Title of Invention	METHOD FOR THE PRODUCTION OF ETHANOL FROM MUNTINGIYA CALABURA
Abstract	Disclosed herein is a method for production of ethanol from Muntingiya calabura comprising the steps of; a) fermenting the pulverized fruit extract of Muntingiya calabura in water medium using at least one microorganism at a temperature range of 25 to 35°C over a period ranging 1 to 7 days; and b) isolating the produced ethanol by steam distillation.

Full Text

Technical field: The present invention relates to a cost effective method for the production of alcohol from Muntingiya calabura in good yield and high purity substantially free from methanol contamination. Background and prior art: Bio fuel can be broadly defined as solid, liquid, or gas fuel consisting of, or derived from biomass. This can be produced from any carbon source that can be replenished rapidly e.g. plants. Many different plants and plant-derived materials are used for biofuel manufacture. Biomass can also be used directly for heating or power. One type of biomass is wood, which is frequently used in industry, either by itself to create energy or with other combustible matter such as coal to bum and create heat. Biofuels are used globally and the biofuel industries are expanding in Europe, Asia and in America. The most common use for biofuels is in automotive transport. In the coming decades biomass will play a critical role in sustainable development, serving as our only renewable resource for carbon-based chemicals, and providing a practical alternative to petrochemical transportation fuels. The utilization of ethanol as oxygenate is the prime need of the country, because, the enormous increase in the number of motor vehicles has been a major cause of air pollution, particularly in Metro Politan cities and big cities. The best way to solve this objective is to utilize ethanol as oxygenate in admixture with petrol/diesel. The distilleries producing alcohol will see a great increase in future as alcohol serves as a basic chemical for a large number of chemical industries. Even the byproducts of alcohol industry i.e. dry ice, acetic acid, butanol, ether, etc. and the alcoholic mash as a valuable fodder (due to its high content of proteins and vitamins) also are important for future needs of country. Inspite of the importance of alcohol, the alcohol industry is showing a decline trend due to the high prices of molasses. The alcohol produced is now utilized in the ratio of 52% for potable liquor and the balance 48% for industrial use. Billions of liters of ethanol is produced annually mostly in Brazil to U.S.A. In our country only Jatropha was considered for Biofuel production for the past few years. But the national consultation of people's moments and NGO's here warned the governments across the country against aggressive promotion of Biofuel crops. (THE HINDU, Dt. 7/12/2007). Recently the petroleum minister stressed the EBP programme (National programme for supply of ethanol blended petrol) of petrol with ethanol launched on November 1^' 2006 (5% blending) is aimed at contributing to energy security through use of alternative fuel sources which also helps the farmers to earn additional income. This initiative is environmental friendly as the oxygenate nature of ethanol ensures greater petrol combustion leading to lesser emission of pollutants. Many different plants and plant derived materials are used for biofuel manufacture. Agricultural products specifically grown for biofuel production include com (12% alcohol) and soybeans primarily in U.S., Rapeseed in Brazil and Jatropha in India. In India, the technology has evolved for processing tropical sugar beet into alcohol, including fuel alcohol by bypassing the production of sugar in the first stage. Therefore, there is a need in the art to develop an alternative process to produce ethanol. Accordingly, the inventor of the present invention have come up with the production of alcohol from Muntingiya Calabura, which is found to be very promising using different microorganism. These are small trees, brenchlets, densely villous, glandular, slightly visical, Leaves oblog, Lanceolate, 3-5 nemed, villous above, wooly below, serrate, acuminate, stipules linear. (Ref: Flowering plants of Chittoor district, AP, India, by Dr. K. Madhva Chetty, K. Sivaji and K. Tulasi Rao, Page No.51-2008), They can be grown in Arid and semi arid zones, dry lands, poor soil, wall crevices, etc. Muntingia calabura, the sole species in the genus Muntingia, is a flowering plant native to southern Mexico, the Caribbean, Central America, and western South America south to Peru and Bolivia. Common names include (English) Jamaican cherry, Panama berry, Singapore cherry, Strawberry tree; (Spanish) bolaina yamanaza, cacaniqua, capulin bianco, nigua, niguito; and (Filipino) aratilis, aratiles, manzanitas. Locally it is referred to as KOMPE tree, rainbow tree and umbrella tree. The plant Muntingiya Calabura belonging to Elaeocarpaces family is generally known in India also as Nakka Regu, Kukka Mogi, Indian cherry, Kompe Chettu etc. Muntingia calabura is a small tree 7-12 meters tall with tiered and slightly drooping branches. The leaves are serrated, evergreen, alternate, lanceolate or oblong 2.5-15 cm long and 1-6.5 cm wide & the flowers borne singly or in 2's or 3's in the leaf axils. The flowers are white, in paler or solitary. Sepals 5, Lanceolati: petals 5, ovate, disc - annular stamens numerous, insuted on the disc. Ovary - half inferior ovules numerous per locale, axile; stigma -sessile, grooned. The flowers are small and white and give rise to 1-1.5 cm light red fruit. The fruit is round(l-1.25 cms), wide, edible, sweet and juicy, and contains a large number of tiny yellow seeds, too fine to be noticed in eating. Fruits are available throughout the year; especially they are more in summer. In India, this plant is distributed throughout the Andhra Pradesh Sate. Abundant in and around Visakhapatnam, Araku and Narsipatnam areas, Tirumala gardens and hills of Tirupathi, S. V. U. Campus, A. U. Campus and Puttaparti etc.. It is a pioneer species that thrives in poor soil, able to tolerate acidic and alkaline conditions and drought. Its seeds are dispersed by birds and fruit bats. It is cultivated for its edible fruit, and has become naturalised in some other parts of the tropics, including southeastern Asia. As a pioneer plant, it could help condition the soil and make it habitable to other plants. It can also be considered as an invasive species. In many countries, the fruits are eaten and sold in markets and also can be processed into jams and the leaves can be used for making tea. In some countries, the trees are planted along river banks. The fruits falling from the tree attract fish that are then caught. In traditional medicine, its flowers can be used as an antiseptic and treat abdominal cramps. The timber from this plant Is reddish-brown. It is compact, durable and lightweight and can be used for carpentry and also can be used as firewood. The bark can be used to produce ropes. Due to its ability to grow in poor soil and its effective propagation by means of bats and birds, it could be used for reforestation projects. In India it is used to grow to attract small fruit eating birds such as the flowerpeckers. It is commonly planted in parking lots. It is also observed growing in wall crevices, hilly areas and rocky soils. Muntingia calabura is a tropical fast growing tree of the slender proportious with spreading horizontal branches belonging to family elaeocarpaceae , abundantly present in and around Visakhaptnam,vizianagaram,srikakulam districts and especially in the agency areas of Araku and Narsipatnam. This produces abundant fruits, throughout the year which are known as the Indian cherries. The fruits are round, smooth, yellow or light brown; contain softy juicy pulp, with fig like flavor filled with. Locally they are called panchadara pandlu. Unlike the energy crops (cereals, sugar cane and beet etc) Muntingiya calabura have no direct food use. This plant is renewable resource yet be fully exploited Although, the growth oiMuntingiya calabura plant is abundant, there are no patents/ literature available on the subject of the present invention. Further, none of the skilled person in the art has studied about this plant and its commercial value/uses. Sugar beet has a growth cycle of 5 months to produce beet and for sugar cane it took 12-months to produce sugar cane, whereas the abundant production of fruits throughout the year, makes Muntingiya calabura as a selective choice of plant to attempt to produce ethanol from the said fruits thus paving the way to meet the global demand in production of biodiesel. Moreover, the production of Pthanol from Muntingiya is cost effective as it can grow in poor soils; and also not incur into shortage of food produce as many people donot consider it as an edible fruit. Therefore, the present invention is an attempt to produce ethanol from the cheaper source i.e. the fruits of Muntingiya in a simple and economical method. Objective of the invention: It is an object of an invention to provide novel source of bio diesel from Muntingiya calabura. Another objective of the invention is to provide a cost effective method for the production of bio diesel from Muntingiya calabura in good yield and high purity substantially free from methanol contamination. Summary of the invention; In accordance with the above objectives, a cost-effective and efficient method for production of ethanol from Muntingiya calabura is disclosed. Accordingly, in a preferred aspect, a method for production of ethanol from Muntingiya calabura comprising the steps of a) fermenting the pulverized fruit extract of Muntingiya calabura in water medium using at least one microorganism at a temperature range of 25 to 35°C over a period ranging I to 7 days; and b) isolating the produced ethanol by steam distillation. The microorganism according to the invention is selected from yeast, bacterial and fungal species The inventive method utilizes solid state fermentation technique. The fermentation method as claimed in claim 1, wherein, said fermentation is carried out at a pHof5. Description of drawings: Fig 1 depicts the flowereing plant oMimtingiya calabura Fig 2 depicts tiie Muntingiya calabura bearing fruits Fig 3 depicts gas ciiromatogram of tlie sample witli Zymomonas ff7oZ>/V/j(Puttaparthi)- 98.85% Fig 4 depicts gas ciiromatogram of the sample with Zymomonas mobilis(Tmat[)-91 .\6% Fig 5 shows gas depicts chromatogram cf the sample with yeast (Visakhapatnam)-88.85% This revealed that the percentage of alcohol does not alter with the conditions in which the plants were grown, but it will be more if the sugar content is more, that is, fully ripened fruits with high sugar content, yield more alcohol. Detailed description: The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated. Accordingly, in a preferred aspect, the invention provides an alternative cheaper source for ethanol from Muntingiya calabura plant. The production of ethanol from the said plant utilizes solid state fermentation method. This solid-state fermentation uses a combination of microbial and enzymatic activity to hydrolyze structural carbohydrates, to produce simple sugars and organic acids that can be recovered and converted to high value products. Thus according to preferred embodiment, the production method for ethanol which comprises a step of fermenting pulverized fruit extract using yeast in water medium at SS^C over a period of one week. The alcohol produced is tested and estimated exactly after 7 days using titrimetric method. Materials and methods: Materials and methods 1) Fruits of Muntingiya Calabura 2) Dry yeast 3) Penicillin and Candida utilis 4) Mucor sps 5) Zymomonas mobilis 6) Inorganic phosphate 7) Tween 80 Method for production of ethanel: In a preferred embodiment. Solid state fermentation was carried out in 500 ml conical flaks each containing 150 g of pulverized fruit extract, 2 g of yeast and 300 ml of water at 35 degrees for one week in an orbital shaker with constant stirring. The experiment was carried out several times (10 times), each time exactly adding double the amount of water (w/v for eg- 250 gms of fruits ground in 500mml of water) and proportionate quantity of dry yeast. The filtered crude extract was subjected to steam distillation to isolate the alcohol. The distilled sample was subjected to presumptive testes and both qualitative and quantitative analysis and found that the process of the present invention results in 80 to 90% of ethanol. The water content in ethanol can be further eliminated using various techniques such as using molecular sieves, ion -exchange resin as an absorbent and the like. Conway Micro diffusion test for alcohol was carried out to know the presence of ethanol. Chromo tropic acid test was conducted to overrule the presence of methanol contamination. Titrimetric analysis (dichromate method) was carried out to know the amount of ethanol present in distilled extract taking pure ethanol as standard. Esterification test and Iodoform test conducted for the samples shown positive results Further, GC of the test sample was confirmed the presence of ethanol as well as the absence of higher alcohols contamination. In another preferred embodiment, the solid state fermentation was carried out not only with yeast but with several other types of bacteria and fungi, such as Penicillin; Candida; Mucor sps; Clostridim and Zymomonas mobilis. Among all these organisms, Zymomonas mobilis was extensively studied as it can tolerate high concentrations of ethanol and acid in the media. The culture obtained from IMTECH, Chandigarh was initially transferred to potato -dextrose medium and after getting profuse growth, it was transferred into nutrient broth with little biotics and kept in the orbital shakes for 24 hours at 28°C. This broth was tiirther utilized for fixing various parameters for the maximum production of ethanol using the fruits of Muntingiya Calabura at 28°C temperature in a medium having a pH 5 for 5 days of incubation period were found to be effective for maximum production. Tween 80 has not altered the production to greater extent. Inorganic phosphate caused unusual foaming during distillation. 50% dilution (w/v i.e. double the amount of water 250g fruit pulp+ 500ml water) was found to be ideal with both the organisms Saccharomyces cerevesial and Zymomonas mobilis. The review of literature revealed that Zymomonas is an unusual bacteria and it ferments glucose anaerobically by Enter - Doudoroff pathway. It is facultatively anaerobic. Chemoorganotrophic, growing on and fermenting Imol of glucose or fructose to 2mol of ethanol. The experiments conducted extensively with different samples collected from different areas using both the species, i.e. Saccharomyces cerevesial and Zymomonas mobilis revealed that the sample produced by distillation contains nearly 80 - 90% ethyl alcohol. Sample from Puttaparthy Sample from Tirupathi Sample from Visakhapatnam The percentage of alcohol was found to vary with the color and ripening state of the fruits. The fruits were analyzed for the total amounts of sugars and the percentage of glucose, fructose and sucrose by standard methods according to Indian pharmacopeia and the results are given below % Total sugars 10.3% % Total Glucose 1,1% % Total Fructose 5.5% % Total Sucrose 3.7% Taking into consideration all these facts and purity of the sample; we can say that nearly 50-55 % of sugars are being converted into pure ethyl alcohol , and the remaining part yields very little amount of methanol and other higher alcohols and c02. Further, the fruit samples from different places of the Andhra Pradesh state were collected and utilized for alcohol production The sediment or the waste can be utilized for the production of vinegar using either chemical or enzymatic methods The optimum conditions of temperature, pH, substrate concentration, effect of inorganic phosphate, effect of surfactants were studied extensively using the same tltrimetric estimation. Other microbial strains like pcnicillium and Candida were not much effective in ethanol production. Nearly 1% sugar concentration, 4 to 6 medium pH, 25°C to 35°C temperature range and 0.3%) of inorganic phosphate concentration were found to be the optimum conditions for the production of alcohol. The process of fermentation became rapid after 48hrs and was found to be maximum after 3days. Inorganic phosphate was added to the media to study Harden and Young effect. But it was found to cause extensive foaming during distillation. 0.1% Tween 80 was found to be more effective as the surfactant in hastening the production process. Addition of inorganic phosphate enhanced the production and reduced the production time, but it caused severe foaming during the distillation process. However, the particular concentration of 0.3% inorganic phosphate was found to be optimum to achieve the targeted results. Though industrially several organisms were used to produce alcohol, they are specific to the sugars present in the media. In addition to the yeast Saccharomycea cerevesiae, the bacterial species Zymomonas mobilis is extensively used industrially for alcohol production. Since yeast is the cheapest source we preferred it. However Initial experiments with Z.mobilis were going on with the same substrate to see the feasibility and cost effectiveness of the process. The Zymomonas mobilis is an alcohol tolerant microorganism, which is potentially useful for the commercial production of ethanol. It is advisable while selecting the microorganism for fermentation process in production of ethanol, whether the microorganism is tolerant to ethanol or not. If the organism is not ethanol tolerant and is ethanol sensitive, then the same will reflects on the total conversion of sugars to alcohols. Therefore, the selection of microorganism is important in fermentation of sugars and the said organism should not be sensitive to high concentrations of ethanol. Other bacterial and fungal species like Thermobanaerobacter ethanolicus, Candida pseudotropicalis, , Pachysoleu tannophilus and Fusarium species were not preferred for fermentation, since they require high content of xylose in the medium. The sediment or the waste can be utilised for the production of vinegar using either chemical or enzymatic methods.This was also confirmed by qualitative analysis. Therefore, the extensive research involved in the present invention and screening of these products (ethanol as well as acetic acid) may change India's goal to achieve energy independence in the coming decades. The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention. Examples Example 1 Charge250 grams with 500ml water of pulverized fruit extract (sample collected from Puttaparthy and SO ml broth of zymomonas mobilis into a 500 ml conical flask. Solid state fermentation was carried out at 35° C for one week in an orbital shaker with constant stirring. After the completion of reaction, the extract was subjected to steam distillation to isolate the alcohol. Yield: 51 % on the basis of % sugar converted purely to ethyl alcohol. Purity: 98.85% by GC Methanol: 0.41% Isopropanol: Not detected Example 2: Charge 500 grams of pulverized fruit extract in 1000 ml of water and (sample collected from Tirupati) 100 ml of broth of zymomonas mobilis and into a 2 It. conical flask. Solid state fermentation was carried out at 35° C for one week in an orbital shaker with constant stirring. After the completion of reaction, the extract was subjected to steam distillation to isolate the alcohol. Yield: Yield: 49.6% on the basis of % sugar converted into alcohol. Purity: 97.16% by GC Methanol: 1.36% Isopropanol: Not detected Example 3: Charge 150 grams of pulverized fruit extract(sample collected from Vishakhapatnam) 2 grams of yeast and 300 ml of water into a 500 ml conical flask. Solid state fermentation was carried out at 35° C for one week in an orbital shaker with constant stirring. After the completion of reaction, the extract was subjected to steam distillation to isolate the alcohol. Yield: Yield: 50.5% on the basis of % sugar converted into alcohol. Purity: 88.85% by GC Methanol: 1.34% Isopropanol: Not detected I claim, 1. A method for production of ethanol from Mimtingiya calahura comprising the steps of a) fermenting the pulverized fruit extract of Muntingiya calabum in water medium using at least one microorganism at a temperature range of 25 to 35°C over a period ranging 1 to 7 days; and b) isolating the produced ethanol by steam distillation. 2. The method as claimed in claim 1, wherein said microorganism is selected from yeast, bacterial and fungal species. 3. The method as claimed in claim 2, wherein said microorganism is selected from bacterial species like Zymomonas mobilis Clostridium thermohydrosulfnricum; closlridiiim therniocelhim: and yeasts such as Sacharomyces cerevesia, pichia slipitis; fimgal species sucfi as Candida tropicalis, Candida species, and kluyveromyces laclis. 4. The method as claimed in claim I, wherein said method is solid state fermentation method. 5. The method as claimed in claim I, wherein said fermentation is carried out at a pH of 5. 6. The method for production of ethanol from Muntingiya calahura as substantially described herein in the accompanying description.

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452-CHE-2008 AMENDED CLAIMS 25-02-2013.pdf

452-CHE-2008 AMENDED PAGES OF SPECIFICATION 25-02-2013.pdf

452-CHE-2008 EXAMINATION REPORT REPLY RECEIVED 25-02-2013.pdf

452-CHE-2008 FORM-1 25-02-2013.pdf

452-CHE-2008 CORRESPONDENCE OTHERS 11-04-2014.pdf

452-CHE-2008 FORM-2 19-02-2009.pdf

452-CHE-2008 ABSTRACT 19-02-2009.pdf

452-CHE-2008 CLAIMS 19-02-2009.pdf

452-CHE-2008 CORRESPONDENCE OTHERS 19-02-2009.pdf

452-CHE-2008 CORRESPONDENCE OTHERS 22-02-2008.pdf

452-CHE-2008 DESCRIPTION (COMPLETE) 19-02-2009.pdf

452-CHE-2008 DESCRIPTION (PROVISIONAL) 22-02-2008.pdf

452-CHE-2008 DRAWINGS 19-02-2009.pdf

452-CHE-2008 FORM -1 22-02-2008.pdf

452-CHE-2008 FORM -1 26-03-2008.pdf

452-CHE-2008 FORM -18 25-02-2009.pdf

452-CHE-2008 FORM -3 22-02-2008.pdf

452-CHE-2008 FORM-5 19-02-2009.pdf

452-CHE-2008 POWER OF ATTORNEY 19-02-2009.pdf