Title of Invention

A FERMENTATION PROCESS FOR THE PRODUCTION OF SECONDARY METABOLITES

Abstract The present invention demonstrates the utilization of halide anions such as iodates, periodates, bromates, chlorates, perchlorates and other forms of halides as supplements in fermentation media for the production of secondary metabolites employing fungal systems such as systems Aspergillus, Rhizopus, Streptomyces etc, Actinomycetes or bacterial systems. The significant aspects of the invention specifically relate to a noticeably reduced oxygen uptake rate without any observed growth inhibition of the fermenting organism.
Full Text FIELD OF THE INVENTION
The present invention demonstrates the utilization of certain forms of halide anions
such as iodates, periodates, bromates, chlorates, perchlorates and other forms of halides as additives in fermentation media for the production of secondary metabolites employing fungal, actinomycetes or bacterial systems such as systems Aspergillus, Rhizopus, Streptomyces etc. The significant aspects of the invention specifically relate to a noticeably reduced oxygen uptake rate without any observed growth inhibition of the fermenting organism.
BACKGROUND AND PRIOR ART OF THE INVENTION
Fungal and bacterial systems have been used for several years to produce a wide range of commercially important products like enzymes, therapeutics and a large number of commodity chemicals like lactic acid and citric acid.
A typical fungal or bacterial fermentation consist of a growth medium in which the micro-organism is propagated and then transferred to a production medium which is designed to produce the product of interest. Even after extensive optimization of the medium and operating conditions, scale-up of processes using fungal cultures and other mycelia forming cultures is a difficult task. The major limitation being the high oxygen demand, which cannot be satisfied in very large scale production fermenters due to limitation's in the oxygen transfer coefficients. The dissolved oxygen levels as measured by the online dissolved oxygen probes are almost zero (below 5%) for high cell density fermentation processes utilizing the above mentioned cultures. Achieving high dissolved oxygen levels in fermenter is desirable for better productivities.
There are reports wherein several methods have been employed to improve the dissolved oxygen availability like improving aeration and agitation, genetic manipulation, partial fed batch fermentation, etc. There are also reports on use of cations and anions as growth-inhibitory agents in fungal cultures.
However the use of inorganic elements for the reduction of dissolved oxygen uptake has not been mentioned in any prior art literature. Nobuyuki K et al, in Agric Biol Chem 48(4), 1984 have mentioned about the antifungal effects of chlorides, bromides and iodides of Li+, Na+, K+, Rb+ and Cs+ and of sodium salts of F", NO3, CIO4" and SCN" and also discussed about the antagonism between different alkali cations in

inhibition of fungal growth for P. citrinum, P. graminearum, A. oryzae, A. niger, F. graminearum, Aureobasidum pullaluns and Paecilomyces lilacinus. Prommart Koohakan et al in Microbes & Environment, vol 17, No. 2, 2002 has discussed about the effects of inorganic elements namely Cu, Ag, Cr, Zn, Iodine, Ni, Fe, Ca, Mg and K, on zoospore release, spore germination, mycelial growth and sporangium formation of Pythium aphanidermatum finding its practical application in hydroponic cultures. Influence of metal ions namely Fe(II), Cu(II), Zn(II) and Mn(II) on pellet formation in Aspegillus niger 3T5B8 fermentation was studied by Sonia Couri, et al (Brazillian Journal of Microbiology, 2003, 34) wherein Fe(II) and Mn(II) were found to induce pelletal morphology. But there have been no mention of the reduced oxygen uptake rate due to any of these factors.
Fermenting cells cultured under various aeration conditions require an optimal dissolved oxygen concentration level, and this impacts several factors of the fermentation process which are of major significance a) yield of the desired fermentation product from the fermentation substrate (expressed in grams product/grams substrate consumed), b) growth rates of the fermenting organism c) specific productivity of the desired fermentation product (expressed in weight product/weight dry cell/unit time) and d) substrate consumption rates (expressed in weight of substrate consumed/unit time).
In large scale fermentation processes the dissolved oxygen levels are below the desired values as a result of poor oxygen mass transfer coefficients because of which achieving high cell densities becomes a matter of concern. The lower dissolved oxygen levels also reduce the specific productivity of the cell mass because of oxygen starvation.
It has been surprisingly found that addition of oxidative forms of halide anions to the fermentation medium results in a significant drop in the oxygen uptake by the fermenting organism without any suppression or inhibition of the cell growth.
The objective of the invention is to provide a method of reducing the oxygen uptake by the fermenting micro-organism. The objective is also to provide a method wherein a more nutritious medium can be used to achieve higher cell growth and thus higher productivities without compromising on the dissolved oxygen levels in the submerged fermentation processes thereby leading to higher yields of the desired end product.

OBJECTIVES OF THE PRESENT INVENTION
The main objective of the present invention is to provide a process of fermentation for production of secondary metabolites.
Another object of the present invention is to provide a method of reducing oxygen uptake by the fermenting organism without hindering the growth of fermenting organism thereby resulting higher productivities.
STATEMENT OF THE INVENTION
Accordingly, the present invention provides for a fermentation process for the production of secondary metabolites, characterized in that the addition of halide anions inhibits reduction of dissolved oxygen content in the fermentation medium.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
FIG 1: Effect of the supplementation of lodate on the production of Mycophenolic acid
FIG 2: Effect of the supplementation of iodate on the production of mycophenolic acid in high nutrient medium.
FIG 3: Effect of the supplementation of lodate on the production of Compactin.
FIG 4: Effect of the supplementation of Iodate on the production of Lovastatin.
FIG 5: Effect of the supplementation of lodate on the production of Pneumocandin.
FIG 6: Effect of the supplementation of Iodate on the production of Tacrolimus.
FIG 7: Biomass- MPA fermentation run with iodate supplementation.
FIG 8: Titer and DO profile - MPA fermentation run with iodate supplementation.
FIG 9: Effect of other oxidative forms of halogen on mycophenolic acid production.

DETAILED DESCRIPTION OF THE INVENTION
The present invention is in relation to a fermentation process for the production of
secondary metabolites, characterized in that the addition of halide anions inhibits
reduction of dissolved oxygen content in the fermentation medium.
In another embodiment of the present invention the addition of halide ions reduces
oxygen uptake of the micro-organism in the fermentation medium.
In yet another embodiment of the present invention the halide anion is utilized as a salt
in combination with its cationic metal component.
In still another embodiment of the present invention the halide anion is selected from a
group comprising iodate, chlorate, bromate, florate perchlorate, perbromate and
metaperiodate or their combination thereof or any of the oxidative forms of halides.
In still another embodiment of the present invention the cationic component is selected
from a group comprising alkali metals, alkaline earth metals and/or their mixtures
thereof.
In still another embodiment of the present invention the cationic component is
Potassium, magnesium or calcium.
In still another embodiment of the present invention the concentration of halide anion is
ranging from of 0.1 - 50g/L.
In still another embodiment of the present invention the concentration of halide anion is
ranging from of 0.1 - lOg/L.
In still another embodiment of the present invention the addition of halide anion
decreases the dissolved oxygen uptake by the fermenting microorganism.
In still another embodiment of the present invention the residual dissolved oxygen level
is increased by at least 10%.
In still another embodiment of the present invention the fermenting organism is a
fungal species.
In still another embodiment of the present invention the fermenting organism is a
bacterial species.
In still another embodiment of the present invention the fermenting organism is an
kctinomycete species.
In still another embodiment of the present invention the productivity is increased by at
least 20%

It is an objective of the present invention to provide a method of reducing oxygen uptake by the fermenting organism without hindering the growth of the fermenting organism thereby resulting in higher productivities.
Surprisingly, the use of certain forms of halide anions significantly reduces the oxygen uptake rates by the fermenting organism acting as a process control parameter, thereby allowing the fermentation process to be optimized, balancing high yields to the desired fermentation product with good production rates that is not hindered by slow growth rates of the organism, the dissolved oxygen content no more acting as a limiting factor.
Utilization of oxidative forms of halide anions as supplements in the fermentation medium can have a positive impact on a) specific productivity of the desired fermentation product (expressed in weight product/weight dry cell/unit time) and b) substrate consumption rates (expressed in weight of substrate consumed/unit time) without hindering the growth rates of the fermenting organism.
According to one aspect of the invention these ions that may be utilized in context of the present invention include, for example and without limitation, iodates, chlorates, bromates. perchlorates. periodates and the like.
According to yet another aspect of the present invention the concentration of the halide anions used is in the range of 0.1 - 50g/L.
According to yet another aspect of the invention, the fermentation medium can be supplemented with said halide anions initially or periodically during the course of fermentation.
All numbers expressing quantities of ingredients and/or fermentation conditions are to be understood as being modified in all instances by the term "about".
In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the

element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".
Yield co-efficient of biomass produced over substrate utilized is herein understood to mean the amount of biomass produced in grams dry weight over the amount of substrate utilized in grams. The yield coefficient of fermentation product produced over substrate may be expressed as units or grams of product produced per Kg of substrate used.
As used herein, the term "dissolved oxygen content" or DO shall mean and refer to the dissolved oxygen content of the medium.
"Secondary metabolites" are organic compounds that are not directly involved in the normal growth, development or reproduction of organisms. A compound that is not necessary for the growth or maintenance of cellular functions but is synthesized, generally for the protection of a cell or microorganism during the stationary phase of the growth cycle.
The rate of consumption of oxygen per unit weight of the product titer can be determined from the amount of dissolved oxygen that is consumed, conveniently determined by measuring the amount of oxygen supplied to and consumed from the fermentation vessel per unit time. The amount of supplied oxygen can be measured by monitoring aeration rates.
It is advantageous to maintain a dissolved oxygen level in the production medium of about 20%-80% of air saturation during the major portion of the fermentation. The ability to achieve a suitable dissolved oxygen level may be enhanced by proper adjustment of the aeration and/or agitation rate. In one aspect of the invention it is desirable to agitate the fermentation medium while being aerated.
The salt anion may be any suitable anion that results in a water soluble oxidizable salt when combined with the cation-containing component- Suitable anions include, for example and without limitation, inorganic halide anions such as iodates. chlorates. perchlorates, periodates and the like. Other suitable anions would be well known to those skilled in the art in view of this disclosure.

The halide anions that may be used in the present invention may be used in their salt forms such as hydrogen, alkali and alkali earth salts of chlorate, bromate, iodate, perchlorate, perbromate and metaperiodate and the like. Particularly preferred metals include sodium, potassium, calcium, and magnesium, alone or in combination.
An object of the present invention is to provide a method for achieving high and efficient production of an objective substance through fermentation using a microorganism having an ability to produce the said objective substance utilizing suitable anions in their salt forms, said addition avoids reduction of dissolved oxygen content in the medium thereby not affecting the growth rate of the microorganism or reduction of productivity of the objective substance.
As used herein, the term "limiting nutrient source" refers to a source of a nutrient (including the nutrient itself) essential for the growth of a microorganism in that, when the limiting nutrient is depleted from the growth medium, its absence substantially limits the microorganism from growing or producing further. However, since the other nutrients are still in abundance, the organism can continue to make and accumulate intracellular and/or extracellular products. By choosing a specific limiting nutrient, one can control the type of products that are accumulated. Therefore, providing a limiting nutrient source at a certain rate allows one to control both the rate of growth of the microorganism and the production or accumulation of desired products
In yet another aspect of the present invention, microorganisms are selected from the group comprising, fungi (including yeasts), actinomycetes, protists, bacteria, or mixtures thereof, wherein the desired fermenting microorganism selected are capable of converting the fermentation substrates under suitable fermentable conditions to produce the desired end product.
The process of the invention can be conducted continuously, batch-wise, or some combination thereof.
Through the judicious selection of nutrients that support microbial growth, the invention provides a medium which allows important secondary metabolites to be produced commercially, in large quantity, using a biological conversion process. The invention provides a low cost alternative to prior art media and methods for its use that

can be easily prepared and sterilized and most importantly results in the production of the desired end product at high yields. It is surprising that the myriad of components with a defined addition of anions such as iodates, chlorates, perchlorates, bromates yields high productivity attributed to the optimal maintenance of the dissolved oxygen levels without sacrificing the quality of microbial growth.
The fermentation medium of the present invention contains in aqueous solution: (a) from about 10 g/1 to about 60 g/1, most preferably from about 20 g/1 to about 40 g/1 of a source of metabolizable carbon and energy, preferably glucose; sucrose; dextrose and the like (b) about 0.1% - 10% of a preferable nitrogen source; (c) from about 1 g/1 to about 10 g/1, preferably from about 1 g/1 to about 7 g/1, of a source of phosphates, sulphates, chlorates, carbonates in their salt forms (d) from about 0.01 g/1 to about 10 g/1, preferably from about 2 g/1 to about 10 g/1 of at least one anion selected from the group consisting of iodate, chlorate, bromate, perchlorate, periodate, perbromate and other halides. The anions are incorporated in the medium as their salt form in combination with a suitable cationic component, such components are selected from the group comprising alkali metals, alkaline earth metals, and mixtures thereof, preferably a potassium, calcium and magnesium.
According to yet another aspect of the invention, the fermentation medium can be supplemented with said halide anions initially or periodically during the course of fermentation.
In one aspect a foam inhibitor is added to the fermentation media in order to prevent the accumulation and build up of foam caused by oxygen sparging of the fermentation broth contained therein.
The process may be operated over any pH or temperature range where the fermenting microorganism can grow and catalyze the desired conversion reaction. The process of this invention may be carried out at a temperature of about 22° C. to about 37° C, preferably about 27°C. Shifts in pH are prevented by addition of bases such as NaOH or acids such as H2SO4 or HCI. The regulating agent is typically a hydroxide or an organic or inorganic acid. Examples of suitable pH regulating agents are potassium hydroxide, sodium hydroxide and hydrochloric acid. An important aspect of the present invention is the control of a number of process parameters to favor the desired reaction

products. Thus, the process or segments of the process can be conducted as continuous operations or various distinct unit operations. The length of time which the fermentation process is allowed to continue depends upon the composition of the fermentation medium, temperature, quantity of inoculum, quantity of product desired, etc.
According to the most significant aspects of the invention, increase in the rate of productivity is at least 10%, at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 35%, more preferably at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, most preferably at least 60%, most preferably at least 65% and most preferably at least 70%. According to yet another aspect of the invention yield conversion obtainable is about 100%.
These and other non-limiting embodiments of the present invention are readily understood by one of ordinary skill in the art upon reading the disclosure and claims provided herein. It is understood that this invention is not limited to the particular methods and processes described, as such desired end products and methods may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
The present invention will be better understood from the examples which follow, all of which are intended for illustrative purposes only, and are not meant to unduly limit the scope of the invention in any way.
The technology of the instant Application is further elaborated with the help of following examples. However, the examples should not be construed to limit the scope of the invention. The following Examples represent preferred embodiments of the present invention.
EXAMPLE 1: Supplementation of iodate in penicillium fermentation for the production of Mycophenolic Acid.
The spore vial for the Penicillium brevicompactum, stored at -85 °C was thawed and inoculated into the growth medium consisting of sucrose 10%, soya peptone 1%, soya flour 2%, magnesium sulphate 0.1% , sodium nitrate 0.2% and small quantities of antifoam. The flask were incubated at 24 °C for 48 h and then transferred to the

production medium. Different levels of iodate ion in the form of potassium iodate were added to flasks. The concentration of potassium iodate tested in this experiment was 0.0, 0.1, 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 7.5 and 10.0 g/L. The production medium consisted of Sucrose 10%, Casein hydrolysate 3.6%, cotton seed flour 2.0%, Potassium phosphate 0.45%, Magnesium Sulphate 0.1%, Potassium chloride 0.15%, calcium carbonate 0.6% and antifoam.
At lower concentrations of iodate, below 2.0 g/L, no significant variation was observed in the growth of the culture. The growth was marginally affected at concentrations of iodate between 2-10 g/L. The productivities were found to be similar in all the flasks with and without potassium iodate concentration. The data also indicates that iodate ion as such does not help increase the titre by acting as a nutrient or a key ingredient. The results are shown in FIG.l.
The effect of iodate is also tested in a concentrated medium where the concentration of carbon source is increased by 50% in the production medium mentioned in Example 1.
As shown in Fig 2, the productivities with supplementation of iodate were higher than the control medium. This clearly indicates that the better dissolved oxygen availability enabled the culture to utilized the extra nutrient and produce the product of interest at higher concentrations. Productivity was less in the medium without the supplementation of iodate ion even though sufficient nutrients were available for the microorganism. The same range of potassium iodate supplementation was tested as in Example 1.
EXAMPLE 2: Supplementation of iodate in penicillium fermentation for the production of compactin.
The similar experiment as mentioned in Example 1 and 2 was carried out for the production of Compactin. The culture used for the experiment, Penicilium brevicompactum, was grown and subsequently transferred to production medium containing varied quantities of potassium iodate.
The growth medium for the experiment was same as that in Example 1. The production medium consisted of sucrose 35%, soya peptone 2%, soya flour 4%, magnesium sulphate 0.1%, sodium nitrate 0.2% and small quantities of antifoam.

It was observed that KI03 doesn't have any deleterious effect on the growth or productivity of compactin using the strain of penicillium rather results in higher productivity of compactin in the concentrated medium wherein otherwise the titers achieved were much lower. The results are graphically represented in FIG. 3
EXAMPLE 3: Supplementation of iodate in Aspergillus fermentation for the production of Lovastatin.
The same strategy was applied for the production of Lovastatin using Aspergillus terreus. In this trial only the concentrated medium was used which was known to create dissolve oxygen limitation. The production medium for Lovastatin consist of starch 2.5%, dextrose mohohydrate 15%, soya flour 13%, peptone 1%, malt extract 1%, citric acid 1.05%, sodium acetate 0.88%, calcium carbonate 0.63%, sodium chloride 0.32%, potassium dihygrogen phosphate 0.075%), magnesium sulphate 0.075% and antifoam.
With the increase in the nutrient concentration, the carbon consumption was expected to increase but due to very low dissolved oxygen availability, the carbon consumption was actually found to decrease. With the addition of potassium iodate, a 35% rise in productivity was observed because of higher oxygen availability and higher carbon uptake rate. As observed in previous examples, the titer stabilized above a certain concentration of potassium iodate. FIG 4.
EXAMPLE 4: Effect on the production of Pneumocandin B0 by Zalerion arboricola.
Pneumocandin B0 is an antibiotic produced by submerged fermentation using the culture Zalerion arboricola.
The growth medium used for this fungal culture consisted of maltodextrin 2.5%, soya flour 1.2%, tryptone 0.5%, peptone 0.1 %, yeast extract 0.2% , potassium phosphate 0.9% and antifoam. The culture inoculated in the growth medium was incubated at 22 -26 °C for 3-4 days for proper growth of the culture. The production medium consisted of the following components - Mannitol 10.0%, Casein 0.825%), potassium phosphate 0.2%, lactic acid 0.75%, corn steep powder 0.15%, soya flour 1.1%, dextrin 2.5%, glycine 0.2%, proline 1.65%, tryptone 0.825% and antifoam. The flasks were incubated for 14-16 days. In the control medium, because of high viscosity of broth and low available oxygen, the carbon uptake was significantly low resulting in considerably

lower titres. Addition of iodate ion was found to have a positive impact on the production of Pneumocandin with the titre rising by almost 43%. The cell growth was estimated to be similar at all levels of iodate supplemented to the medium.
EXAMPLE 5: Effect of iodate on Streptomyces sp in submerged fermentation for the production of Tacrolimus.
Tacrolimus is produced by fermentation using Streptomyces sp. in submerged cell cultivation.
Potassium iodate was found to have similar effects on Streptomyces cultures as in Fungal cultures and the productivities were found to increase with the increase in iodate concentration and then stabilized. The growth medium used in this experiment consisted of the following components - Starch 1.0%, Dried Yeast 0.5%, Dextrose 0.5 %, glycerol 1.0%, Cotton seed flour 1.0%, corn steep liquor 0.5%, calcium carbonate 0.1% and antifoam. The culture was grown for 48-56 hours in the growth medium at 22-30 °C and then transferred to the production medium containing Starch 10.0%, Dried Yeast 4.5% , Calcium Carbonate 0.1% and antifoam. The concentration of iodate at which the effect stabilized, was found to be much less in Streptomyces as compared to in fungal strains (shown in Example 1, 2 and 3). The results are shown in FIG 6.
EXAMPLE 6: Two 50 L fermentor batches were run using Penicillium aerenicola for the production of Mycophenolic acid. One batch (Experiment #1) is run as a reference batch where iodate was not supplemented in the medium. The maximum product achieved was about 4.8 g/L and the maximum biomass concentration achieved was about 48% v/v when centrifuged at 10000 rpm for 10 min. The second trial (Experiment #2) was taken with 3 g/L iodate ion added to the production medium. The fermenter was operated under the exactly similar condition as of Experiment #1. The experimental run #2 resulted in the final titre of about 6.29 g/L which was about 30% higher than that in the control trial. The biomass generated during the course of fermentation in Experiment #2 was maximum of 44% v/v which later dropped to 39.5% at the end of fermentation. The fermentation time for both the fermenter trials was about 10 days. It was found that because of lower levels of dissolved oxygen in the control trial, the productivity was low. There was no significant difference in the growth of biomass within the two trials.

Biomass, product concentration and dissolved oxygen profiles resulting from the above experimentation are represented in FIG. 7 & 8 respectively
EXAMPLE 7:
Since iodate ion was found to improve the available dissolved oxygen, the same experiment was tried with another oxidative form of a halogen, which is potassium chlorate. For this trial, Penicillium aerenicola was cultivated in the growth medium and then transferred to the production medium for Mycophenolic acid containing different levels of iodates and chlorate to compare the effects of the two anions.
Both the anions were found to have a similar effect on the productivity. The concentrated medium for mycophenolic acid as used in Example 2, showed the same titer profile with both anions. The results are shown in FIG. 9.
The above description and examples have been given for ease of understanding only. No unnecessary limitations should be understood therefrom, as modifications will be obvious to those skilled in the art who will recognize that the invention can be practiced with modifications and variations within the spirit of the appended claims.
WE CLAIM:
1. A fermentation process for the production of secondary metabolites, characterized in that the addition of halide anions inhibits reduction of dissolved oxygen content in the fermentation medium.
2. The process as claimed in claim 1, wherein the addition of halide ions reduces oxygen uptake of the micro-organism in the fermentation medium.
3. The process as claimed in claim 1, wherein the halide anion is utilized as a salt in combination with its cationic metal component.
4. The process as claimed in claim 1, wherein the halide anion is selected from a group comprising iodate, chlorate, bromate, florate perchlorate, perbromate and metaperiodate or their combination thereof or any of the oxidative forms of halides.
5. The process as claimed in claim 3, wherein the cationic component is selected from a group comprising alkali metals, alkaline earth metals and/or their mixtures thereof.
6. The process as claimed in claim 5, wherein the cationic component is Potassium, magnesium or calcium.
7. The process as claimed in claim 1, wherein the concentration of halide anion is ranging from of 0.1 - 50g/L.
8. The process as claimed in claim 1, wherein the concentration of halide anion is ranging from of 0.1 - 10g/L.
9. The process as claimed in claim 1, wherein the addition of halide anion decreases the dissolved oxygen uptake by the fermenting microorganism.
10. The process as claimed in claims 1 to 9, wherein the residual dissolved oxygen level is increased by at least 10%.
11. The process as claimed in claim 9, wherein the fermenting organism is a fungal species.

12. The process as claimed in claim9, wherein the fermenting organism is a bacterial
species.
13. The process as claimed in claim 9, wherein the fermenting organism is an
Actinomycete species.
14. The process as claimed in claims 1 to 13, wherein the productivity is increased by at
least 20%
15. The fermentation process for the production of secondary metabolites is
substantially as herein described along with accompanying drawings and examples.



Documents:


Patent Number 248816
Indian Patent Application Number 1175/CHE/2008
PG Journal Number 36/2011
Publication Date 09-Sep-2011
Grant Date 26-Aug-2011
Date of Filing 14-May-2008
Name of Patentee BIOCON LIMITED
Applicant Address 20TH KM, HOSUR ROAD, ELECTRONIC CITY,BANGALORE-560 100.
Inventors:
# Inventor's Name Inventor's Address
1 SAURABH GARG H.NO. 15- ASHIRVAD ENCLAVE, STREET NO.10 CHAKRATA ROAD, DEHRADUN,UTTARANCHAL-248001.
2 BIMALKUMAR PLOT NO. A3/15, PURNENDU NAGAR, KHOJA IMALI MAZAAR, PHULWARI SHARIF. PATNA 801508.
3 SANJAY TIWARI 20/A, 11TH CROSS, 6TH A MAIN. JP NAGAR 3RD PHASE, BANGALORE-560 078.
4 ANUJ GOEL H. NO. 72, 10TH CROSS, 29TH MAIN, 1ST PHASE, JP NAGAR, BANGALORE-560 078.
5 CHITTNALLI RAMEGOWDA NAVEEN KUMAR HOUSE NO. 108, 2ND CROSS, HMT LAYOUT, MATHIKERE, BANGALORE-560054.
6 HARISH IYER 5C-221, 3RD MAIN ROAD, HRBR 3RD BLOCK, BANGALORE 560043.
PCT International Classification Number C12P1/02
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA