Abstract:
The present invention provides an improved process for the production of gibberellic acid (GA 3 ), achieving a yield over 225 g/kg of GA 3  with solid substrate fermentation, and over 15 g/L by 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 GA 3 , as the process has a surprisingly high yield of product, achieves the maximal yield in shorter time than other techniques, consumes less energy, and works with very inexpensive substrates. In all, the manufacturing costs are significantly reduced.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of Indian provisional application No. 1473/MUM/2006, filed on Sep. 15, 2006, the disclosure of which is incorporated by reference in its entirety. 
     FIELD OF THE INVENTION 
     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. 
     BACKGROUND OF THE INVENTION 
     Gibberellic acid (GA 3 ) is the most important gibberellin, a class of diterpenoid acids that function as plant growth regulators (Jefferys, E. G.,  Adv. Appl. Biol.  13:283-316 (1970)). GA 3  affects stem elongation, elimination of dormancy, flowering, sex expression, enzyme induction and leaf and fruit senescence. GA 3  is a high-value, industrially-important, biochemical with various applications in agriculture (L. M. Pastrana et al., “Interactions affecting gibberellic acid production in solid-state culture: A factorial study”; Enzyme and Microbial Technology 17:784-790 (1995), citing to Kumar, P. K. R., and Lonsane, B. K.,  Appl. Microbiol. Biotechnol.  34:145-148 (1990)). However, its high cost has restricted its use to premium crops (Jefferys, E. G.,  Adv. Appl. Biol.  13:283-316 (1970)). 
     GA 3  has been obtained by chemical synthesis, extraction from plants and by microbial fermentation. Stereospecific chemical synthesis involves multiple steps and the use of expensive reagents. To date, chemical synthesis is not economically competitive with fermentation techniques. Direct extraction of GAs from higher plants is not economically feasible due to extremely low concentration of GAs in plant tissue. A large number of bacteria, actinomycetes and yeast cultures have been reported to produce GA 3  or GA 3 -like substances, but most do not produce GA 3  at commercially-feasible levels. Today, GA 3  is produced by fermentation, mostly from fungi. 
     Fermentation Methods 
     GA 3  production in fungal culture includes liquid surface fermentation, submerged fermentation, and solid state fermentation. 
     Liquid Surface Fermentation (LSF) 
     Liquid surface fermentation (LSF), often referred to as surface fermentation, was employed in earlier years for the production of GA 3  but suffered from low yield (e.g. 3.3-9.2 g of crude powdered GA 3  from 130 liters of medium) problems with scalability, contamination and more, and was discontinued (P. K. R. Kumar and B. K. Lonsane, “Microbial production of Gibberellins: state of the art” in  Advances in Applied Microbiology.  34: 29-140 (1989)). 
     Submerged Fermentation (SmF) 
     Currently, GA 3  is largely produced by submerged fermentation (SmF) of the fungus  Gibberella fujikuroi . The fungus known as  Fusarium moniliforme  is also used;  F. moniliforme  is the anamorph (asexual stage) of  Gibberella fujikuroi . GA 3  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. 
     The initial pH values generally employed by various workers are within the range 3.5-5.8, especially around pH 5.5. Id. pH was not usually controlled during fermentation and thus resulted in a final pH differing from the starting pH, with reports of the final pH of 3.9-5.2, 1.8-1.9, or even slight alkalinization. Id. 
     The effect of temperature on the production of GA 3  is dependent on the strain employed. The optimal temperatures reported for the production of GA 3  using  G. fujikuroi  or  F. moniliforme  include 25° C.; 28.5-29.5° C.; 30° C.; 34° C.; 29 ±  0.5° C.; 27° C.; and 28° C. (See Id.) 
     Different workers have used a variety of carbon sources for the production of GAs, as reported by different authors. A combination of readily and slowly metabolizable carbon sources gave a higher yield of GA 3 . Id. Use of molasses led to decreased but economically useful yields. Id. There was a 49% decrease in the yield of GAs with stearic acid as compared to sucrose. Use of dairy waste as carbon source resulted in 0.75 g GA 3 /liter in 12 days. The production was completely inhibited by 1 ppm geraniol due to total inhibition of cell growth. A direct proportional relationship has been shown between the initial concentration of nitrogen supplied in the medium and the rate of product formation as well as the amount of metabolite produced. Id. See also Jefferys, E. G.,  Adv. Appl. Biol.  13:283-316 (1970) 
     Trace elements such as Fe, Cu, Mn, Zn, Al and Ca are required in the fermentative production of GAs. Id. Trace elements may be expressly added in excess or in combination, as in Raulin-Thom medium, or sufficient trace elements may be present as impurities in other ingredients of the fermentation medium. Id. 
     Other growth factors such as vitamins or Yeast extract may improve yield; while other heavy metals or the use of lines steel or stainless steel fermentation tanks can decrease yield. Id. 
     Various processes for production of GA 3  are described in U.S. Pat. Nos. 2,842,051, 2,865,812, 2,906,671, 2,906,673 and 3,021,261. U.S. Pat. No. 2,865,812 reported a yield of 630 mg/L in 664 hrs. Eleazar et al. (“Optimization of gibberellic acid production by immobilized  Gibberella fujikuroi  mycelium in fluidized bioreactors”, Journal of Biotechnology, 76, 147-155 (2000)) reported yields by submerged fermentation as high as 2.862 g/L. 
     Solid Substrate Fermentation (SSF) 
     SSF (Solid Substrate Fermentation) is defined as any fermentation process performed on a non-soluble material that acts both as physical support and source of nutrients, in the absence of free flowing liquid. No free-moving water is present, but there is enough moisture present for the growth and metabolism of the microorganism. The low moisture content means that fermentation can only be carried out by a limited number of microorganisms, mainly yeasts and fungi, although some bacteria have been used. Work on the production of GAs using SSF technique was initiated in the early 1980s and the initial studies gave variable yields. No information was available on the economics of the process until early 1987. Three cases were examined wherein the yield of GA 3  under the SSF technique were 0.825, 1.05 and 1.54 g/kg of Dry Mouldy Bran. The yield of GA 3  in the fed-batch SSF Process was 1.54 g/kg of Dry Mouldy Bran as compared to 1.05 g/kg of Dry Mouldy Bran in the batch SSF process. Id. One group reported a yield of 3 g/kg by SSF (Bandelier, S., Renaud, R., and Durand, A., “Production of Gibberellic acid by Fed-batch solid state fermentation in an aseptic pilot-scale reactor”, Process Biochemistry, 32:141-145 (1997)). B. Tudzynski (“Biosynthesis of gibberellins in  Gibberella fujikuroi : biomolecular aspects”; Appl. Microbiol Biotechnol (1999) 52:298-310) reported that German Patent Number DD252000 also described processes for GA 3  by submerged fermentation, and reported yields of 8 g/kg. 
     Art-Known Fermentation Techniques Remain Expensive 
     Despite advances in fermentation technology, the cost of production of GA 3  has been a deterrent to its widespread use. Additional cost considerations include problems in the downstream processing and, given the potency of GA 3  to plants, removal and disposal of contaminated wastewater. 
     SUMMARY OF THE INVENTION 
     The inventors of the present invention have developed a process to manufacture GA 3  with  Fusarium moniliforme  by submerged and solid state fermentation and have been successful in obtaining yields over 15 g/L in submerged fermentation, and over 200 g/kg in solid state fermentation. 
     In one embodiment of the present invention, GA 3  is produced by solid state fermentation using various substrates such as wheat bran and Jatropha seed cake, 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 analyzed for GA 3 . GA 3  is isolated by adjusting the aqueous dilution of the mixture to acidic pH, and extracting using an organic solvent. The organic solvent is distilled and the GA 3  obtained is dissolved in ethanol. 
     In another embodiment of the present invention, GA 3  is produced by submerged fermentation by culturing  F. moniliforme  in Czapek-Dox media containing a carbon source, and incubating for 10 days. 
     The present invention has provided industrially-viable processes for the manufacture of GA 3  by fermentation processes such as solid-state fermentation or submerged fermentation. The present invention has in particular provided an improved, cost-effective process for the manufacture of GA 3 , as the process has a surprisingly high yield of product, achieves the maximal yield in shorter time than other techniques, consumes less energy, and works with very inexpensive substrates. In all the manufacturing costs are significantly reduced. 
     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 3  produced either by submerged or solid-state fermentation (SSF) technique. In certain embodiments, 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 , including  Gibberella fujikuroi  or  Fusarium moniliforme.    
     Production of GA 3  is influenced by cultural conditions. In certain embodiments, high yields of GA 3  are obtained by varying factors, such as pH, temperature, incubation time, and other conditions such as optimization of the fermentation media. 
     In another embodiment, the present invention may use humid solid matrices substrates such as wheat bran or Jatropha seed cake. 
     In another embodiment, the production process of GA 3  by submerged fermentation involves incubating the fungi in liquid media containing various minerals, sodium nitrate as the nitrogen source, and sucrose as the carbon source. 
     In another embodiment, the present invention has optimized the pH conditions for submerged fermentation. The process of the present invention provides an optimal pH in the range of 5-8. In one embodiment, the pH is pH 7.0 
     In another embodiment, the present invention has optimized the temperature conditions for submerged fermentation. The present invention provides an optimal temperature ranging between 25° C. (room temperature) to 37° C. In one embodiment, the temperature is 30° C. 
     In another embodiment, the present invention has optimized the carbon source for submerged fermentation. In one embodiment, the carbon source is sucrose or glucose. 
     In another embodiment, the present invention has optimized the media for submerged fermentation. In one embodiment, the medium is reconstituted Czapek-Dox medium. 
     In still another embodiment, the present invention provides a process for production of GA 3  by solid-state fermentation. In one embodiment, this involves growing fungi on wheat bran supplemented with a mineral salts solution. 
     In the case of GA 3  production in SSF, factors that affect yield include temperature, pH, moisture, substrate particle size, light, autoclave timing, incubation period, amount and age of inoculum. 
     GA 3  was produced by submerged or solid-state fermentation (by free or immobilized cells, respectively). We observed that the yield from solid-state fermentation was as high as 39 g/kg using wheat bran as a substrate, and 237.2 g/kg, when using of Jatropha seed cake as substrate. To the best knowledge of the inventors, the GA 3  yields of the present invention are higher than any reported, by any mode of fermentation. For example, only one previous report provides a yield as high as 19.3 g/kg of GA 3 , which was obtained using starch (corn flour) as substrate (Gelmi et al. (“Solid substrate cultivation of  Gibberella fujikuroi  on an inert support”;  Process Biochemistry.  35: 1227-1233 (2000), citing to Qian et al., ( World J. Microbiol Biotechnol.  10:93-98, (1994))). 
     A comparison of the present invention with conventional methods was performed. GA 3  produced by the present invention by submerged is greater than 3-fold higher than the highest yield reported in the literature; and by solid-state fermentation greater than 10-fold higher. The present process has demonstrated a yield greater than 15 g/L by submerged fermentation, and greater than 225 g/kg by solid state fermentation. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       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. 1 : Production of GA 3  production (g/kg) by  F. moniliforme  in SSF using Jatropha seed cake as the substrate. 
         FIG. 2 : Illustrates GA 3  production in g/L and biomass, as determined by OD at 600 nm, for  Fusarium moniliforme  grown by submerged fermentation in commercial Czapek-Dox broth. 
         FIG. 3 : Illustrates the effect of pH on GA 3  production (g/L) and biomass (OD at 600 nm) by  F. moniliforme  in submerged fermentation in commercial Czapek-Dox broth. 
         FIG. 4 : Illustrates the effect of temperature on GA 3  production by  F. moniliforme  in submerged fermentation in Czapek-Dox broth. 
         FIG. 5 : Illustrates the effect of carbon source on GA 3  production by  F. moniliforme  in submerged fermentation in Czapek-Dox broth. No growth was observed when acetic acid was used as the carbon source. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Definitions 
     As used herein the term GA 3  or gibberellic acid refers to gibberellic acid with the molecular weight of 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. This is also known as “solid substrate fermentation. 
     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 mineral salts solution used in the present invention comprises copper sulphate, ferric chloride and zinc sulphate. 
     Czapek-Dox (Himedia-M076) media contains the following components (per liter) 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 sucrose 
                 30 
                 g 
               
               
                   
                 sodium nitrate 
                 3 
                 g 
               
               
                   
                 dipotassium phosphate 
                 1 
                 g 
               
               
                   
                 magnesium sulphate 
                 0.5 
                 g 
               
               
                   
                 potassium chloride  
                 0.5 
                 g 
               
               
                   
                 ferrous sulphate 
                 0.01 
                 g 
               
               
                   
                 distilled water 
                 1000 
                 ml 
               
               
                   
                   
               
             
          
         
       
     
     In one embodiment, the Czapek-Dox media is reconstituted rather than commercially purchased. Reconstituted Czapek-Dox media is prepared by adding all components individually in water at the same concentrations as that of commercially available media. 
     One substrate used in the present invention is Jatropha seed cake. Jatropha is grown for its oil, which is extracted after crushing the seeds. Typically, seeds are ground to uniform sized particles and oil is extracted by Soxhlet extraction using Hexane as a solvent. Jatropha seed cake “with oil” means that the seeds have been crushed, but before the oil has been extracted. Seed cake “without oil” refers to seed cake left over after oil extraction, from which more than about 95% of the oil has been removed. 
     Solid State Fermentation 
     The present disclosure provides an improved process for the production of GA 3  either by submerged fermentation or solid state fermentation. 
     In one embodiment, GA 3  production via solid state fermentation comprises the following steps: 
     a) Preparation of solid substrate mixture; 
     b) Inoculation with  F. moniliforme  culture; 
     c) Incubation; 
     d) Extraction of GA 3 ; and 
     e) Purification. 
     In certain embodiments, the solid-state substrate used in the present invention is wheat bran (50 g) or Jatropha seed cake (5 g) to which is added mineral salt solutions (30 ml and 8 ml, respectively) under high moisture content and then autoclaved in the flasks. In one embodiment, autoclaved substrate is inoculated with  F. moniliforme  culture in Czapek-Dox broth. 
     In one embodiment, incubation of the substrate is done at temperatures ranging between 25-37° C. and analyzed for GA 3  content, at periodic intervals, using a spectrophotometric method (Berrios et al. (Spectrophotometric method for determining Gibberellic acid in fermentation broths. Biotechnology Letters, 26: 67-70 (2004)), high-pressure liquid chromatography (HPLC) and/or thin-layer chromatography (TLC). In TLC the culture filtrate obtained after fermentation was subjected to extraction and purification as described above. The residue obtained was dissolved in ethanol and separated by thin layer chromatography 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 an oven at 80° C. for 10 min. GAs fluoresce and appear as greenish spot under UV light, allowing their detection. (D. Puchooa and R. Ramburn; “A study on the use of carrot juice in the tissue culture of  Daucus carota : African Journal of Biotechnology, Vol. 3(4), pp. 248-252, April-2004). 
     The inventors of the present invention extracted GA 3  by diluting the above mixture with water, and acidification with concentrated HCl, to pH 2.5. 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 5 ml of ethanol and the amount of GA 3  was determined. To a 1 ml aliquot of the sample in ethanol, 8 ml of 3.75N HCl was added and O.D. was measured at 254 nm after 2 min., following the protocol of Berrios et al. (2004). 
     Various substrates have been used for solid state fermentation in the literature. Machado et al. reported a yield of 0.925 g of GA 3 /kg of biomass using coffee husk or cassava bagasse as a medium (Machado, C. M. M., Oishi, B. O., Pandey, A., and Soccol, C. R.,  Biotechnol. Prog.  20:449-1453 (2004)). Gelmi et al. (“Solid substrate cultivation of  Gibberella fujikuroi  on an inert support”;  Process Biochemistry.  35: 1227-1233 (2000)) reported that Qian et al., ( World J. Microbiol. Biotechnol.  10:93-98, (1994)) achieved a yield of 19.3 g GA 3 /kg of dry fermented substrate after 18 days of cultivation using corn flour as a substrate (although Gelmi suggested that the yield was actually less, when taking into account degradation of the substrate). 
     Gelmi, ibid. also reported that other workers achieved yields of 3.8 g GA 3 /kg vermiculite and of 6.8 g GA 3 /kg initial dry mass over 190 h, using a wheat bran culture medium. A yield of 8 g/kg of GA 3  was obtained on rice as a substrate (German Patent Number DD 252000). Prema et al.,  Indian J. Microbiol.  28:1-2 (1988)) reported very low yields of GA 3  (1.14 g/kg) and Kumar, P. K. R., and Lonsane, B. K.,  Appl. Microbiol. Biotechnol.  34:145-148 (1990), reported 1.2 g/kg of GA 3  using wheat bran as a medium. 
     The present invention has obtained GA 3  using wheat bran, and using Jatropha seed cake with oil and without oil. The present invention has found that the GA 3  yields were as high as 237.2 g/kg using Jatropha seed cake with oil on the 6 th  day, which is more than ten fold higher than the previously reported possible yield of 19.3 g/kg (Gelmi, ibid.). The GA 3  yield obtained by using Jatropha seed cake as a substrate was also five fold higher than that obtained by wheat bran under the present optimized conditions (see Examples). 
     In certain embodiments, the yield of GA 3  in the present invention is further improved by having a 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.,  Indian J. Microbiol.  28:1-2 (1988), or Pastrana et al., Enzyme and Microbial Technology, 17:784-790, 1995 (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 3  was only 3 g/kg (Bandelier et al.,  Process Bioche . Vol. 32 2:141-145 (1997)). 
     Submerged Fermentation 
     In one embodiment, 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 3 ; and 
     4. Purification. 
     In one embodiment, the inoculum is prepared by culturing and maintaining  F. moniliforme  in Czapek-Dox broth medium, in the presence of a carbon source, such as sucrose or glucose. 
     In the present invention, various conditions such as pH, temperature, carbon source, and the media may be optimized for high yield. 
     One group has previously reported a yield of GA 3  of 5 g/L by submerged fermentation, using a fed-batch cultivation mode under conditions of nitrogen limitation using genetically improved strains (P. K. R. Kumar and B. K. Lonsane, “Microbial production of Gibberellins: state of the art” in  Advances in Applied Microbiology.  34: 29-140 (1989)). 
     General Comments 
     The following examples are included to demonstrate certain exemplary 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 exemplary 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. 
     EXAMPLES 
     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. Unless otherwise indicated, the media used for production of gibberellic acid was Czapek-Dox (Himedia-M076) and the culture was incubated at 30° C. for 10 days on as shaker incubator at 150 rpm. 
     Example 2 
     Solid Substrate Fermentation with Wheat Bran 
     To 50 g wheat bran, 30 ml of a mineral salts solution (copper sulphate, 0.007 g; ferric chloride, 0.007 g; and zinc sulphate, 0.007 g, dissolved in 1 liter of 0.2N HCl) 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 a 45° angle. Yield was determined by the method of Berrios et al., as described below. 
     
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                   
               
               
                 Yield of GA 3  from wheat bran under SSF, over time. 
               
             
          
           
               
                   
                   
                 GA 3  concentration (g/kg) 
               
               
                   
                 Days of 
                 in solid substrate 
               
               
                   
                 incubation 
                 fermentation (wheat bran) 
               
               
                   
                   
               
             
          
           
               
                   
                 5 
                 29 
               
               
                   
                 6 
                 32 
               
               
                   
                 7 
                 33 
               
               
                   
                 8 
                 34 
               
               
                   
                 9 
                 39 
               
               
                   
                 10 
                 39.4 
               
               
                   
                   
               
             
          
         
       
     
     Example 3 
     Analytical Procedures 
     GA 3  was determined spectrophotometrically by the method described by Berrios et al. (Biotechnology Letters 26: 67-70, (2004)) at 254 nm. GA 3  was also determined by HPLC at 206 nm using a C18 column with methanol:water (3:1) as the mobile phase at 1 ml/min flow rate, as described by Sharma et al. (Biotechnol. Appl. Biochem., 39, 83-88 (2004)). The retention time of GA 3  is 3 minutes under these conditions. 
     GA 3  was also detected by TLC as described by Puchooa et al. (African Journal of Biotechnology, 3(4): 248-252 (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 an oven at 80° C. for 10 min. GA 3  fluoresce and appear as a greenish spot under UV light. 
     Example 4 
     Extraction of GA 3  from the Wheat Bran Solid Substrate 
     GA 3  was extracted from the solid substrate by adding 100 ml 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 3  concentration spectrophotometrically. 
     Example 5 
     Purification of GA 3    
     Isolation of GA 3  from the solid substrate extract was done by the method described in Ergun et al. (Turk J. Bot., 26: 13-18 (2002)). Briefly, to 5 ml of the water extract from the solid substrate, 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 with concentrated HCl the solution was extracted thrice with 15 ml of ethyl acetate. The ethyl acetate phase was collected and evaporated to dryness. The dried material was dissolved in 5 ml of ethanol and GA 3  was determined, using the protocol of Berrios et al. This method of purification may also be adapted to GA 3  from submerged fermentation. 
     Example 6 
     Solid Substrate Fermentation with Jatropha Seed Cake 
     Jatropha seed cake, one of cheapest substrates available, was employed as the substrate for the production of GA 3  Production of gibberellic acid using Jatropha as the substrate has never been previously reported. Both Jatropha seed cake with oil and without oil were used as the substrate. Five flasks (5 g of Jatropha, 8 ml of MSS (copper sulphate 0.007 g; ferric chloride 0.007 g; zinc sulphate: 0.007 g; dissolved in 1 liter of 0.2N HCl) and 3.5 ml of inoculum)) were inoculated with 3.5 ml of four day old inoculum of  F. moniliforme , and were incubated at room temperature. Growth was observed within 24 hours of inoculation. Maximum yield was obtained on the 6 th  day of incubation and the yield was found to decrease on the 8 th  and 10 th  day. 
     As depicted in  FIG. 1 , the maximum yield of GA 3  obtained was 237.2 g/kg by SSF. This is so far the best-reported yield of GA 3  obtained by SSF. The GA 3  extracted from the SSF migrated on TLC as a single spot similar to the standard and showed an R f  value of 0.74, consistent with GA 3 . 
     Example 7 
     Production of GA 3  in Jatropha Seed Cake without Oil 
     The production of gibberellic acid was determined every 48 hours. Five flasks (5 g of Jatropha seed cake without oil, 8 ml of mineral salts solution MSS and 3.5 ml of inoculum) were inoculated with 3.5 ml of four day old inoculum ( F. moniliforme ) and was incubated at room temperature. Maximum yield was obtained on the 6 th  day of incubation and the yield was found to decrease on the 8 th  and 10 th  day. 
     
       
         
               
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 Days of 
                 GA 3  Concentration 
               
               
                   
                 incubation 
                 g/kg 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 2 
                 122 
               
               
                   
                 4 
                 136 
               
               
                   
                 6 
                 213 
               
               
                   
                 8 
                 145 
               
               
                   
                 10 
                 159 
               
               
                   
                   
               
             
          
         
       
     
     Example 8 
     Comparison of Yield from Jatropha Seed Cake with Oil and without Oil 
     Gibberellic acid was produced by using Jatropha seed cake with oil and without oil as the substrates. A very slight difference was observed in the yield, however yield from Jatropha seed cake with oil (149.72 g/kg) was found to be slightly higher than the yield obtained from Jatropha seed cake without oil (140.22 g/kg) on day 10 after inoculation. 
     Example 9 
     Submerged Fermentation 
       Fusarium moniliforme  culture was inoculated from PDA slants into 250 ml of commercial Czapek-Dox broth at an initial pH of 7.0, and incubated at 30° C. for 10 days on a shaking incubator at 150 rpm. Cell growth was monitored by collecting 1 ml of culture from fermented broth every 24 h, and centrifuged at 13200 rpm for 10 min. The supernatant was used for GA 3  estimation, and the pellet, washed thrice with saline, was used for determining cell growth. The growth and GA 3  production patterns of  F. moniliforme  culture in Czapek-Dox broth is shown in  FIG. 2 . 
     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. 2 ). Under these conditions, the yield of GA 3  reached the highest on the 8 th  day. 
     
       
         
               
               
               
             
               
               
               
             
               
               
               
             
           
               
                   
               
               
                 Days of 
                 
                   F. moniliforme inoculum 
                 
                   
               
             
          
           
               
                 Incubation 
                 GA 3 (g/L) 
                 Biomass 
               
               
                   
               
             
          
           
               
                 1 
                 0.09 
                 1.50 
               
               
                 2 
                 — 
                 — 
               
               
                 3 
                 1.92 
                 14.70 
               
               
                 4 
                 2.76 
                 19.44 
               
               
                 5 
                 3.29 
                 23.84 
               
               
                 6 
                 3.35 
                 24.36 
               
               
                 7 
                 3.71 
                 27.20 
               
               
                 8 
                 4.21 
                 22.24 
               
               
                 9 
                 — 
                 — 
               
               
                 10 
                 4.02 
                 24.56 
               
               
                   
               
             
          
         
       
     
     Example 10 
     Optimization of pH in Submerged Fermentation 
     Optimization studies were carried out to increase the yield of GA 3 . To determine the optimal pH for the growth and production of gibberellic acid,  Fusarium moniliforme  culture was inoculated into 100 ml of broth in 250 ml flasks, with flasks having different initial pH values (5, 7 and 8). The cultures were incubated at 30° C. for 10 days on a shaking incubator at 150 rpm. Sampling of 1 ml of broth was done every 24 h throughout the incubation period, to determine cell growth and GA 3  concentration. Initial pH did not have any significant effect on the GA 3  yield ( FIG. 3 ) although the highest yield (6.5 g/L) was obtained at an initial pH of 7.0. 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Days of 
                 pH 5 
                 pH 7 
                 pH 8 
               
               
                   
                 Incubation 
                 GA 3 (g/L) 
                 GA 3 (g/L) 
                 GA 3 (g/L) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 1 
                 0.10 
                 0.14 
                 0.11 
               
               
                   
                 2 
                 — 
                 — 
                 — 
               
               
                   
                 3 
                 1.15 
                 1.32 
                 1.24 
               
               
                   
                 4 
                 2.43 
                 2.99 
                 2.57 
               
               
                   
                 5 
                 3.59 
                 4.45 
                 3.48 
               
               
                   
                 6 
                 4.25 
                 4.86 
                 3.79 
               
               
                   
                 7 
                 4.50 
                 5.31 
                 4.22 
               
               
                   
                 8 
                 5.10 
                 6.50 
                 4.73 
               
               
                   
                 9 
                 — 
                 — 
                 — 
               
               
                   
                 10 
                 5.46 
                 5.87 
                 5.02 
               
               
                   
                   
               
             
          
         
       
     
     Example 11 
     Optimization of Temperature in Submerged Fermentation 
     The optimal 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 room temperature of 23-25° C.) for 10 days on a shaking incubator at 150 rpm. Sampling was done after every 24 hrs for determination of cell growth and GA 3  concentration. Incubation at 30° C. was the optimal condition for maximizing the production of GA 3  ( FIG. 4 ). 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                 Days of 
                 25° C. 
                 30° C. 
                 37° C. 
                 23° C. 
               
               
                 Incubation 
                 GA 3 (g/L) 
                 GA 3 (g/L) 
                 GA 3 (g/L) 
                 GA 3 (g/L) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 0.10 
                 0.11 
                 0.024 
                 0.10 
               
               
                 2 
                 — 
                 — 
                 — 
                 — 
               
               
                 3 
                 1.14 
                 1.32 
                 0.092 
                 1.21 
               
               
                 4 
                 2.33 
                 2.97 
                 0.10 
                 2.73 
               
               
                 5 
                 3.0 
                 4.06 
                 0.29 
                 3.33 
               
               
                 6 
                 3.10 
                 4.34 
                 0.33 
                 3.86 
               
               
                 7 
                 3.34 
                 4.76 
                 0.32 
                 4.57 
               
               
                 8 
                 3.88 
                 5.77 
                 0.23 
                 5.01 
               
               
                 9 
                 — 
                 — 
                 — 
                 — 
               
               
                 10 
                 3.65 
                 5.42 
                 0.2 
                 1.92 
               
               
                   
               
             
          
         
       
     
     Example 12 
     Optimization of Carbon Source in Submerged Fermentation 
     To determine the carbon source, which gives optimal yield of GA 3  production, the following carbon sources were used: sucrose, glucose, galactose, xylose, glacial acetic acid and methanol.  Fusarium  culture was inoculated into reconstituted Czapek-Dox medium containing the aforementioned carbon sources and incubated at 30° C. for 10 days on a shaking incubator at 150 rpm. Sampling was done every 24 hrs throughout the incubation period for determining the cell growth and the GA 3  concentration. Sucrose and glucose were found to be the best source of carbon giving a yield of over 15 g/L ( FIG. 5 ). Galactose and xylose also gave yields higher than 8 g/L. 
     
       
         
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 GA 3  (g/L) 
               
             
          
           
               
                   
                   
                   
                   
                   
                 Glacial 
                   
               
               
                 Days of 
                   
                   
                   
                   
                 Acetic 
               
               
                 Incubation 
                 Glucose 
                 Galactose 
                 Xylose 
                 Methanol 
                 acid 
                 Sucrose 
               
               
                   
               
             
          
           
               
                 1 
                 0.28 
                 0.23 
                 0.64 
                 0.026 
                 No 
                 0.43 
               
               
                   
                   
                   
                   
                   
                 Growth 
               
               
                 2 
                 1.12 
                 0.89 
                 0.66 
                 0.028 
                 No 
                 1.72 
               
               
                   
                   
                   
                   
                   
                 Growth 
               
               
                 3 
                 3.07 
                 2.57 
                 1.61 
                 0.018 
                 No 
                 5.57 
               
               
                   
                   
                   
                   
                   
                 Growth 
               
               
                 4 
                 5.63 
                 4.89 
                 3.51 
                 0.015 
                 No 
                 7.69 
               
               
                   
                   
                   
                   
                   
                 Growth 
               
               
                 6 
                 12.14 
                 10.93 
                 7.81 
                 0.014 
                 No 
                 13.85 
               
               
                   
                   
                   
                   
                   
                 Growth 
               
               
                 7 
                 14.02 
                 12.17 
                 9.25 
                 0.001 
                 No 
                 14.6 
               
               
                   
                   
                   
                   
                   
                 Growth 
               
               
                 8 
                 13.82 
                 12.35 
                 11.08 
                 0.0 
                 No 
                 14.76 
               
               
                   
                   
                   
                   
                   
                 Growth 
               
               
                 9 
                 15.07 
                 12.64 
                 11.58 
                 0.023 
                 No 
                 15.37 
               
               
                   
                   
                   
                   
                   
                 Growth 
               
               
                 10 
                 15.28 
                 13.3 
                 12.45 
                 0.018 
                 No 
                 15.91 
               
               
                   
                   
                   
                   
                   
                 Growth 
               
               
                   
               
             
          
         
       
     
     Example 13 
     Optimization of Submerged Fermentation 
     A  Fusarium moniliforme  culture was inoculated into reconstituted Czapek-Dox medium, followed by incubation at 30° C. at an initial pH of 7.0, using sucrose as the carbon source, and was 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 3  concentration. As shown in the following table, the yield reached 15.9 g/L at 10 days. 
                                                           Days of               incubation   GA 3 (g/L)                                        1   0.43           2   1.72           3   5.57           4   7.70           5   —           6   13.85           7   14.60           8   14.76           9   15.37           10   15.91                        
Discussion: Yield from Submerged Fermentation and Solid Substrate Fermentation.
 
     The GA 3  yield was determined in both submerged fermentation and solid substrate fermentation. The yield obtained from solid substrate fermentation reached as high as 237.2 g/kg when using Jatropha seed cake as a substrate, and as high as 39.4 g/kg when using wheat bran as a substrate. The yield obtained from submerged fermentation reached as high as 15.9 g/L. 
     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.