Title of Invention


Abstract The present invention provides an improved process for the production of gibberellic acid (GA3), achieving a yield over 225 g/kg of GA3 with solid substrate fermentation, and over 15g/L submerged fermentation. The method also provides novel substrates, including the use of Jatropha seed cake. The present invention has in particular provided an improved, cost-effective process for the manufacture of GA3, as the process has a surprisingly high yield of product, achieves the maximal yield in shorter time than other techniques, consumers less energy, and works with very inexpensive substrates. In all, the manufacturing coats are significantly reduced.
Full Text FORM 2
(39 of 1970)
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an Indian Company having its Registered office at
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The following specification describes and ascertains the nature of this invention and the manner in which it is performed

The present invention relates to an improved process for the production of Gibberellic acid by fermentation techniques. The present invention in particular relates to the production and optimization of Gibberellic acid with strains of Fusarium moniliforme by submerged or solid state fermentation.
Gibberellic acid (GA) is the most important gibberellin, a class of diterpenoid acids that function as plant growth regulators (Jeffery's, 1970). It affects stem elongation, elimination of dormancy, flowering, sex expression, enzyme induction and leaf and fruit senescence and is a high valued industrially important biochemical with various applications in agriculture (Jeffery's, 1970; Kumar & Lonsane, 1989). However, its high cost has restricted its use for premium crops (Jeffery's, 1970).
GA is presently produced by submerged fermentation techniques using the strains of Fusarium moniliforme or Gibberella fujikuroi. Currently, GA is largely produced by submerged fermentation of the fungus Gibber ella fujikuroi on an industrial scale. It is also synthesized from several bacteria such as Azotobacter, and Azospirillium in culture medium and from wild strains of fungi such as Sphaceloma sp., Phaeosphaeria sp., and Neurospora sp. Production of GA is considerably influenced by cultural conditions. Some of the important factors in obtaining high yields of the GA include pH, temperature, incubation time and conditions such as optimization of the fermentation media.
Various processes for production of Gibberellic acid are described in US patent numbers 2842051, 2865812, 2906671, 2906673 and 3021261 and German Patent Number DD252000. The above stated patents have focused on the submerged production and a maximum yield of 8 g/kg has been demonstrated in the patents.

The cost of production of GA has been a deterrent to widespread production of this plant growth regulator. Recently, there have been advances in fermentation techniques in the production of GA. The isolation of GA3 produced in submerged fermentation and its presence in dilute form has lead to higher costs of downstream processing and disposal of wastewater. All these factors have accounted for high cost of GA3 in present market scenario.
Looking to the dire need of the hour, the scientists of the present have developed a improved process of producing GA, with Fusarium moniliforme either by solid state or submerged fermentation technique.
According to the present invention, GA is produced by solid state fermentation using wheat bran as a solid substrate to which mineral salts are added under high moisture content. The mixture is further inoculated with F. moniliforme and incubated for 10 days and the content of the mixture is analysed for GA. GA is isolated by adjusting the aqueous dilution of the mixture to acidic pH and extracted using the organic solvent. The organic solvent is distilled and the crude GA obtained is purified using ethanol.
According to the present invention, GA is produced by submerged fermentation by culturing F. moniliforme in the Czapek- Dox media containing a carbon source and incubated for 10 days.
The present invention has provided industrially viable processes for the manufacture of GA by fermentation processes such as solid-state fermentation or submerged fermentation. The present invention has in particular provided an improved cost effective process for manufacture of GA in terms of high yield, low energy consumption having commercial viability. The present process developed by the inventors is efficient, cost effective and less cumbersome as compared to the conventional process.

The present invention has focused on the various fermentation processes, which gives better yields. The inventors of the present invention has provided a process to manufacture GA by submerged or solid-state fermentation and has been successful in obtaining higher yields as high as 15-20gm/Kg compared to the conventional methods (8gm/Kg)
It is the object of the present invention to provide a commercially viable process for the production of Gibberellic acid GA by submerged or solid-state fermentation.
It is the object of the present invention to provide an improved process by submerged or solid-state fermentation of GA with low energy consumption.
It is the object of the present invention to provide a cost effective process giving rise to high yields of GA.
The present invention relates to the improved process for manufacture of GA. In the present invention, the higher yields of GA is attributed to the optimization of the process conditions and this has also helped in reduction of cost of the manufacturing process of GA.
In one embodiment, the present invention has focused on production and optimization of Gibberellic acid by Fusarium moniliforme using various fermentation techniques.
The present disclosure provides GA produced either by submerged or solid-state fermentation (SSF) technique. In the present invention, the submerged fermentation is carried out in reconstituted Czapek-dox broth whereas solid-state fermentation is done on a humid solid matrix.

In another embodiment, the present invention relates to the use of microorganism belonging to the genus Gibberella. Particularly the preferred microorganism used in the present invention is Gibberella fujikuroi or Fusarium moniliforme.
In another embodiment, the production process of GA by submerged fermentation involves incubating the fungi in liquid media containing various minerals, sodium nitrate as nitrogen source and sucrose as carbon source.
In another embodiment, the present invention has optimized the pH conditions for the submerged fermentation. The process of the present invention provides an optimum pH in the range of 5-8 and the preferred pH being pH 7.0
In another embodiment, the present invention has optimized the temperature conditions for the submerged fermentation. The present invention provides an optimum temperature ranging between 25°C/ room temperature to 37°C and the preferred temperature is 30 °C.
In another embodiment, the present invention has optimized the carbon source for submerged fermentation. The preferred carbon source is sucrose or glucose.
In another embodiment, the present invention has optimized the media for submerged fermentation and the preferred media is reconstituted Czapek- Dox medium.
In still another embodiment, the present invention provides a process for production of GA by solid-state fermentation. This process preferably involves growing fungi on wheat bran supplemented with mineral salts solution.
A comparison was done for the GA of the present invention, produced by submerged or solid-state fermentation (by free and immobilized cells). It was observed that by solid-state fermentation the yields of GA was as high as 15 - 20g/kg. To the best of the knowledge of the inventors, GA yielded of the present invention is found to be the

highest as compared to that available in the market by any mode of fermentation. Till date only one report provides a yield of 20g/kg of GA obtained using starch as substrate (Qian etal.,1994, W. J. Microbiol. Biotechnol.)
A comparative study was done by the scientists of the present invention on the conventional methods of production of GA, with that of the process developed under the present invention. The scientist of the present invention observed that GA produced by the present invention either by submerged or solid-state fermentation is 3-fold higher than the highest yield reported in the literature. The present process can produce GA with a yield of 16g/l, by submerged fermentation. Besides, irrespective of the fermentation process employed, the yields obtained using the strain of this present invention was consistently higher.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Fig.l: Illustrates GA and OD at 600nm on Czapek - dox broth
Fig 2: Illustrates the effect of pH on GA production by submerged fermentation
Fig. 3: Illustrates the effect of temperature on GA production by F. moniliforme
Fig. 4: Illustrates the effect of carbon source on GA production by F. moniliforme in
submerged fermentation. Fig 5: Illustrates Comparison of GA production in commercial and reconstituted
Czapek-Dox media

As used herein the term GA or GA3 or gibberellic acid refers to Gibberellic acid with
molecular weight 346.38 g/mol
As used herein the term "solid state fermentation (SSF) " includes a process wherein the microbial growth and formation of product on and inside a humid solid matrix is in the absence of free water.
As used herein the term "submerged fermentation" includes a process wherein microbe is grown in a liquid medium and the product is secreted into the media.
The present disclosure provides an improved process for the production of GA either by submerged fermentation or solid-state fermentation in preferred substrate, wheat bran.
In the present invention process GA produced by for solid state fermentation comprises of the following steps :
a) Preparation of solid substrate mixture
b) Inoculation with F. moniliforme culture
c) Incubation
d) Extraction of GA
e) Purification.
The solid-state substrate used in the present invention is wheat bran to which is added mineral salt solutions under high moisture content and then autoclaved in the flasks.
The autoclaved wheat bran is inoculated with F. moniliforme culture from Czapek_Dox broth.
Incubation of the wheat bran is done at optimum temperature ranging between 25-3 7°C and analysed for GA content at periodic intervals using TLC and HPLC.

The extraction of GA is done by diluting the above mixture with water and acidified with acid. The GA is then extracted using organic solvents suich as ethylacetate and the organic solvent is distilled off.
GA is purified using organic solvent such as ethanol.
Various substrates have been used for solid state fermentation in the literature. Cristina et al (2004), have reported a yield of 0.925 mg of GA/g of biomass using coffee husk, cassava bagasse as a medium whereas, after 18 days of cultivation, Qian et al(1994), reported a yield of 19.3 mg GA/ g of dry fermented substrate. However, this was partly due to high degradation of the corn flour substrate.
Agosin et al (1997) achieved yields of 3.8 mg GA/ g vermiculite and of 6.8 mg GA/g initial dry mass over 190 (h), using a wheat bran culture medium. A yield of 8 g/kg of GA was obtained on rice as a substrate (German Patent Number DD 252000). Prema et al, 1988 has reported very low yields of GA: 1.14 g/kg and Kumar et al 1990 has reported 1.2 g /kg of GA using wheat bran as a medium
The improvement in the yield of GA, in the present invention is due to the higher initial moisture level of 60% and a lower incubation temperature of 23 C as compared to moisture level of 50% at 28°C or 30°C as reported in (Prema et al 1988, Pastrana et al, 1995). There does exist one report wherein the temperature was decreased from 28°C to 22°C as the fermentation progressed. However, the yield of GA was only 3 g/kg (Bandelier et al, 1997).
The present invention provides a process for submerged fermentation which comprises the following steps :
1. Preparation of the inoculum
2. Incubation in medium containing sugars
3. Extraction of GA
4. Purification

The preparation of the inoculums is done by culturing and maintaining the culture of F.moniliforme in Czapek-Dox broth medium.
The inoculum is incubated with carbon source. The preferred source is sucrose or glucose.
In the present invention, various conditions were optimized for high yield such as pH, temperature, carbon source and the media.
There is only one report that claims a higher yield of 5 g/L of GA by submerged fermentation (Bu'Lock 1982) using a fed-batch cultivation mode under conditions of nitrogen limitation using genetically improved strains. Duran-Paramo et al have reported a yield of 0.12 g/L
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLE 1: Organism
Fusarium moniliforme NCIM 1100 was obtained from National Collection of Industrial Microorganisms, Pune, India. The strain was cultured and maintained on sterile Potato dextrose agar (PDA) slants. The media used for production of Gibberellic acid was Czapek - Dox (Himedia-M076).

Solid Substrate Fermentation
Wheat bran (50g) was used as a solid substrate and mineral salts solution (30ml) was added to maintain the moisture at 60%. To wheat bran, 30 ml of mineral salts solution (copper sulphate, 0.007g; ferric chloride, 0.007g; and zinc sulphate, 0.007g were dissolved in 1 liter of 0.2N HC1) was added and mixed well. The mixture was then distributed equally into five flasks and sterilized at 15 PSI for 30 min. The autoclaved wheat bran in each flask was inoculated with 10 ml of 6 days old F. moniliforme culture from Czapek - Dox broth, mixed thoroughly and were incubated at 30°C for 10 days at 45° angle.
EXAMPLE 3 Analytical procedures
GA was estimated spectrophotometrically by the method described by Berriso et al. (2004), at 254nm and also by HPLC at 206 run using CI8 column with methanol: water (3:1) as the mobile phase at 1 ml/min flow rate as described by Sharma et al (2004). The retention time of GA is 3 min under these conditions.
GA was also detected by thin layer chromatography (TLC) as described by Puchooa et al (2004). The culture filtrate obtained after fermentation was subjected to extraction and purification as described below. The obtained residue was dissolved in ethanol and separated by TLC using isopropanol - ammonia - water (10:1:1, v/v/v) as mobile phase. The plates were sprayed with 3 % sulphuric acid in methanol containing 50 mg ferric chloride and heated in oven at 80°C for 10 min. GA fluoresce and appear as greenish spot under UV light.
Extraction of Gibberellin from the SSF
Gibberellins were extracted from SSF by adding 100ml of distilled water to moldy bran in each flask and were kept on shaking incubator at 150 rpm for 2 hrs. The slurry from each flask was filtered through muslin cloth and the volume of the filtrate was made to

100 ml. Filtrate was centrifuged at 10000 rpm for 10 min at 28°C. Supernatant was collected and analyzed for GA concentration spectrophotometrically.
Purification of Gibberellin from the SSF extract
Isolation of GA from the SSF extract was done by the method described in Ergun et al (2002). Briefly, to 5 ml of broth, 60 ml of methanol: chloroform: 2 N ammonium hydroxide in the ratio of 12:5:3 and 25 ml of distilled water was added. The mixture was shaken well in a separating funnel. The bottom chloroform layer was removed and methanol in the upper aqueous layer was evaporated. After adjusting the pH of the remaining solution to 2.5, the solution was extracted thrice with 15 ml of ethyl acetate each time. The ethyl acetate phase was collected and evaporated to dryness. The dried material was dissolved in 5ml of ethanol and GA was estimated.
As depicted in Fig. 2, the maximum yield of GA obtained was 20 g/kg by SSF using a 6 days old inoculum. This is so far the best-reported yield of GA3 obtained by SSF. The GA extracted from the SSF migrated on TLC as a single spot similar to the standard and showed an R/value of 0.74 thereby indicating its purity.
EXAMPLE 6: Submerged fermentation
Fusarium moniliforme culture was inoculated from the PDA slants into 250 ml of Czapek - Dox broth and incubated at 30°C for 10 days on a shaking incubator at 150 rpm. Cell growth was monitored by collecting 1ml of culture from fermented broth every 24h and centrifuged at 13200 rpm for l0min. The supernatant was used for GA estimation and the pellet washed thrice with saline was used for determining cell growth.
The growth and GA production patterns of F. moniliforme culture in Czapek - Dox broth is shown in Fig.l. After an initial lag, there was an exponential increase in growth. This log phase continued up to 5 days before reaching a plateau indicating the

commencement of stationary phase. Production of GA was found to start increasing during the late exponential phase (5 days) and extending well into the stationary phase before reaching a plateau (Fig.l). The yield of GA reached the highest on the 8th day. This is so far the highest yield of GA obtained by submerged fermentation.
Optimization of pH in Submerged Fermentation
Optimization studies were carried out to increase the yield of GA. To determine the optimum pH for the growth and production of Gibberellic acid, Fusarium moniliforme culture was inoculated into three different Czapek-Dox broth flasks having different initial pH values (5, 7 and 8) and was incubated at 30°C for 10 days on a shaking incubator at 150 rpm.
Sampling was done every 24hrs throughout the incubation period for determining the cell growth and the GA3 concentration. Initial pH did not have any significant effect on the GA yield (Fig. 2) although the highest yield (5.8 g/L) was obtained at an initial pH of 7.0.
Example 8
Optimization of Temperature in Submerged Fermentation:
The optimum temperature for the growth and production of Gibberellic acid by Fusarium moniliforme was evaluated. The F. moniliforme culture was inoculated into four different Czapek-Dox broth flasks, which were incubated at different temperatures (25°C, 30°C, 37°C and RT) for 10 days on a shaking incubator at 150 rpm and sampling was done after every 24hrs for determination of cell growth and GA concentration. Incubation at 30°C was the optimum condition for maximizing the production of GA (Fig. 3).
Example 9
Optimization of Carbon Source in Submerged Fermentation:

To determine the carbon source which gives optimum yield of GA production, the following carbon sources viz. sucrose, glucose, galactose, xylose, glacial acetic acid and methanol were used. Fusarium culture was inoculated into reconstituted Czapek-Dox medium containing the aforementioned sugars and incubated at 30°C for 10 days on a shaking incubator at 150 rpm.
Sampling was done every 24hrs throughout the incubation period for determining the cell growth and the GA concentration. Sucrose and glucose were found to be the best source of carbon giving a yield of over 15 g/L (Fig. 4).
Example 10
Optimization of Media in Submerged Fermentation:
A comparison study was done between commercial Czapek-Dox medium and reconstituted Czapek-Dox medium under optimized conditions of GA production. A Fusarium moniliforme culture was inoculated in the two culture media followed by incubation at 30°C at an initial pH of 7.0 using sucrose as carbon source. The flasks were incubated for 10 days on a shaking incubator maintained at 150 rpm.
Sampling was done every 24 h throughout the incubation period for determining the cell growth and the GA concentration. The GA yield was almost 3-fold higher in the reconstituted media as compared to the commercially available medium (Fig. 5).

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4) Sharma, R., Iyer, J.P., Chakraborti, A.K., and Banerjee, U.C. 2004. Determination of gibberellins in fermentation broth produced by Fusarium verticilliodes MTCC 156 by high-performance liquid chromatography tandem mass spectrometry. Biotechnol. Appl. Biochem. Vol. 39: 83-88
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6) Ergun, N., Topcuoglu, F., and Yildiz, A. Auxin (Indole-3-acetic acid), Gibberellic acid (GA3), Abscisic acid (ABA) and Cytokinin (Zeatin) production by some species of mosses and lichens. Turkish Journal of Botany, 2002, vol. 26: 13-18
7) Bu'Lock JD (1982) Useful metabolites of Fusarium. In: Moss MO, Smith JE (eds) The applied mycology of Fusarium. University press, Cambridge, pp 1-12
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All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically or physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Dated this 15 day of September, 2006
For Reliance Life Sciences Pvt. Ltd.

The present invention provides improved process for production of Gibberellic acid (GA) that is commercially viable. The present invention describes that solid-state fermentation is most economical method for GA production giving a yield as high as 20g/kg of GA. The present invention also described optimized conditions for production of high yield of GA by submerged fermentation.






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Patent Number 245442
Indian Patent Application Number 1473/MUM/2006
PG Journal Number 03/2011
Publication Date 21-Jan-2011
Grant Date 19-Jan-2011
Date of Filing 15-Sep-2006
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PCT International Classification Number C12P27/00
PCT International Application Number N/A
PCT International Filing date
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