Title of Invention

"AN IMPROVED PROCESS FOR MANUFACTURE OF INDIGO FROM INDIGO PLANTS"

Abstract

This invention relates to an improved process for manufacture of indigo from plants using anoxic extraction and subsequent oxidation for use as a dye and as a pigment. In this process Indigofera plant material is steeped with water containing microbes and carbonaceous and nitrogenous nutriments. The reduced indigotin formed under anoxic conditions of the extract is precipitated by air oxidation and recovered by known processes. The process is organic, eco-friendly, simple, fast, produces better quality indigo and can be easily implemented in loco on rural farms since all material needed for the process is conveniently available.

Full Text

Field of Invention: This invention relates to an improved process for manufacture of indigo by steeping the indigo plants with water to which is added microbes and carbonaceous and nitrogenous matter that it can easily assimilate under anoxic conditions, and the subsequent oxidation of the extracted reduced indigotin for use as a dye and as a pigment. Background and Prior Art: Indigo has been prepared from time immemorial by a fermentation process. Plants of the Indigofera, Isatis and Polygonum genera containing the glucoside - indican - are fermented to enable the endogenous indimulsin break down the glucoside under anaerobic conditions. The indoxyls formed due to the enzymatic action join together in pairs to form leuco-indigotin that is water soluble in the prevalent reduced environment. This leuco-indigotin diffuses out of the plant material into the fermentation liquor. The anoxic fermented liquor is separated from the plant material and oxidised by air to obtain indigo blue that is insoluble in water. The conventional processes for extraction of indigo from Indigofera have been reviewed in the Wealth of India [The Wealth of India: A Dictionary of Indian Raw Materials and Industrial Products. Raw Materials, Part V, (1997) pp 173-174. New Delhi: CSIR]. According to the review the wet process is universally adopted in the extraction of indigo. In this process, freshly harvested plants are steeped in water in a specially constructed vat; the water is warmed if necessary to 32*C. Fermentation sets in and the glucoside breaks down by the action of indimulsin, naturally present in the plant, to glucose and indoxyl. The fermentation is completed in 10-15 hours. The yellow liquor is fun into beating (oxidising) vats equipped with paddle wheels; when the wheels are rotated, the liquor is thrown up into the air as a fine spray and the indoxyl gets oxidised into indigo, which settles down as a fine blue mud at the bottom of the vat. The supernatant water (seeth water) is then drawn off. Better yields are obtained by fermenting the leaves under acid condition and effecting oxidation by blowing ammonia, steam and air simultaneously into the fermented liquor by means of an injector. Addition of sodium nitrate or Dhak gum (from Butea monospenna) facilitates the settling of the indigo mud. The oxidised product is stirred with boiling \vatej containing a little dilute sulphuric acid, allowed to settle and the clear liquor decanted: it is then washed with belling water filtered through cloth and pressed. The product is cut into 3 inch cubes and air dried to about 6% moisture. The cubes are brushed to remove surface mould growth, if any, polished and packed in wooden chests. The average yield is 25% of the total extractable colouring matter. In Europe, indigo has been extracted from Isatis (woad) as described by Hurry [Hurry, Jamieson B (1930) The Woad Plant and its Dye. (3rd ed. 1973) Clifton, N.J.: Augustus M. Kelly, pp 24-27]. The ancient process to make indigo from Isatis (woad) is as follows: The leaves are crushed, drained, partly dried and kneaded into balls. The balls are dried slowly and powdered. The powder is sprinkled with water, fermented for 9 weeks at 125°F, after which it emits an odour of ammonia. The resultant clay-like material is thoroughly dried and packed for use by dyers. Recently a new process for preparing indigo from woad has been developed and described by Garfield [Garfield, Simon (2000) Mauve. London: Faber and Faber, p 143]. This method involves making of an extract of leaves with hot water to burst the cells and liberate the precursors into the extraction liquid. The extract is then cooled, made alkaline by adding lime and aerated. The ancient process of drying and fermentation is then followed. The indigo produced by the prior art processes described above is of low quality. It contains many impurities such as indigo red, indigo brown, indigo gluten, efc., in appreciable amounts. The prior art process is time consuming. Process variables such as pH, temperature and redox potential levels affect the quality of the final product. These were never controlled in the prior art process leading to substantial variation in the quality (hue) of the product. If the leuco-indigotin comes in contact with air within or outside the plant material, it would get oxidised and precipitate therein, thereby lowering yields of indigo blue. The Coventry lime process was used, where slaked lime was added to the fermenting liquor to neutralize the effects of the acidity and improve the quality of indigo [Rawson C (1899) Report on the cultivation and manufacture of indigo in Bengal, p 25. For The Indigo Defence Association, Ltd.]. In a US patent USS077201 (1991) a method to produce indigo from the mycelia of a mutant Morel mushroom is described. The Fungus Morchella rotunda run. ES-1 is grown in a fermenter under submerged conditions in a medium containing a carbon and nitrogen substrate. The indigo accumulates in the fungal mycelia after the carbon source is exhausted. The fungal mycelia time after the carbort source is exhausted and the mycelia are macerated and the indigo extracted with solvents such as chloroform, ether, ethyl acetate, acetone, hexane, benzene, ethanol, methanol or sulphuric acid. The solubilized indigo is separated from the mycelial tissue and the indigo recovered by crystallization. In this process the indigo is produced under axenic and submerged conditions in a fermenter. Growth conditions are complex, expensive and capital intensive. Blue (oxidised) indigotin is found in the mycelia and the extraction methods of indigotin use hazardous solvents and acids. Object: The principle objective of the present invention is to improve the prior art anoxic process of indigo extraction from indigo plants so that indigo of a good quality can be produced in a shorter time and with a better recovery. Another object of the present invention is to improve the process in a manner that is easy to employ by farmers in loco. Summary of the invention: An improved process for manufacture of indigo from indigo plants comprising following steps: . (i) placing the young twigs or cut plants of Indigofera, Tsatis, Polygonum, Wrightia genera or any other plant that synthesises indican in a fermentation tank in such a manner as they will remain submerged in the nutrient enriched fermentation liquor with which the tank will be filled in the next step; (ii) filling the said tank containing plants with warm water enriched with 0.01-0.5% w/v assimilable nitrogenous and 0.5-10% w/v carbonaceous nutriments at 2-10 litres per 1 kg of plant material, along with (at 10-150 mg compacted wet weight per litre) aerobic or facultatively anaerobic, mesophilic or thermophilic microorganisms capable of aerobic respiration in such a manner as to avoid any air getting entrapped in the system; (iii) fermenting the plants, microbes and the nutriments in the said water under submerged condition for about 15 minutes to 2 hours at the optimal growth temperature of the microbe added to the fermentation liquor at 25-70"C and pH 5.5-10, and then continuing the fermentation either at the same temperature or at 50-70°C for a further periol- 2-8 hours; (iv) separating and clarifying the fermented liquor from the plants and its suspended matter; (v) precipitating indigo by adding oxidizing agents to the clear fermented liquor obtained at the end of step (iv); (vi) allowing the fermented liquor to precipitate as oxidation proceeds and settle indigo, with or without addition of flocculating agents; (vii) separating said precipitated indigo from said fermented liquor, and purifying it if necesary. Description of the invention: The mesophyll cells in the leaf of Indigofera tinctoria (Indigofera sumatrana) and Indigofera arrecta contain the ß-glucoside 'indican' which is the precursor of indigo [Leake HM (1905) The Localization of the Indigo-Producing Substance in Indigo-yielding Plants. Annals of Botany XIX no. LXXIV: 298-31]. Indimulsin (the ß-glucosidase enzyme) that cleaves the indican into glucose and indoxyl is also present in the leaves [Bergtheil, Cyril (1904) The Fermentation of the Indigo-plant. JCS.T 85, vol. II: 870-892; and Perkin AG, Bloxam WP (1907) Indican Part I. JCS.T, 91, 1715]. In the plant Polygonum tinctorium, the ß-glucosidase is present in the stroma of the chloroplast and the indican is present in vesicles of the same cells [Yoshiko, M., et al (1997) Intracellular localization of beta-glucosidase and indican in Indigo plant. Plant Biology '97, Abstract No. 679]. In the present process of manufacture of indigo plants of any of the genera Indigofera, Isatis, Polygonum, Wrightia and/or other plants containing indican may be used. Preferably the species are Amorpha fruticosa, Asclepias tingens, Baptisia tinctoria, Galega tinctoria, Gymnema tingens, Indigofera arrecta, Indigofera articulata (I. argentea; I. caerulea), Indigofera disperma, Indigofera g la bra, Indigofera hirsuta (L glauca), Indigofera leptostycha, Indigofera longeracemosa, Indigofera pseudo-tinctoria, Indigofera suffruticosa (I. anil), Indigofera tinctoria (I. sumatrana), I satis alpina, Isatis indigotica, Isatis leptostycha, Isatis tinctoria, Justicia tinctoria, Marsdenia tinctoria, Polygonum tinctorium, Ruellia indigotica, '. Saphora tinctoria, Scabosa succisa, Spilanthes tinctoria, Tephrosia apollinea, Tephrosia purpurea, Tephrosia tinctoria, Wrightia tinctoria (including plants with synonymic names). The cut plant-s or plant parts of any of the species are tied in bundles with all branchies facing in a single direction and transported to the fermentation tranks. Allternatively young twigs of the above plants comprising of four expanded leaves and a bud can be plucked and transported to the fermentation tanks. The presence of indican in the plants is ascertained by subjecting the leaves to the action of membrane disrupting agents (such as chloroform, diethyl ether or ice-salt mixture) or physical conditions (such as freeze-thawing) that allow the indican and ß-glucosidase that are normally apart to interact and produce indigo in situ in the leaves. For this purpose the young leaves are immersed for 5-10 seconds in membrane-solubilizing organic solvents such as chloroform or diethyl ether. The leaves are immediately rinsed in water to remove the organic solvent and incubated in a moist atmosphere. Alternatively the leaves are kept at below -15°C (in freezing solutions such as ice-common salt, mixture) for 15 min. The leaves are then thawed and washed to remove traces of the freezing solution and incubated in a moist atmosphere. The leaves are repeatedly treated with 1 N caustic soda to remove coloured soluble substances such as caratenoids and the blue colouration of the leaf is ascertained using a colour chart. The depth of colour of the pinna is proportional to the indigo content of the leaf. In the present invention the bundles of the plant material are arranged in a vertical manner (with the tips of the branches directed upwards) in a tank which may have a drain valve fitted at the bottom. The tank is fitted with an arrangement of pipes to enable introduction of water at multiple sites from the bottom to maintain temperature uniformity within a small range. The tank may also be fitted with an air-tight cover having an outlet that can be connected to a vacuum pump. In order to keep the plants submerged, the bundles are fastened in place or weighed down. Warm water free from heavy metals (such as mercury, copper, zinc, cadmium, and organic amines at concentrations that affect indimulsin or ß-glucosidase activity) to which is added (at 10-150 mg compacted wet weight per litre) aerobic or facultatively anaerobic, mesophilic or thermophilic microorganisms and capable of aerobic respiration such as brewers or bakers yeast, and enriched with 0.01-0.5% w/v ammonium chloride or other assimilable nitrogen sources such as ammonium sulphate, potassium nitrate, plant extracts, seed extracts, animal tissue extracts, stale urine (prepared by keeping urine from dairy and/or draught animals or humans for 2-3 days), microbial extracts, e.t.c.. and 0.1-10. w/v assimilable carbon ,suree such as molasses, sugar, corn .sleep liquor, whey, coin .syrup. starch) hydrolysate. etc., is filled into the tank from the bottom so that the rising water can sweep the air upwards (the branches of the plant should be tied in a manner that facilitates the escape of air); through a multiple arrangements of inlets, so as to submerge the plant material. The temperature of the water should not exceed the thermal tolerance level of the selected microbe. The introduced microorganism are initially cultured on nitrogen and carbon sources used in the fermentation water to reduce the lag phase of growth. About 100-500 g of fresh plant material is used per litre of fermentation liquor. The leaves of Indigofera leaves have hair growth on the undersurface that trap air bubbles when water is poured over the plant to submerge/steep it. During the filling process, a low pressure may be applied to the air outlet attached to the cover. This will cause the bubbles adhering to the leaf hairs to get detached and will increase the area of contact between the liquid and leaf. This will hasten the diffusion of indican and indimulsin out of the plant tissue. After the filling process is complete, the air-tight cover may be removed and a floating sheet of plastic placed over the surface of the liquor to prevent air-exchange. The addition assimilable nitrogen source prevents the wild microbes from consuming the indoxyl as a source of nitrogen and thereby does not adversely affect the yield of indigo blue. The temperature of the fermentation liquor is first maintained for \ to 2 hours at the optimal growth temperature of the selected microbial strain so that the oxygen can be consumed at the expense of the added carbonaceous nutriment. It may be then raised to 50-70*C and maintained at that temperature for a further 2-8 hours, to assist extraction of indican and reduced leuco indigotin and also to increase the activity of the ß-glucosidase. This may be done insulating the tank and heating the fermentation liquor by radiative heaters, immersion heaters, passing oxygen free steam through the steeping water, or by drawing the water from under the surface, circulating the water through heat exchangers and reintroducing it at the bottom. All these operations are done with abundant caution to avoid introduction of air in the system. - The plant material is steeped in the water for 2-8 hours and the pH maintained between 5.5-10 preferably between 6.5-8 by the addition of alkali or acid. The alkali substances can be an organic or inorganic base or a salt of a weak acid with a strong base. The alkali substance can be chosen from a group of .substances such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, ammonia, ammonium hydroxide, extract of plant ash that are alkaline, stale urine (prepared by keeping the urine of dairy or draught animals or humans for 2-3 days at room temperature), organic amines, etc., and mixtures thereof. The acid can be chosen from acids such as sulphuric acid, muriatic acid, phosphoric acid, nitric acid, acetic acid, lactic acid, propionic acid but not arsenic acid or tartaric acid which adversely affect indigo production, and salts of strong acids and weak bases chosen from a group of salts such as ammonium sulphate, etc., and mixtures thereof; but not salts of aluminium, arsenates or tartarates. A small amount of liquid is collected from the drain valve from time to time, aerated and the quantity of indigo estimated. When there is no further increase in the quantity of indigo precipitate, the steeping is complete, the liquid is separated from particulate plant matter. The reduced leuco indigotin in this liquid extract is then completely oxidised to indigo blue by adding oxidising agents. Oxidising agents selected from a group of substances such as air, oxygen, hydrogen peroxide, ammonium persulphate, sodium peroxide, sodium perborate, etc. In case air is used aeration is done by conventional methods such as by beating the drained liquid, using a rotating paddle/arrangement of aerators, sparging compressed air through it, ejecting the liquid into the air as a fountain or pumping the liquid as a spray back into the tank. Appropriate quantity of acid or alkali may be added to adjust the pH to 5.5-10 preferably between 6.5-8 so as to assist the formation of indigo blue. To aid settling of the indigo blue, flocculating agents, at a concentration of around 1% W/V1 are added to the extract. The flocculating agents selected from a group of substances comprising natural gums such as dhak gum, xanthan gum, guar gum, gum acacia, gum ghatti, tragacanth gum and/or polymers such as polyethylene glycol, polyvi'nylpyrollidone and/or settling salts such as sodium nitrate, aluminium chloride, aluminium sulphate, aluminium potassium sulphate. The extract may be heated up to 70°C to aid flocculation. The use of flocculating agents will be influenced by the end use of the product. For example the presence of aluminium in the indigo will be toxic to the organisms used to reduce indigo in the dye application step. The indigo precipitate is separated from the seet liquor. The separation is brought about by conventional separation methods such us settling and decautalion (draining), fiiltration and/or centrifagation. The settled indigo is collected and transferred to a vessel where it is purified by heating/boiling with dilute sodium or potassium hydroxide, followed by washing with water, then boiling in dilute muriatic or sulphuric acid and washing with water and processing in a desired form such as powder, paste or cake. Examples: The invention will now be illustrated with the help of examples. The examples are by way of illustration only and in no way restrict the scope of the invention. Note: All chemicals used unless indicated otherwise were of LR grade, the water used was distilled water. Drawings: Figure 1 - shows absorbance spectra of natural indigo obtained in the example 2. The spectra of synthetic indigo is also shown in the same figure. Example 1: In this process indigo was prepared from fresh leaves of Indigofera tinctoria. a) Four 500 mL conical flasks marked A, B, C and D were cleaned and to each flask was added 60 g leaves of Indigofera tinctoria were added. b) To the flask A 400 mL water at 37*C was added. c) To the flask B 400 mL water at 37*C containing 0.4 g ammonium chloride was added. d) To the flask C 400 ml water at 37 °C containing 0.4 g sugar (commercial food grade) and 0.1 g of wet bakers yeast (tower brand) was added. e) To the flask D 400 mL water at 37 *C containing 0.4 g ammonium chloride, 0.4 g sugar {commercial food grade) and 0.1 g of wet bakers yeast (tower brand) was added. f) Each of these flasks were plugged with cotton wool and incubated at 37*C in an incubator for 7 hours. g) The mouth of flask A was covered with a cloth and the liquid extract in it was collected by inverting over another flask marked A'. The leaves were discarded. h) The pH of flask A' was measured and found to be 7.6-7.9. i) Air was bubbled through the fermented liquor in flask A' with the help of a compressor, j) The oxidised liquor in flask A' was transferred to a centrifuge tube marked A and centrifused. The supernatant was discarded. As the capacity of the centrifuge tube was small the process was repeated till all the contents of flask A' was ceiitrifuged. k) The precipitated indigo In tube A was suspended in 5 of hot (70- 80°C) 0.1% w/v NaOH, mixed for 5 rain cooled and centrifuged. 1) The precipitated and alkali washed indigo in tube A was suspended in 5 mL of hot (70-80°C) 0.1% v/v sulphuric acid, mixed for 5 min cooled and centrifuged. m) The precipitated indigo in tube A was suspended in 5 mL of 0.1% w/v sodium carbonate and centrifuged. n) The precipitated indigo in tube A was suspended in 5 mL of water and centrifuged. o) The precipitate in tube A was dried at 50°C in an oven and weighed. The process (g)-(o) was repeated for flasks B, C and D. The results are shown in Table 1. Table 1: Indigo obtained from 60 g fresh leaves of Indigofera tinctoria. (Table Removed) This result shows that maximum yields of indigo are obtained when the fermenting liquor contains assimilable nitrogen, assimilable carbon and a respiring microbe. Example 2: In this process indigo was prepared from fresh leaves of Indigofera tinctoria on a rural farm. a) 150 L of water was warmed to 42°C in a aluminium vessel placed on a gas stove. A blanket was wrapped around a covered 200 L HDPE drum for purpose of insulation. b) 25 kg fresh leaves were packed in the HDPE drum. c) 150 g of sugar from the grocery store, 1 L of stale (3 days old) cows and buffalo urine and 10 g of bakers yeast procured from the local bakery was dissolved/dispersed in the 150 L warm water. d) The 150 L warm water containing the additives was poured into the drum, the leaves were submerged with the help of a pole and a circular thermocole sheet was placed over the surface of the liquid and the drum covered and placed in the sun for warmth. e) The drum was incubated for 8 hours at ambient temperature (25-40°C). f) The cover was lifted off the drum and the mouth was covered with a cloth and the drum was inverted to collect the drained extract. The leaves were discarded, the drum was washed and refilled with the liquor. g) A pump fitted with a suction pipe and footvalve on the intake side and pipe and shower on the outlet side was used to oxidise the liquor. With the aid of this device the liquor was sucked and sprayed back into the tank from a height of 1 foot (above liquid surface). The redox potential was continually monitored. The spraying was terminated when the redox potential reached 100 mV. h) The liquor was kept standing over night and the supernatant siphoned out. i) The indigo blue precipitate was washed in a LDPE bucket with 25 L water allowed to settle, k) The precipitate was stirred with 1 L hot (at 70-80*C) 0.1% v/v sulphuric acid and allowed to settle, m) The precipitate was stirred with 1 L 0.1% w/v sodium carbonate. The pH was adjusted to neutrality with 10 N caustic soda and the indigo allowed to settle. n) The indigo blue precipitate was washed in a bucket with 2 L water allowed to settle, o) The precipitate was dried away from sunlight in a stream of hot air. Yield was 170.5 g. Examination of the product: 10 nig of precipitate was dissolved in 1 mL concentrated sulphuric acid and diluted with 99 mL of glacial acetic acid. The absorbance was measured at 600 nm. 10 mg of synthetic indigo procured from BASF was used as the standard. This shows "0!" purity. The spectral absorbance of synthetic indigo and the extracted natural indigo is depicted in Fig.l. 10 ing of indigo was dissolved in 1 mL concentrated sulphuric acid and diluted with 99 mL of glacial acetic acid and analyzed. The graph in Fig.l indicates, that the absorbance of natural indigo is about 70% that of the synthetic. The absorbance curve of the natural indigo is broader than that of synthetic indigo in the violet region indicating that natural indigo contains some impurities of orange-red colouration. It also has a substantial absorbance in the UVA region (350-400 nm).The example shows that organic indigo can be produced in loco on the farm. Advantages of the invention: The process described above is simple, takes a shorter time and produces natural indigo of a quality better than what is presently available. All material needed to manufacture the indigo are easily available in rural settings. With judicious choice of additives organic indigo can be produced. Claims: We Clam 1. .An improved process for manufacture of indigo from indigo plants comprising following steps: (i) placing the young twigs or cut plants of Iridigofera, Isatis, Polygonum, Wrightia genera or any other plant that synthesises indican in a fermentation tank in such a manner as they will remain submerged in the nutrient enriched fermentation liquor with which the tank will be filled in the next step; (ii) filling the said tank containing plants with warm water enriched with 0.01-0.5% w/v assimilable nitrogenous and 0.5-10% w/v carbonaceous nutriments at 2-10 litres per 1 kg of plant material, along with (at 10-150 mg compacted wet weight per litre) aerobic or facultatively anaerobic, mesophilic or thermophilic microorganisms capable of aerobic respiration in such a manner as to avoid any air getting entrapped in the system; (iii) fermenting the plants, microbes and the nutriments in the said water under submerged condition for about 15 minutes to 2 hours at the optimal growth temperature of the microbe added to the fermentation liquor at 25-70°C and pH 5.5-10, and then continuing the fermentation either at the same temperature or at 50-70*C for a further period 2-8 hours; (iv) separating and clarifying the fermented liquor from the plants and its suspended matter; (v) precipitating, indigo by adding oxidizing agents to the clear fermented liquor obtained at the end of step (iv); (vi) allowing the fermented liquor to precipitate as oxidation proceeds and settle indigo, with or without addition of flocculating agents; (vii) separating said precipitated indigo from said fermented liquor, and purifying it if necesary. 2. An improved process for manufacture of indigo from indigo plants as. claimed in claim 1, wherein said plants are of Amorpha fruticosa, Asclepias tingens. Baptisia tinctoria. Galega tinctoria, Gvmanma tingens Indigofera arrecta, Indigofera articulata (I. argentea; I. caerulea), Indigofera disperma, Indigofera glabra, Indigofera hirsuia (I. glauca), Indigofera leptostycha, Indigofera longeracemosa. Indigofera pseudo-tinctoria, Indigofera suffruticosa (I. anil), Indigofera tinctoria (I. sumatrana), Isatis alpina. Isatis indigotica, Isatis Icptostycha, Isatis tinctoria, Justicia tinctoria, Marsdenia tinctoria, Polygonum tinctorium, Ruellia indigotica, Saphora tinctoria, Scabosa succisa, Spilanthes tinctoria, Tephrosia apollinea, Tephrosia purpurea, Tephrosia tinctoria, Wrightia tinctoria {including plants with synonymic names). 3. An improved process for manufacture of indigo from indigo plants as claimed in claim 1 or 2, wherein said nutriments added to the warm water are chosen from a group of substances that provide assimilable nitrogen such as ammonium chloride, ammonium sulphate, potassium nitrate, plant extracts, seed extracts, animal tissue extracts, stale urine, microbial extracts, etc., and assimilable carbon source such as molasses, sugar, corn steep liquor, whey, corn syrup, starch hydrolysate, etc. 4. An improved process for manufacture of indigo from indigo plants as claimed in any claim 1-3, wherein the temperature of the fermentation liquor at step (iii) the temperature of the fermentation liquor at step (iii) is maintained by using radiative heat, internal heaters, passing oxygen free steam through the fermentation liquor and/or circulating said liquor through external heat exchangers without introducing any air in the system. 5. An improved process for manufacture of indigo from indigo plants as claimed in any claim 1-4, wherein the pH of the fermentation liquor at step (iii) is maintained between 6.5-8 by addition of acid or alkali. The alkali substances can be an organic or inorganic base or a salt of a weak acid with a strong base. The alkali substance can be chosen from a group of substances such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, ammonia, ammonium hydroxide, extract of plant ash that are alkaline, stale urine (prepared by keeping the urine of dairy or draught animals; or humans for 2-3 days at room temperature), organic amines, etc., and mixtures thereof. The acid can be chosen from acid such as sulphuric acid, muriatic acid, phosphoric acid, nitric acid, acetic acid, lactic acid, propionic acid but not arsenic acid or tartaric acid which adversely affect indigo production, and salts of strong acids and weak bases chosen from a group of salts such as ammonium sulphate, etc., and mixtures thereof; but not salts of aluminium, arsenates or tartarates. 6. An improved process for manufacture of indigo from indigo plants as claimed in any claim 1-5, wherein the microbial culture used for inoculation is Brewers yeast, Bakers yeast. 7. An improved process for manufacture of indigo from indigo plants as claimed in any claim 1-6, wherein said aeration is done by any of the conventional processes such as rotating paddles, arrangement of aerators, sparging compressed air, ejecting liquid in to air as a fountain, pumping said liquid as a spray back into the tank. 8. An improved process for manufacture of indigo from indigo plants as claimed in any claim 1-7, wherein flocculating agents added at step (vi) are selected from a group of flocculating agents comprising natural gums such as dhak gum, xanthan gum, guar gum, gum acacia, gum ghatti, tragacanth gum and/or polymers such as polyethylene glycol, polyvinylpyrollidone and/or settling salts such as sodium nitrate, aluminium chloride, aluminium sulphate, aluminium potassium sulphate. 9. An improved process for manufacture of indigo from indigo plants as claimed in any claim 1-8, wherein the precipitated indigo is purified by heating/boiling in dilute sodium or potassium hydroxide, followed by washing with water, then boiling in dilute muriatic or sulphuric acid and washing with water and processing in a desired form such as powder, paste or cake. 10. An improved process for manufacture of indigo from indigo plants substantially as herein described in the text, in the examples and in the drawings.

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909-del-2002-abstract.pdf

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