WO2010103548A2

WO2010103548A2 - Improved method for producing lactic acid and derivative thereof

Info

Publication number: WO2010103548A2
Application number: PCT/IN2010/000146
Authority: WO
Inventors: Samir S. Somaiya; Sangeeta Srivastava; Martin Annette; Amol Dive
Original assignee: Godavari Biorefineries Ltd.
Priority date: 2009-03-13
Filing date: 2010-03-12
Publication date: 2010-09-16
Also published as: WO2010103548A3

Abstract

The present invention provides a mutagenized strain of Bacillus coagulans, MTCC 5454, and discloses its use in the production of Lactic acid. The invention also provides an efficient, rapid method for increased production of lactic by using the strain MTCC 5454. The present invention uses inexpensive natural sugars as carbon source for conversion to lactic acid.

Description

TITLE

IMPROVED METHOD FOR PRODUCING LACTIC ACID AND DERIVATIVE THEREOF.

FIELD OF THE INVENTION:

The present invention relates to an improved method for producing lactic acid. In particular, the invention also relates to the mutagenized strain of microorganism, Bacillus coagulans (MTCC 5454). More in particular, the invention relates to rapid and increased production of lactic acid. The lactic acid produced by the method and strain of the current invention is optically pure.

BACKGROUND OF THE INVENTION:

Lactic acid (2-hydroxypropanoic acid), also known as milk acid, is known to play an essential role in several biochemical processes. It is also used in many industries for various purposes. In the food industry it is commonly used as a food additive, as an acidulant and preservative. Similarly it is used in the chemical industry as oxychemicals, green solvents, and specialty chemicals for deliming, metal etching, cosmetic and textile applications. The L (+)-lactic acid isomer is used in the production of dairy products, as an acidulating agent in the food industry, as a mordant in wool dying and, as an intermediate for the production of plasticizer agents, adhesives and pharmaceutical product.

Currently there is growing interest in the production of lactic acid for use in synthesis of biodegradable polymer materials. The lactic acid is used as a monomer for producing polylactic acid (PLA) for biomedical applications. The polylactic acids have potential applications in the medical field as prosthetic devices and devices for controlled drug delivery in humans. It is also used in food industry for packaging. The polylactic acids are l also used for synthesis of ethanol, a cost efficient alternative fuel source. Moreover the plastics made from lactic acids can be substituted for conventional plastics produced from petroleum oil owing to their lower emission of carbon dioxide thus reducing global warming.

In view of the benefits described above, the interest in lactic acid have raised progressively. Efforts are evident from the various studies that have focused on the production of lactic acids using various methods and microorganisms. Lactic acid is commercially produced by fermentation of carbohydrates using microorganisms. Various lactic acid-producing microorganisms have been reported and include lactic acid bacteria of the genera Lactobacillus, Lactococcus, Bacillus, Escherichia coli; yeasts of the genus Saccharomyces, and filamentous fungi of the genus Rhizopus. However, the optical purity of the lactic acid produced using lactic acid bacteria is low. With other microorganisms the yield is generally low, due to large amount of the by-products formed. For example, it is observed that both native and recombinant yeast strains produce large amount of ethanol, whereas filamentous fungus produces large amount of glycerol, ethanol and fumaric acid. Although production of Lactic acid from various sugars is also known in the art, the methods described are more expensive, require very long period for fermentation and productivities are low.

Based on the current status of production, supply and demand of Lactic acid, there is a need for an alternative and more efficient lactic acid producing strain of microorganism and method for production of lactic acid. Furthermore there is urgent need in the art for the production of purer forms of lactic acid. The current invention overcomes the problems described above and provides a new and improved strain of bacterium for increased and rapid production of lactic acid. Further the lactic acid produced by the method and strain of the current invention is purer. The invention further discloses the use of inexpensive natural sugar sources, such as sugarcane juice and molasses, as substrate for the production of Lactic acid.

DISCLOSURE OF THE INVENTION:

It is the object of the present invention to provide a method of producing lactic acid and/or derivative thereof by the use of the mutagenized strain of Bacillus coagulans (MTCC 5454).

It is further the object of the present invention to provide a rapid and increased production of lactic acid and/or derivative thereof.

It is yet another object of the invention to produce optically pure form of lactic acid and/or derivative thereof.

A further object of the current invention related to the use of inexpensive natural sugar sources as substrates for the production of lactic acid.

SUMMARY OF THE INVENTION:

The present invention relates to a new and improved method for producing lactic acid and/or derivative thereof by use of the mutagenized strain of Bacillus coagulans (MTCC 5454). The invention also relates to rapid and increased production of lactic acid and/or derivative thereof. The invention further relates to production of lactic acid and/or derivative thereof that is optically pure and can be obtained from inexpensive sources. BRIEF DESCRIPTION OF THE FIGURES;

Figure 1, 2 and 3 represent the percentage of lactic acid (LA) produced from reducing sugar (RS) at various time points by Bacillus coagulans MTCC 5454.

Figure 4 represents the percentage of lactic acid (LA) produced from reducing sugar at various time points by native wild type Bacillus coagulans.

DETAILED DESCRIPTION OF THE INVENTION:

The present invention provides a method of production of lactic acid and/or derivative thereof, by the microorganism Bacillus coagulans (MTCC 5454) comprising culturing the micro-organism coagulans (MTCC 5454) in a nutrient medium and/or fermentation medium according to the invention.

The lactic acid "derivative" as used herein refers to any compound modified in at least one chemical element and/or group compared to Lactic acid of the invention. However, the derivative expresses a biological activity similar to and/or the same as that of Lactic acid. The Lactic acid derivative may be obtained by further modifying the microorganism strain of the present invention and/or modifying at least one step of the process of Lactic acid preparation. Accordingly, any reference to Lactic acid encompasses any possible Lactic acid derivative and/or similar compound.

According to one aspect of the current invention there is provided a micro-organism, Bacillus coagulans (MTCC 5454).

According to another aspect of the current invention there is provided a culture comprising of a micro-organism of species Bacillus coagulans (MTCC 5454), wherein the Bacillus coagulans (MTCC 5454) is mutagenized strain. The strain may be capable of producing lactic acid and/or derivative thereof in an aqueous nutrient culture medium and/or fermenting medium. The culture comprising mutagenized Bacillus coagulans (MTCC 5454) may be capable of higher production of Lactic acid and/or derivative thereof compared to wild type strain.

The microorganism used in the current invention was isolated from soil samples collected from the vicinity of Sakarwadi distillery, located at, Ahmednagar, Maharashtra, India and cultured by standard culture procedures known in the art. The strain was cultured in a medium appropriate for the production of lactic acid. The microorganism was identified as Bacillus coagulans based on the taxonomic characteristics. The isolated microorganism is Gram-positive, spore-forming, motile rods, 0.9 micrometer by 3.0 micrometer to 5.0 micrometer in size, aerobic to microaerophilic as all other species of its genus. S. coagulans forms endospores, resistant to chemical and physical agents. B.coagulans was mutagenized by ultra violet mutation (dark repair mechanism). Any other methods mutagenesis known in the art may also be used and will fall within the scope of the invention. The ultra violet method of mutagenesis was chosen in the current invention for introducing random mutations. Mutagenized isolates are subjected to enrichment, biochemical characterisation and productivity of lactic acid was checked.

After several generations of mutations, a strain capable of producing more lactic acid as analyzed by HPLC method was selected and subjected to several generations of culture passages to attain stability. The yield of Lactic acid was analyzed by HPLC however, other methods for analysis of the lactic acid yield known in the art may also be used and shall fall within the scope of the current invention. The new strain thus selected was deposited with Microbial Type Culture Collection and gene bank (MTCC), at Institute of Microbial Technology (IMTECH), Sector39-A, Chandigarh, India. The deposit number is MTCC 5454.

The new mutagenized strain Bacillus coagulans MTCC 5454 provided by the invention was identified based on biochemical test (Table I).

TABLE 1

According to yet another aspect of the invention there is provided a method for production of lactic acid and/or derivative thereof, comprising

a) Culturing the microorganism Bacillus coagulans (MTCC 5454) in an aqueous nutrient culture medium and/or fermenting medium; and

b) recovering lactic acid from the fermentation broth.

The aqueous nutrient culture medium and/or fermenting medium may comprise

a) at least one carbon source; and

b) at least one nitrogen source; and/or

c) micro-nutrients and/or trace minerals; The "culture medium" may be the conventional media used for Bacillus spp. or any known media in the field. The said medium may comprise carbon source and nitrogen source and/or carbon source. The "fermentation medium" may further contain other organic and/or in-organic materials so as to accelerate growth of the microorganism and conversion of carbon to lactic acid to increase the rate of lactic acid production. The carbon source may be selected from the group consisting of glucose, maltose, fructose, glycerol, starch, lactose, galactose, sugarcane juice, jaggery, molasses, sugarbeet, wheat, potatoes, rice and sweet sorghum, wherein the carbon source may be 20%.. The Nitrogen source may be selected from the group consisting of peanut, powder of soybean, corn, yeast powder, peptone, whey, yeast extract, ammonium nitrate, and/or ammonium chloride, casein, malt extract and Soya, wherein the nitrogen source may be atleast 0.1%(w/v), atleast 0.25%(w/v), atleast 0.5% (w/v), atleast 1% (w/v).

Sugar medium such as sugarcane juice, sugar and/or molasses are the most preferred carbon source as they are easily available for lactic acid production. Sugar cane juice may be used being a natural and inexpensive starting material. Therefore aqueous nutrient and/or fermentation medium of the present invention is highly economical compared to sugars used in conventional methods. In other embodiments, the growth substrate that may be used, may be selected from but not limiting to inexpensive materials, such as peptone, soy, corn, whey, potatoes, rice, molasses of beet-sugar corn steep liquor and yeast extract, can be used as side starting materials. The concentration of proteinaceous and/or micronutrients in fermentation media may be about 1% to 2%. The fermentation media may also contain pH regulators, such as acids preferably, sulphuric acid, alkali such as calcium carbonate, calcium hydroxide, caustic soda. The fermentation medium may further comprise inorganic nutrients like phosphate, magnesium salts such as magnesium sulphate, ferric salts such as ferrous sulphate, ferric chloride, potassium salts such as potassium di- hydrogen phosphate but not limited to these. The fermentation medium may further comprise, but not limited to, trace minerals, such as boric acid, potassium iodide, cobalt dichloride, zinc sulphate, manganese sulphate. The fermentation medium may also comprise, inducers, such as methyl jasmonic acid, arachidonic acid, aminocitrate, ferric ammonium nitrate, potassium permanganate, pyruvic acid, p-coumaric acid, vanadic sulfate, hippuric acid, α-naphthylacetic acid, 6-benzylamine purine, silver nitrate, and cinnamic acid but should not be considered to be limiting to these. The fermentation medium may also comprise but not limited to precursors, such as phenylalanine, benzamide, sodium benzoate, sodium acetate, propionamide, benzoic acid, and acetamide.

The aqueous nutrient culture medium and/or fermenting medium may further comprise glucose, maltose, fructose, glycerol, starch, lactose, galactose, powder of peanut, powder of soybean, corn steep solid, yeast powder, peptone, yeast extract, ammonium nitrate, and/or ammonium chloride for growth of Bacillus coagulans (MTCC 5454). .

The method of production of Lactic acid and/or a derivative thereof may be by fermentation and/or conversion of carbon source. In particular the fermentation may be carried out at 40-50 ° C and at a pH of 5 - 7. More in particular, the fermentation may be carried out for about 30 hours.

The culture may be subjected to conventional and/or known fermentation conditions in the art. The fermentation may be under anaerobic conditions at the temperature of at least 40°C. In particular the temperature may be in the range of at least 40-50°C. The fermenting pH value may be at least 5.0. In particular the pH may be in the range of 5.0-7.0. The pH value may be adjusted in the mid and late phases of fermentation.

Bacillus coaaulans (MTCC 5454) may be cultured by batch fermentation methods for production of lactic acid. The fermentation may be carried in stirred tank reactor and/or use of known apparatus, conditions and/or methods known in the art, for example, oscillating the bottle under a speed rate known for the particular apparatus, condition and/or method. The fermentation may also be carried out in conventional fermentation tanks, such as the 7L and 50L tanks. As Bacillus coaaulans (MTCC 5454) is facultative anaerobe (microaerophilic) so it does not require strict anaerobic conditions, it grows well in stirred tank reactor. The fermentor may be inoculated with an established culture of microorganism Bacillus coaaulans (MTCC 5454).

The Lactic acid and/or derivative thereof may be recovered by any methods known in the art. In particular membrane separation, ion exchange, solvent extraction, electro dialysis and/or precipitation of lactate salts methods may be used. The productivity of Lactic acid and/or derivative thereof, obtained using the current strain and/or methods of the present invention may be in the range of 4-6g/L/Hr. In particular the lactic acid productivity may be at least 4.5 g/L/Hr. In particular, the Lactic acid and/or derivative thereof thus obtained may be at least 99% pure. The optically pure lactic acid and/or derivative thereof may be L - Lactic acid.

The method according to the invention is of shorter cycle, lower cost, and higher production rate. Further, it allows an efficient mass production of Lactic acid if it is used in industry. Its industrial application cannot only protect natural resources, but satisfies the requirement of the Lactic acid demand as well. An essential feature of the invention is that, the strain Bacillus coagυlans MTCC 5454 in culture is capable of higher conversion of the carbon sources, thus capable of producing higher yield of lactic acid. Therefore the current invention solves an essential problem of low yield and offers an advantage over microbial fermentation methods available in the prior art. The technology of the current invention may be characterized by a short cycle, low cost, and high fermentation rate. The method gives increased yield in 30 hrs. Therefore the lactic acid production method described herein may be applied for industrial production. The use of natural sugar sources as substrate for conversion to lactic acid, contributes to protecting the natural environments yet being able to meet the demand.

While the invention has been described with particular reference to certain embodiments thereof, it will be understood that various modifications can be made to the above- mentioned embodiments without departing from the spirit and scope of the present invention. The examples and the particular proportions set forth are intended to be illustrative only.

EXAMPLES

EXAMPLE 1:

The Bacillus coagulans_(MJCC 5454) mutant strain was first grown onto agar slants. The microorganism was then inoculated into three screw cap tubes containing 10 -15ml of MRS broth. The tubes were incubated at a temperature ranging from 40-50⁰C for 24-36 hrs in a shaker at 150 rpm. 50ml of the above inoculum was added to a 1000 ml flask containing 450 ml of fermentation media prepared by conventional acid hydrolysis of molasses with an initial reducing sugar (RS) content of 15.1% along with micronutrients 1% peptone, 0.05%

MgSO₄, 0.02% MnSO₄, and 6.5% CaCO₃ and incubated at 40° C for 35 hrs. The pH was maintained at 5.5. The concentration of lactic acid produced was 14.4%. Lactic acid (LA) thus produced was isolated and analyzed by the standard practice. The volumetric productivity of lactic acid was found to be 4.5g/L/Hr. Lactic Acid was purified by conventional method for obtaining 99.9% optically pure L-lactic acid.

EXAMPLE 2:

The Bacillus coagulans (MTCC 5454) mutant strain was first grown onto agar slants. The microorganism was then inoculated into screw cap tubes containing 5 ml of MRS broth. The tubes were incubated at a temperature ranging from 40-50⁰C for 24-36 hrs in a shaker at 150 rpm. The 45 ml of the above inoculum was added to a 1000 ml flask containing 450 ml of fermentation media prepared by conventional acid hydrolysis of molasses with an initial reducing sugar (RS) content of 15.6% along with micronutrients 1% yeast extract, 0.5% MgSO₄ ,0.2% MnSO₄, 2% Ca(OH)₂ and 6.5% CaCO₃ and incubated at 46° C for 31 hrs. The pH was maintained at 5.5. The concentration of lactic acid produced was 15.4%. Lactic acid thus produced was isolated and analyzed by the standard practice. The volumetric productivity of lactic acid was found to be 4.9g/L/Hr. Lactic Acid was purified by conventional method for obtaining 99.9% optically pure L-lactic acid.

EXAMPLE 3:

The Bacillus coagulans (MTCC 5454) mutant strain was first grown onto agar slants. The microorganism was then inoculated into screw cap tubes containing 5 ml of MRS broth. The tubes were incubated at a temperature ranging from 40-50⁰C for 24-36 hrs in a shaker at 150 rpm. The 45 ml of the above inoculum was added to a 1000 ml flask containing 450 ml of fermentation media prepared by conventional acid hydrolysis of molasses with an initial reducing sugar (RS) content of 15.8 % along with micronutrients 1% yeast extract, 0.5% MgSO₄, 0.2% MnSO₄, 2% Ca(OH)₂ and 6.5% CaCO₃ and incubated at 48° C for 30 hrs. The pH was maintained at 5.5. The concentration of lactic acid produced was 15%. Lactic acid thus produced was isolated and analyzed by the standard practice. The volumetric productivity of lactic acid was found to be 5g/L/Hr. Lactic Acid was purified by conventional method for obtaining 99.9% optically pure L-lactic acid.

EXAMPLE 4: (Native wild type strain):

The Bacillus coagυlans wild type strain was first grown onto agar slants. The microorganism was then inoculated into screw cap tubes containing 15 ml of MRS broth. The tubes were incubated at a temperature ranging from 40-50⁰C for 24-36 hrs in a shaker at 150 rpm. The 45 ml of the above inoculum was added to a 1000 ml flask containing 450 ml of fermentation media prepared by conventional acid hydrolysis of molasses with an initial reducing sugar (RS) content of 11.57% along with micronutrients 1% peptone, 0.05% MgSO₄, 0.02% MnSO₄, 2% Ca(OH)₂ and 6.5% CaCO₃ and fermented at 49° C for 45 hrs. The pH was maintained at 5.5. The concentration of lactic acid was found to be 11.5 %. The volumetric productivity of lactic acid was 2.6 g/L/Hr.

Claims

1. A micro-organism, Bacillus coagulans MTCC 5454.

2. A culture comprising of a micro-organism Bacillus coagulans (MTCC 5454), capable of producing lactic acid and/or derivative thereof in an aqueous nutrient culture medium and/or fermenting medium.

3. The culture according to claim 2, wherein the Bacillus coαgu/αns_(MTCC 5454) is mutagenized and is capable of higher production of Lactic acid and/or derivative thereof compared to wild type strain.

4. The culture according to claim 2 wherein, the aqueous nutrient culture medium and/or fermenting medium comprises

a) at least one carbon source; and

b) at least one nitrogen source; and/or

c) micro-nutrients and/or trace minerals;

5. The culture according to claim 2, wherein the aqueous nutrient culture medium and/or fermenting medium further comprises glucose, maltose, fructose, glycerol, starch, lactose, galactose, powder of peanut, powder of soybean, corn steep solid, yeast powder, peptone, yeast extract, ammonium nitrate, and/or ammonium chloride for growth of Bacillus coagulans (MTCC 5454).

6. The culture according to claim 4, wherein the carbon source is selected from the group consisting of glucose, maltose, fructose, glycerol, starch, lactose, galactose, sugarcane juice, jaggery, molasses, sugarbeet, wheat, potatoes, rice, jowar and sweet sorgham.

7. The cell culture according to claim 4, wherein the Nitrogen source is selected from the group consisting of peanut, powder of soybean, corn, yeast powder, peptone, whey, yeast extract, ammonium nitrate, and/or ammonium chloride, casein, malt extract and Soya.

8. A method for production of lactic acid and/or derivative thereof, comprising

a) Culturing the organism of claim 1 in an aqueous nutrient culture medium and/or fermenting medium; and

b) recovering lactic acid from the fermentation broth.

9. The method according to claim 8 wherein, the aqueous nutrient culture medium and/or fermenting medium comprises

a) at least one carbon source; and

b) at least one nitrogen source; and/or

c) micro-nutrients and/or trace minerals;

10. The method according to claim 8, wherein the aqueous nutrient culture medium and/or fermenting medium further comprises glucose, maltose, fructose, glycerol, starch, lactose, galactose, powder of peanut, powder of soybean, corn steep solid, yeast powder, peptone, yeast extract, ammonium nitrate, and/or ammonium chloride for growth of Bacillus coagulans (MTCC 5454).

11. The method according to claim 9, wherein the carbon source is selected from natural sources comprising glucose, maltose, fructose, glycerol, starch, lactose, galactose, sugarcane juice, jaggery, molasses, sugarbeet, wheat, potatoes, rice and sweet sorghum.

12. The method according to claim 9, wherein the nitrogen source is selected from the group consisting of peanut, powder of soybean, corn, yeast powder, peptone, whey, yeast extract, ammonium nitrate, and/or ammonium chloride, casein, malt extract and Soya.

13. The method according to claim 8, wherein the production of lactic acid and/or a derivative thereof is by fermentation and/or conversion of carbon source.

14. The method according to claim 13, wherein, the fermentation is carried out at 40-50 ° C and at a pH of 5- 7.

15. The method according to claim 13, wherein the fermentation and/or conversion of carbon source is carried out at for about 30 hours.

16. The method according to claim 8, wherein the Lactic acid and/or derivative thereof is recovered by membrane separation, ion exchange, solvent extraction, electro dialysis and/or precipitation of lactate salts.

17. The method according to claim 16, wherein the productivity of Lactic acid and/or derivative thereof, obtained is at least 4.5 g/L/Hr.

18. The method according to claim 17 wherein the Lactic acid and/or derivative thereof is at least 99% optically pure.

19. The method according to claim 18 wherein the optically pure Lactic acid and/or derivative thereof is L-Lactic acid.

20. A method for production of lactic acid and/or derivative thereof as described herein above and illustrated by drawings and example.