used between 1 % and 25 %.

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

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

DETAILED DESCRIPTION OF THE INVENTION

Acid hydrolysis of banana pseudostem waste
Incubation at varying conditions

The lignocellulosic feedstock optionally milled was used for further studies. One
part of lignocellulosic waste treated with 5-10 parts of mild mineral acid at
different concentrations (1 - 25) under pressurized (15 lb) at different
temperature (80 - 130 degree C) for 5-150 minutes of residence time. 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 sugar production was regularly increased from initial
residence time and above but initiation of maximum sugar release occurred in 8
concentration mild mineral acid and it reduced dramatically in all other concentrations of C2H2O4. Since maximum initiation of sugar release has occurred in even mild mineral acid at diluted concentrations of C2H2O4, 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 total and reducing sugars. The resulting hydrolyzed fractions from dry milled lignocellulosic waste are then subjected to fermentation as described below. The hydrolyzed product was found to be maximum 53.5 percentages. The hydrolyzed aqueous material was fermented by both reference strain and isolated yeast under anaerobic condition.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Schematically illustrate the effect of dosage of acid, thermal conditions and the sugar yield of the process of the present invention on lignocellulosic feedstocks of banana pseudostem waste, 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 banana pseudostem waste 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- banana pseudostem waste 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 cerew's/ae MTCC 170: AB - banana pseudostem acid hydrolysate: A- pH and cell number of little millet hydrolysate fermented broth, B-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 lignocellulosic feedstock was used for the preparation of hydrolysates. 8 concentrations of oxalic acid and banana pseudostem 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 5-150 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 lignocellulosic feedstocks of banana
pseudostem waste

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 technology, the ethanol production was preceded by treating the samples with optimized concentrations of mild mineral acid. Two different reference and isolate yeast strains were used as fermenting microorganisms for conversion of lignocellulosic feedstock hydrolyzed product to ethanol. The final ethanol yield from separate hydrolysis and fermentation 18.65g / 100g (w/w) of lignocellulosic feedstock at solid basis and with urea supplementation 22.77g / 100g (w/w) of lignocellulosic feedstock at solid basis.

Ethanol fermentation efficiencies were between 69.72 percentage and 84.67 percentage.

In this present invention, lignocellulosic based ethanol production was invented to decide the various parameters essential for maximum ethanol recovery. The time for maximum ethanol production was also reduced in urea supplementation from 168 hrs to 72 hrs in banana pseudostem 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 4.5 in both the hydrolysates with and without urea supplementation. The yeast growth also regularly increased initially and decreased in the later part of the process. The yeast cell number was high (42 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 invention the dry milled banana pseudostem samples were used for ethanol production. The first step was the hydrolysis of the banana pseudostem to release maximum amount of sugars. This was done using physical, chemical and thermal reactions in the occurrence of dilute oxalic 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 banana pseudostem hydrolysate was 22.65g / 100g (Figure. 3). Maximum ethanol production was noticed on day 2 in banana pseudostem hydrolysate. The ethanol yield was comparably equal in the native isolate, /. orientalis (MTCC 10641) based fermentation of S. cerevisiae (MTCC 170) based fermentation. The pH was found to regularly reduce from 5.1 to 4.65 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 (14.9 log 5) in the same (Figure. 3A).

Selected citations and bibliography

Anonymous (2008). Agriculture Statistical Information of Tamil Nadu.Commissionerate of Agriculture, Tamil Nadu, India.

Khali), H.P.S.A., Alwani, M.S., Omar, A.K.M. (2006). Chemical composition, anatomy, lignin distribution, and cell wall structure of Malaysian plant waste fibers. Bioresources. 1: 220-232.

Saeed, A.A., Qazi, J.I. (2005) Simultaneous saccharification and ethanol fermentation of apples, bananas and potatoes by Issatchenkia orientalis. Mycopath. 3: 73-78.

Shafique, S., Asgher, M., Sheikh, M.A., Asad, M.J. (2004). Solid State Fermentation of Banana Stalk for Exoglucanase Production, Int. J. Agri. S/0/.6: 488-491

Sharma, N., Kalra, K.L., Oberoi, H.S., Bansal, S. (2007). Optimization of fermentation parameters for production of ethanol from kinnow waste and banana peels by simultaneous saccharification and fermentation. Ind. J. Microbiol. 47:310-316.

Uma, S., Kalpana, S., Sathiamoorthy .S. (2003). Banana Fibre, Niseema printers, Cochin.
Zuluaga, R., Putaux , G.L., Restrepo, A., Mondragon, I., Ganan.P. (2007). Cellulose microfibrils from banana farming residues: isolation and characterization. Cellulose. 14: 585-592.

What is claimed is:

1. A method of making an ethanol product comprising: hydrolyzing carbohydrates under acidic condition with mild diluted mineral 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 oxalic 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 10 or 100 min.

6. A method of making hydrolyzed product comprising: hydrolyzing banana pseudostem lignocellulose under acidic conditions with mild mineral diluted 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 lignocellulosic feedstock Poovan (var. Mysore AAB); wherein hydrolyzing the lignocellulose is of banana pseudostem.

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 manure.

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=qNrQQ0fUww30oqjDrESwOA==&loc=egcICQiyoj82NGgGrC5ChA==

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Patent Number

278966

Indian Patent Application Number

387/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

SIVASANKARI.S

Applicant Address

49A/6, MAIN ROAD, KATCHIPERUMAL (PO), UDAYARPALAYAM (TK) ARIYALUR DT., PIN - 621 804.

Inventors:

#	Inventor's Name	Inventor's Address
1	SIVASANKARI.S	49A/6, MAIN ROAD, KATCHIPERUMAL (PO), UDAYARPALAYAM (TK) ARIYALUR DT., PIN - 621 804.
2	SIVAGURUNATHAN.P	49A/6, MAIN ROAD, KATCHIPERUMAL (PO), UDAYARPALAYAM (TK) ARIYALUR DT., PIN - 621 804.

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