FORM - 2
THE PATENTS A C T, 1970 (39 OF 1970)
COMPLETE SPECIFICATION
(SEC-10, RULE 13)
PATENT APPLICATION NO. 1912/MUM/2009 "ETHANOL PRODUCTION FROM SOME CULINARY FRUITS"
ABELLON CLEANENERGY LIMITED,
10th Floor, Sangeeta Complex,
Nr. Parimal Crossing, Ellisbridge,
Ahmedabad-380 006, Gujarat State, India,

Title: Ethanol production from some culinary fruits. Background of the invention
The Govt, of India Ministry of Petroleum and Natural Gas, 'Gasohol Program' of blending 5% ethanol in petrol has given an assured scope for ethanol industry in the country. For 5% blend in gasoline, requirement on all India basis is 500 million liters per annum (http://www.ethanolindia.net/ethanol_demand.htm). The present invention relates to production of Ethanol from culinary fruits like Musa sp., and Carica sp. Ethanol fuel is ethanol (ethyl alcohol), the same type of alcohol found in alcoholic beverages. It can be used as a fuel, mainly as a biofuel alternative to gasoline. Anhydrous ethanol (ethanol with less than 1% water) can be blended with gasoline in varying quantities up to pure ethanol (El00), and most modern gasoline engines will operate well with mixtures of 10% ethanol (El 0).
Ethanol production through fermentation may provide an economical competitive source of energy by its incorporation in to gasoline. Among a crucial factor affecting ethanol fermentation, culture conditions play significant role on growth of yeast as well as ethanol production (Naresh Sharma, etal Indian J. microbiol. December 2007, 47: 310-316).
The increasing demand for ethanol for various industrial purposes such as alternative source of energy, industrial solvents, cleansing agents and preservatives, has necessitated increased production of this alcohol. Ethanol production is usually accomplished by chemical

synthesis of petrochemical substrates and microbial conversion of carbohydrates present in agricultural products. Owing to the depleting reserves and competing industrial needs of petrochemical feed stocks, there is global emphasis in ethanol production by fermentation process. Increased yield of ethanol production by microbial fermentation depends on the use of ideal microbial strain, appropriate fermentation substrate and suitable process technology,
Corn, sweet sorghum, sugar beet, sugarcane and cassava are among the several alternative sources of plant materials from which bioethanol is produced today. Ethanol or ethyl alcohol is a clear colourless liquid, biodegradable, low in toxicity and causes very little environmental pollution when burnt. Ethanol is a high octane fuel and has replaced lead as an octane enhancer in petrol. With an octane rating of 129, compared to about 91 for fossil-petrol, engines fired on ethanol can run at a much higher compression ratio without the octane-boosting additives. Bioethanol-blended fuel burns more completely because the ethanol molecule contains oxygen, the result, carbon-dioxide and carbon-monoxide emissions can be reduced by nearly 90% as compared to engines run exclusively on fossil-fuels. Vehicle engines require no modifications to run on 10 % blends of bioethanol (E10) nor are vehicle warranties affected either. Flexible fuel vehicles can run on 85% ethanol and 15% petrol blends (E85) (http://www.archeangroup.com/greenergy/bioethanol.html,).
Banana is the common name for a type of fruit and also the herbaceous plants of the genus Musa which produce this commonly eaten fruit. They are native to the tropical region of Southeast Asia. Bananas are likely to have been first domesticated in Papua New Guinea.

Today, they are cultivated throughout the tropics. Banana plants are of the family Musaceae. They are cultivated primarily for their fruit, and to a lesser extent for the production of fiber and as ornamental plants. As the banana plants are normally tall and fairly sturdy they are often mistaken for trees, but their main or upright stem is actually a pseudostem (literally "fake stem"). For some species this pseudostem can reach a height of up to 2—8 m, with leaves of up to 3.5 m in length. Each pseudostem can produce a bunch of yellow, green or even red bananas before dying and being replaced by another pseudostem (http://en.wikipedia.org/wiki/Banana, visited on 18th May 2009).
Banana is the most important fruit crop of India having great socio-economic significance. It contributed 31% of the total food production in India. World production of banana is estimated at 48.9 million tonnes out of which 10.4 million tonnes, is contributed by India. India ranks No.l in production followed by Brazil (5.5 million tonnes), Indonesia (2.3 million tonnes), Philippines (3.8 million tonnes), China (1.9 million tonnes) and Australia (1.8 million tonnes). In India, Maharashtra state is the largest banana producing state followed by Tamil Nadu, Gujarat, Assam, Andhra Pradesh and others. In Tamil Nadu banana is cultivated in 83,308 ha with an annual production of 2,782 million tonnes. The productivity in Tamil Nadu is 33.39 tonnes/ha compared to 38.1 tonnes/ha in Gujarat (http://www.indiaagronet.com/indiaagronet/Market_upQ7banana.html
Papaya (Carica papaya) is a large tree-like plant, the single stem growing from 5 to 10 meters tall, with spirally arranged leaves confined to the top of the trunk; the lower trunk is

conspicuously scarred where leaves and fruit were borne. The leaves are large, 50-70 cm diameter, deeply palmately lobed with 7 lobes ( http://en.wikipedia.org/wiki/Papaya, visited on 26th May , 2009). The papaya is a short-lived, fast-growing, woody, large herb, it generally branches only when injured. All parts contain latex. The hollow green or deep purple trunk is straight and cylindrical with prominent leaf scars ( http://www.crfg.org/pubs/ff/papaya.html).
Papaya cultivation had its origin in South Mexico and Costa Rica. Total annual world production is estimated at 6 million tonnes of fruits. India leads the world in papaya production with an annual output of about 3 million tonnes. Other leading producers are Brazil, Mexico, Nigeria, Indonesia, China, Peru, Thailand and Philippines. The fruit being perishable in nature poses problem in marketing. Development of infrastructure facilities for transport to primary markets, standardization of packaging techniques are aspects which need special attention. Processing facilities also need to be created in the major producing states for value addition. (http://nhb.gov.in/Horticulture%20Crops°/o5CPapaya%5CPapayal.htm). Papaya being a tropical fruit grows well in the mild sub-tropical regions of the country up to
1,000 m. above sea level. Night temperature below 12° to l4°C for several hours during winter season affects its growth and production severely. It is very much sensitive to frost, strong winds and water stagnation. Deep, well drained sandy loam soil is ideal for cultivation of papaya.
The area under papaya cultivation in India increased by 63% from 45.2 thousand hectare in 1991-92 to 73.7 thousand hectare in 2001-02 and the production increased from

800000 tonnes to 2600000 tonnes, as per data from National Horticulture Board, Ministry of Agriculture, Govt, of India. Papaya is mostly cultivated in the states of Andhra Pradesh, Karnataka, Gujarat, Orissa, West Bengal, Assam, Kerala, Madhya Pradesh and Maharashtra in India.
Caricaceae is a family of flowering plants in the order Brassicales, The family comprises five genera such as Carica, Cylicomorpha, Jacaratia, Jarilla, and Vasconcellea. In India papaya varieties cultivated in different states are Honey Dew, Coorg Honey Dew, Washington, Solo, Co-1, Co-2, Co-3, Sunrise Solo, Taiwan, Ranchi selection, Pusa Delicious & Pusa Nanha, Coorg Green.
It is desirable to produced bioethanol from fruit of Musa Sp. and Carica sp. pulp, for use as a liquid fuel.
Object of the Invention
The main object of the present invention is to produce fuel grade bioethanol, from fermentation of fruit of Musa Sp. and Carica sp. pulp, by suitable micro organism such as but not limited to yeast. The yeast could be any suitable alcohol producing strain such as but not limited to Saccharomyces cerevisiae, Schizosaccharomyces pombe or indigenous yeast present in respective fruit pulp.
Further object of the invention is to produced bioethanol, a liquid fuel, which is used in blending with petrol and used in transport sector.

Detail Description of invention
In the present invention production of bioethanol as liquid bio-fuel is done from fruit of Musa Sp. and Carica sp. pulp.
The Musaceae are large, often treelike perennial herbs comprising two genera and about fourty five species. Fruit pulp of banana is used for fermentation from Musa sp. Such as but not limited to Musa velutina, Musa paradisiaca ssp, Musa rosea, Musa troglodytarum , Musa alinsanaya, Musa bauensis, Musa boman, Musa bukensis , Musa exotica.
In the present invention fruits of Musa Sp. And Carica sp. fresh, rotton, or ripe are used for production of bioethanol.
The embodiment of the invention are directed to use of fruits, either freshly collected or stored, processed, unprocessed, including all wild, cultivated, sub-varieties, cultivars, hybrids and genetically modified species of Musa Sp. and Carica sp..
Another embodiment of the invention is to hydrolyze starch of unripe Musa Sp. and Carica sp. fruits into fermentable sugars by suitable process such as but not limited to acid hydrolysis using sulphuric acid or hydrochloric acid, enzymatic hydrolysis using enzymes such as amylase as such or in combination with pectinase and cellulase.
Another embodiment of the invention is to produce bioethanol by either solid state fermentation or submerged fermentation carried out in batch, fed batch, or continuous mode.

Yet another embodiment of the invention is to produce bioethanol by fermentation of unripe fruits of Musa Sp. using combination of microorganism such as amylase producer fungus or bacteria and ethanol producer yeast.
Yet another embodiment of the invention is to produce bioethanol by fermentation of fermentable sugars of Musa Sp. and Carica sp. by indigenous isolated yeast from respective fruit pulp. Such a yeast can be adapted to high rate of fermentation by serial cultivation in dilute pulp or juice of respective fruits.
Further such embodiment of the invention is to separate solid mass of fermented hydrolyze starch sugars or fermented pulp sugar to recover liquid fraction containing ethanol and solid residue by suitable separation process such as filtration or centrifugation. Ethanol present in liquid fraction is further concentrated by distillation.
The main process steps of the present invention may in one embodiment be described as separated into the following main process stages: (a) Chopping (b) Slurry formation (c) Yeast inoculation (d) fermentation (e) filtration (f) distillation. The individual process steps of alcohol production may be performed batch wise or as a continuous flow. For the invention processes where all process steps are performed batch wise, or processes where all process steps are performed as a continuous flow, or processes where one or more process step(s) is(are) performed batch wise and one or more process step(s) is(are) performed as a continuous flow, are equally preferred.

In a preferred embodiment of the process of the invention, the sugar containing materialis Musa Sp. and Carica sp, and the process comprises a step of chopping the fruits before step (b), in other words, the invention also encompasses processes of the invention, wherein the sugar containing material is obtainable by a process comprising chopping of fruit, preferably manual cutting by knife or electrical chopper. Seeds were removed from Carica sp. By pasing through vibrating sieves of 2 - 5 mm. The chopped peaces of fruit used in the remaining part of the process.
The slurry comprising the pulp sugar containing material and distilled water. The distilled water may be heated to a suitable temperature prior to being combined with the sugar containing material in order to achieve a mash temperature of 60 to 70 °C, preferably of 50 to 60°C. Typically the wet solids % in the slurry tank (containing pulp of fruit) is in the range from 25 - 90%.
The cooled slurry comprising the mesocarp sugar containing material and distilled water is inoculating with appropriate micro organism, preferably yeast, more preferably Saccharomyces cerevisiae, Pichia stipitis. The slurry containing fermenting organism 0.5 % to 25 % w/w, more preferably 1 to 5 % w/w. Suitable nutrient source was added to slurry for proper growth of yeast, preferably substance containing Nirogen, more specifically Urea, DAP and MgSO4. The nutrient percentage in slurry is 0.0001 % to 0.1 %, preferably 0.001 % -0.010%.

The pH of slurry comprising the mesocarp sugar containing material, distilled water, fermenting organism and nutrient is preferably set to 4 to 6.5 pH, more specifically 4.5 to 4.9 pH. Fermentation temperature was set to constant temperature 26 to 30°C, more specifically 29°C, The fermentation is ongoing until the desired amount of fermentation product is produced; in a preferred embodiment wherein the fermentation product is ethanol to be recovered this may, e.g., befor 24-96 hours, such as 48-96 hours for Musa sp. and 24 - 48 hours for Carica sp. Another embodiment of the invention is to produce ethanol by either solid state fermentation or submerged fermentation carried out in batch, fed batch, or continuous mode.
In another embodiment wherein the fermented broth separated into solid and liquid by centrifugation, or Alteration by cloth and followed by centrifugation In such embodiment filtration clothe could be cotton cloth, plastic net, steel net, of 100 - 500 mesh size, more preferably 150 -300 mesh size.
The process of the invention may further comprise recovering of the fermentation product, i.e. ethanol; hence the alcohol may be separated from the filtrate and purified. Distillation temperature was increase by rate of 2 °C/min and distilled liquid called ethanol collected.
In embodiments wherein the fermentation product is ethanol, the ethanol obtained by the process of the invention may be recovered from the fermented filtrate and used as, e.g., fuel

ethanol; drinking ethanol, i.e., potable neutral spirits, or industrial ethanol, including fuel
additive.
Example 1: Indigenous yeast isolation from Musa Sp. and Carica sp.
Naturally ripe Musa Sp. and Carica sp. were used as sample for yeast isolation. Surface of both pods were sterilized by 0.001% solution of HgCl2 for five minutes followed by continuous wash with sterile distilled water. Remove the skin by sterile blade and transfer approx one gram in 10 ml of sterilized water separately for each fruit. Crush and allow it to settle for 15 minutes, clear solution was used as sample for yest isolation by method of Ameh et al. (1989). The last two dilutions were plated in duplicates using pour plate technique on potatoto dextrose aga (PDA), as described by Madigan et al. (1997). Confirmation of isolate as yeast were done by plating isolate on RBC (rosebengal chloramphenicol) agar plate. Results for presence /absence of yeast are shown in table -1. Table 1: Isolation of indiginous yeast from Musa sp. and Carica sp

Sr.
No Medium used for Isolation Musa sp. Carica sp
1 PDA Positive Positive
2 RBC Positive Positive
Example 2: Revival of Saccharomyces cerevisiae
Fermented broth containing Saccharomyces cerevisiae was collected from sugar factory
Mahua. Culture suspension was prepared using sterile d/w and loop full culture was streak on

YPD agar plates (table 3). Plates were incubated under 28 ± 1°C, and kept till proper growth appeared. From this plate Isolated colony was picked up and transferred to liquid YPD medium and incubated under shaking (60 rpm) at 28 ± 1°C. After 72 h cells were harvested by centrifugation and used in fermentation. Table 3: YPD medium composition

Medium component Amount
Yeast extract l.0g
Peptone 2.0 g
Glucose 2.0 g.
Distilled water 100 ml
Agar agar powder 1.2 g
pH 6.5-6.7
Example 3: Fermentation of Musa sp.
Ripe/rotten fruits were collected from fruit market. Remove the peel and chopped in to small pieces . Banana (500 g) was made to slurry by addition of distilled water (120 ml). 10 mg each of urea, DAP (di ammonium phosphate) and MgS04 were added to support yeast growth and pH was adjusted to 4.5 Slurry was poured into two liter capacity glass flask with went facility. Whole flask was sterilized at 121°C for 10 min in autoclave. After cooling, flask was inoculated with 1 g yeast cells, mixed it properly and incubated under static condition at 29°C temp. Fermentation process was monitored by estimation of the sugar and ethanol (Table 4).

After completion of fermentation, bioethanolwas recovered by distillation. Purity of distilled bioethanol was confirmed with gas chromatogram (GC).
Table 4: Fermentation of pulp of Musa sp.

Fermentation parameter Batch I Batch II Batch III
Pulp 500 g 500 g 500 g
Water 120m 120ml 120ml
Nutrients Urea= 10 mg Urea =10 mg Urea = 10 mg

DAP=10mg DAP = 10 mg, DAP=10mg

MgSO4 = I0mg MgSO4 = 10mg MgS04= 10 mg
Yeast type Indigenous Yeast Sacchcromyces cerevisiae Pichia stipitis
Amount of yeast (w/w) 1 gm 1 gm 1 gm
PH of fermentation broth 5.0 5.0 5.0
Incubation temp 29° C 29° C 29° C
Batch type Static Static Static
Sugar at 0 br 15% 15 15
Alcohol at 0 hr 0.0 0.0 0.0
Sugar at 24 hr 14.58 10.44 13.14
Alcohol at 24 hr 0.0 0.0 0.0
Sugar at 48 hr 9.8 8 8.89
Alcohol at 48 hr 8.44 5.99 8.5

Sugar at 72 hr 9.0 ~3 -3
Alcohol at 72 hr 8.6 5.95 6.3
Sugar at 96 hr 6.35 NA NA
Alcohol at 96 hr 8.0 NA NA
Volume recovered by distillation 5 ml 5ml 5ml
Purity by GC 85% 85% 87%
Example 4: Fermentation of Carica sp pulp
Ripe fruits of carica were collected from the fruit market of ahmedabad. Remove the peel and chopped fruit in to small pieces. Weight (300 g) of fruit pulp was made to slurry by addition of distilled water (750 ml). 10 mg each of urea, DAP (di ammonium phosphate) and MgSO4 were added to support yeast growth and pH was adjusted to 4.5 Slurry was poured into two liter capacity glass flask with went facility. Whole flask was sterilized at 121°C for 10 min in autoclave. After cooling, flask was inoculated with 1 g yeast cells, mixed it properly and incubated under static condition at 29°C temp. Fermentation process was monitored by estimation of the sugar and ethanol (Table 4). After completion of fermentation, bioethanol was recovered by distillation. Purity of distilled bioethanol was confirmed with gas chromatogram (GC). Table 4: Fermentation of Carica sp. pulp.

Fermentation parameter Batch I Batch D Batch HI
Mesocarp 400 g 400 g 400 g
Water 50 ml 50 ml 50 ml
Nutrients Urea = 10 mg Urea = 10 mg Urea =10 mg

DAP=10mg DAP=10mg DAP=10mg

MgSO4 = 10mg MgSO4 = 10mg MgSO4=10mg
Yeast type Indigenous Yeast Saccharomyces cerevisiae Pichia stipitis
Amount of yeast (w/w) 1 gm 1 gm 1 gm
PH of fermentation broth 5.6 5.6 5.6
Incubation temp 29° C 29° C 29° C
Batch type Static Static Static
Sugar at 0 hr 6.81 6.81 6.81
Alcohol at 0 hr - 0.0 0.0
Sugar at 24 hr 6.2 5.6 6.2
Alcohol at 24 hr 4.55 4.4 5
Sugar at 48 hr 3.56 <2 2.7
Alcohol at 48 hr 3.9 4.5 5.7
Volume recovered by distillation 7ml 7 ml 7ml
Purify by GC 78 % 85 % 80%

We Claim:
1. A process to produce ethanol as liquid biofuel from fresh, rotton, or ripe fruit of Musa Sp. and Carica sp. Pulp of Musa Sp. and Carica sp. such as but not limited to Musa velutina, Musa paradisiaca ssp, Musa rosea, Musa troglodytarum , Musa alinsanaya, Musa bauensis, Musa boman, Musa bukensis , Musa. exotica is hydrolyzed to convert into fermentable sugars, followed by fermentation using microorganism followed by separating solid mass of fermented hydrolyze starch sugars or fermented pulp sugar from liquid fraction containing ethanol and where one or more process step(s) is (are) performed batch wise and one or more process step(s) is (are) performed as a continuous flow, are equally preferred, the said process comparising following steps,
(a) Chopping; comprising chopping of fruit, preferably manual cutting by knife or electrical chopper. Seeds were removed from Carica sp. By pasing through vibrating sieves of 2 - 5 mm. The chopped peaces of fruit used in the remaining part of the process.
(b) Slurry formation: comprising the pulp of sugar containing material and distilled water.
(c) Yeast inoculation: comprising the pulp sugar containing material and distilled
water is inoculating with appropriate micro organism.

(d) fermentation; Suitable nutrient source was added to slurry for proper growth of yeast, fermentation is ongoing until the desired amount of fermentation product, ehtanol is produced
(e) filtration: fermented broth separated into solid and liquid by centrifugation, or Alteration by cloth and followed by centrifugation.
(f) distillation: fermentation product, i.e. ethanol present in liquid fraction is separated
from the filtrate and purified and concentrated through distillation to produce purified
useable ethanol.
2. A process as claimed in 1 wherein the distilled water may be heated to a suitable temperature prior to being combined with the sugar containing material in order to achieve a mash temperature of 60 to 70 °C, preferably of 50 to 600C.
3. A process as claimed in 2 wherein the wet solids percent in the slurry tank (containing pulp of fruit) is in the range from 25 - 90%.
4. A process as claimed in 1 wherein the cooled slurry comprising the pulp sugar containing material and distilled water is inoculating with appropriate micro organism, preferably yeast, more preferably Saccharomyces cerevisiae, Pichia stipitis.
5. A process as claimed in 1 wherein the slurry containing fermenting organism between 0.5 % to 25 % w/w, more preferably 1 to 5 % w/w.

6. A process as claimed in 5 wherein suitable nutrient source was added to slurry for proper growth of yeast, preferably substance containing Nirogen, more specifically Urea, DAP and Sulphate more specifically MgS04
7. A process as claimed in 6 wherein the nutrient percentage in slurry is 0.0001 % to 0.1 %, preferably 0.001 % - 0.010 %.
8. A process as claimed in 6 and 7 wherein the pH of slurry comprising the pulp sugar containing material, distilled water, fermenting organism and nutrient is preferably set to 4 to 6.5 pH, more specifically 4.5 to 4.9 pH:
9. A process as claimed in 8 wherein fermentation temperature was set to constant temperature 26 to 30°C, more specifically 29°C.
10. A process as claimed in 9 wherein the fermentation is ongoing until the desired amount of fermentation product, ehtanol is produced.
11. A process as claimed in 10 wherein the fermentation product is ethanol recovered in between 48-96 hours for Musa Sp. and 24 - 48 hours for Carica Sp,
12. A process as claimed in 1 wherein ethanol is produced by either solid state fermentation or submerged fermentation carried out in batch, fed batch, or continuous mode.
13. A process as claimed in 1 wherein filtration clothe could be cotton cloth, plastic net, steel net, of 100 - 500 mesh size, more preferably 150 -300 mesh size.

14. A process as claimed in 1 wherein recovering of the fermentation product, i.e. Ethanol is separated from the filtrate and purified under increased distillation temperature by rate of 2 °C/min and distilled liquid called ethanol collected.
15. As claimed in 1 wherein the Musaceae Sp. are large, often treelike perennial herbs comprising two genera and about forty five species.
16. As claimed in 1 wherein the fruit pulp of banana is used for fermentation from Musa sp. such as but not limited to Musa velutina, Musa paradisiaca sp, Musa rosea, Musa troglodytarum , Musa alinsanaya, Musa bauensis, Musa boman, Musa bukensis , Musa exotica.
17. As claimed in 1 wherein fruits, either freshly collected or stored, processed, unprocessed, including all wild, cultivated, sub-varieties, cultivars, hybrids and genetically modified species of Musa Sp. and Carica sp. are used to produce ethanol.
18. A process as claimed 1 wherein hydrolysis is carried out to convert starch of unripe Musa Sp. and Carica sp. fruits into fermentable sugars by process such as but not limited to acid hydrolysis using sulphuric acid or hydrochloric acid, enzymatic hydrolysis using enzymes such as amylase as such or in combination with pectinase and cellulase.
19. A process as claimed in 1 wherein ethanol is produced by fermentation using combination of microorganism such as amylase producer fungus or bacteria and indigenous isolated yeast.

20. A process as claimed in 1 wherein yeast can be adapted to high rate of fermentation by serial cultivation in dilute pulp or juice of respective fruits.
21. As claimed in 1 wherein the ethanol produced can be used as fuel ethanol; consumable alcohol, potable neutral spirits, or industrial ethanol, including fuel additive.
22. A process for production of ethanol such as herein described with reference to foregoing examples.