THE PATENTS ACT, 1970
COMPLETE SPECIFICATION
Section 10
"Process for Efficient Production of 1,3-Propanediol"
Tata Chemicals Pvt. Limited, Leela Business Park, Andheri KInula Road, Andheri (E) Mumbai, India.
The following specification particularly describes the nature of this invention and the manner in which it is to be performed:
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Field of the invention
The instant invention provides for a novel process for production of 1,3-propanediol. More particularly, the invention pertains to an environmentally friendly procedure for the production of 1,3-propanediol using Citrobacter sp.
Background of Invention
1,3-Propanediol is a three-carbon diol. It is also known as Trimethylene glycol. Recent application of 1,3-propanediol as a monomer for novel polyester and biodegradable plastics has lead to strong market need for production of this bulk chemical preferably by biological route. The main advantage of this biodegradable polyester compared to polyhydroxy alkanoate or polylactides (two bio degradable polyesters from renewable sources) is the possibility that the physical properties of the polyester can be easily modified to meet the manufacturing specifications for the final article made from the plastic. Realizing the importance of 1,3- propanediol and its application in different industries it is desirable to develop methods, for maximum production of this diol.
Traditionally, this important diol has been produced by chemical routes using propylene or ethylene derived petroleum. However, these methods make use of nonrenewable resources (i.e capital intensive) and generate a lot of pollution. Therefore, today there is a paramount interest in producing 1,3-propanediol using microorganisms by fermentation processes focusing on the environmental aspect, industrial safety and renewable nature of this mode of production.
1,3 -propanediol is generally produced by chemical method such as by reduction of ethylglycidate with lithium aluminium hydride, by reduction of ethtylene oxide in presence of phosphine, water, CO, H2, air or by hydration of acrolein followed by reduction. All these methods possess lacunas as these are either capital intensive or/ and. result in the production of hazardous and toxic byproducts. Thus, chemical methods cannot be used for routine production of 1,3- propanediol. The present invention was done and an eco-friendly and cost-effective procedure for production of 1,3-propanediol
was developed to harness the undoubted commercial potential of anaerobic microorganisms, which produce this important diol.
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Realizing the importance of 1,3- propanediol and its application in different industries, the biological methods for the production of 1,3- propanediol is of great importance. In this direction, it is claimed that the procedure developed by us could successfully be used for production of 1,3-propanediol through biological route. This method is environmentally friendly and cost effective 'green technology'. It is worth patenting procedure as the procedure is 100% environmentally benign. The development of this technology may give the industries new ways to make valuable chemicals and as a result there may be strategic shift of the major chemical companies in to this area.
1,3-propanediol is a typical product of glycerol fermentation and can not be produced microbially by the conversion of any other organic substrate. Only very few organisms, all of them bacteria, are able to form it. In earlier times, high price of glycerol was prohibitive for the production of 1,3-propanediol through biological means. A lot has. changed since on both the glycerol and the l,3propanediol site. Glycerol, being a constituent of natural fats and oil can be regarded as re-growing resource available in surplus.
Biotechnological method to produce 1,3-propanediol is important because it is favorable for environmental protection and its raw material is cheap. The biotechnological method can also be the most appropriate method for the production of 1,3-propanediol if petroleum resources become exhausted.
The production of 1,3-propanediol using anaerobic bacteria has been found to not very efficient and recombinant bacteria have been tried to produce 1,3-propanediol from glycerol. US6953684 and US 6136576 discloses method for the recombinant production of 1,3-propanediol. WO 9821341 provides for a method for the recombinant production of 1,3-propanediol using protein X, protein 1, protein 2, and protein 3 for dehydratasereactivation. However, the use of recombinant bacteria, increases the initial cost and also requires a complicated procedure to maintain the bacteria. Also recombinant bacteria, not being stable, . further add to maintenance cost.
In view of the above, the present invention discloses an ecofriendly and cost effective and simple biological method for the production of 1,3-propanediol using renewable resources.
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Summary of the Invention:
The production of 1,3-propanediol from glycerol using anaerobic fermentation in presence of Citrobacter by using a novel medium composition which allows optimum production has been obtained. The process allows intake of high concentration of glycerol as raw material for natural bacteria without harming the 1,3-propanediol production. The use of a simple process for efficient production of 1,3-propanediol in less incubation time has been envisaged in the instant invention.
The invention provides for a process for the maximum production and optimization of a glycerol from renewable resources in the presence of a suitable microbe/microorganism and reaction medium either comprising of organic or inorganic medium maintaining pH, temperature, agitation rate, inoculum density, incubation period and nutritional factors like substrate concentration, nitrogen source and its concentration, carbon source and its concentration, metal used as additive.
Brief Description of Drawings
Figure.l: Experiments at different pH values showing corresponding production of 1,3-propanediol.
Figure 2: Experiments at different temperature showing corresponding production of 1,3-propanediol.
Figure 3: Experiments at different concentration of glycerol showing corresponding production of 1,3-propanediol
Figure 4: Experiments with different carbon additives showing corresponding production of 1,3-propanediol
Figure 5: Experiments at different concentration of selected carbon additive showing corresponding of 1,3-propanediol production.
Figure 6: Experiments with different organic and inorganic nitrogen sources showing corresponding production of 1,3-propanediol
Figure 7: The use of Tryptone as nitrogen source and amount of 1,3-propanediol production.
Figure 8: Experiments with different metal ion showing corresponding production of 1,3-propanediol
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Figure 9: The use of Magnesium (Mg ) metal ion and corresponding production of 1,3-propanediol
Figure 10: Experiments with different inoculum density showing corresponding production of 1,3-propanediol.
Detailed Description of the invention:
In the present invention, production and process optimization of 1,3-propanediol from glycerol was carried out using Citrobacter- sp. The production of 1,3-propanediol from glycerol using anaerobic fermentation in presence of Citrobacter by using a novel medium composition which allows optimum production in less incubation time. The process allows intake of high concentration of glycerol as raw material by the natural bacteria without harming the 1,3-propanediol production.
The invention is herewith explained with the help of different experiments, the details of the same being described below:
Screening and selection of 200 anaerobic and facultative anaerobic bacteria was carried out to evaluate ability to produce 1,3-propanediol from glycerol. Production medium contained the following medium components. Glycerol, Glucose, K.2HP043H20, KH2P04, (NH4)2SO4, MgS04.7H20, CaCl2.2H20, CoCl2.6H20, Yeast Extract, Bactopeptone, Beef extract. The bacterial isolates CB-1 (a Citrobacter sp.), was selected for developing the process for 1,3-propanediol .
Experiments for physiological factors such as pH and temperature of the production medium, agitation rate, inoculum density and incubation period used for maximum production of 1,3-propanediol were as follows:
The pH of the medium was used in the range of 6.0-12.0 adjusted with different buffers. Production of 1,3-propanediol was carried out at different temperatures ranging from 20°C to 50°C.
Example 1: the process was carried out at pH 7.0 and 30°C and 24 h of incubation. The production of 1, 3-propanediol during such condition was found to be 2.48 g/L (Fig .1).
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Example 2: 2.50 g/L of 1,3- propanediol was produced at 30° C in 24 h incubation time (Fig .2).
Nutritional factors such as substrate concentration, type of carbon source and its concentration, nitrogen source and its concentration, metal ions, etc were optimized for maximum production of 1,3- propanediol.
Experiments were carried out at different concentrations of glycerol viz. 0.5 - !0.0 %. The best mode of carrying out the experiment is by using 2.0 % concentration of glycerol.
This is one of the major achievements of the invention, wherein it is discovered that such high concenteration of glycerol does not affect the 1,3-prppandiol production.
Example 3: The anaerobic fermentation containing 2% glycerol resulted in high yeids. Citrobacter sp. produced 3.18g/l of 1,3-propandiol when the medium contained 20 g/L of glycerol (Fig .3).
Example 4: Citrobacter sp. produced 3.20g/l of 1,3-propandiol when the medium contained 20g/Lof glycerol (Fig .4).
Selection of other carbon additive: glycerol was used as the main carbon substrate , other carbon sources such as glucose, fructose, lactose, sucrose, maltose, cane molasses and whey were used as additives at the concentration of 0.1 % . Glucose is the most preferred additive.
Example 5: A preferred range of concentration of glucose viz. 0.05% - 0.3% was used in several experiments, the result showed that 0.05% - 0.2% were preferred, 0.15 % being the optimum concentration of glucose. Citrobacter sp. produces 3.4 g/1 of 1,3-propandiol when the medium contained 0.15 % of glucose and 20 g/L of glycerol (Fig .5)..
Different organic nitrogen sources such as peptone, tryptone, beef extract, yeast extract, malt extract, corn steep liquor (CSL).and inorganic nitrogen sources such as ammonium sulphate, sodium nitrate and urea were used in different experiments for their effect on propanediol production(Fig .6). Tryptone was selected as the most preferred nitrogen source.
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Example 6: Different concentration of tryptone viz. 1 - 5% were tried. Citrobacter sp. produces 4.50 g/1 of 1,3-propandiol when the medium contained 2% is the optimum concentration of tryptone (Fig .7).
Example 7: Metal ions like Na, Mg, Ca, Fe, Mn, K, Zn and Co were used as additives at 1.0 % concentration on 1,3-propanediol production. 5.20 g/1 of 1,3-propandiol was produced when the medium contained 1.0 % of Mg ions (Fig .8).
Example 8: Different salts like chloride, carbonate, sulphate and phosphate of magnesium ion were used at 1.0 % concentration 5.80 g/1 of 1,3-propandiol was obtained when MgCO3 (1.0%) was used (Fig .9).
Example 9: Production of 1,3-propanediol was examined at different agitation rates i.e. 100,150, 200, 250 and 300 rpm. Maximum propanediol production i.e..5.80 g/1 was observed at 150 rpm.
Example 10: Different inoculum densities ranging from 1% to 8 % was tested for 1,3-propanediol production. 4.0 % inoculum density produced 7.0 g/1 1,3-propanediol (Fig. 10).
Example 11: Production of 1,3-propanediol was carried out after different incubation period. Maximum production of 1,3-propanediol was obtained with in 24h (Table 1).
Table 1: Different experiments carried out at different incubation times

Incubation time (h) 1,3- propanediol (g/L)
0 -
6 0.8
12 2.0 '
18 4.6
24 7.0
30 7.0
36 7.2
42 6.8
48 6.9
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Response surface methodology (rsm) for optimization of 1,3-propanediol production was carried out for the parameters found useful for 1,3-propanediol production.
Although "one at a time" approach leads to a significant enhancement in 1,3-propanediol production, the interaction of the optimized factors collectively may also influence further production. Keeping in view the interactions between the most influential physical and nutritional factors of the "one at a time" optimized medium, maximum propanediol production was obtained by using a statistical design approach - Face Centered Central Composite Design (FCCCD), falling under the response surface methodology (RSM).
The FCCCD incorporates replication of medial point (000). In this method, the concentration of the components - Glycerol, tryptone, MgC03, inoculum density and incubation period whose influences were found significant by "one at a time" approach,
were further optimized. The ranges of these variables were decided according to the optimum value determined by the "one variable at a time" method. Other variables were set at their optimum levels. The minimum and maximum ranges selected for the medium components are presented in the Table 2 and the design matrix of 32 experiments were generated using Design Expert software 6.0 (Stat Ease Inc., Minneapolis, USA). The details of the experimental design are presented in Table 3.
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Table 2: Experimental range and levels of the five independent variables used in RSM in terms of actual and coded factors
Variables Actual Coded Actual Actual Coded
(zero)
Glycerol (%) 1.0 2.0 3.0 +1
Tryptone (%) 1.5 2.0 2.5 +1
Magnesium Carbonate (%) 1.0 1.5 2.0 +1
Inoculum density (%) 3.0 4.0 5.0 +1
Incubation period 12 24 36 +1
Thus, the optimum composition obtained by the response surface methodology wherein the concentration of glycerol 2.0 %, tryptone 1.5 %, MgCO3 2% , inoculum density 3% at agitation rate of 150 rpm and incubation period of 24h. This resulted in the production of 11.51 g/L of 1,3-propanediol, which is an approximately 1.5-fold increase in the propanediol production as obtained through "one at a time" method (7.2 g/L).
Subsequently, the production was carried out in 10 L fermentor with a working volume of 7.5 under conditions optimized by using RSM.
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Table 3; Experimental design matrix efface centered central composite design for 1,3-PD production (For experiments R-1 to R-32)

Successful and Efficient scale up has also been achieved in bioreactors, some of the examples are illustrated below:
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Example 12: Large-scale production of 1,3-propanediol production by Citrobacter sp. was carried out in a 10 L fermentor (New Brunswick Scientific, USA) with 7.5 L of the optimized production medium. The optimized medium and production conditions as obtained by "Reponse surface Methodology" in 500 ml bottles were translated identically in a 10 L bioreactor. Other operating parameters such as were pH. 7.0; agitation rate, 150 rpm; temperature 30° C, air flow rate at 0.6 vvm were kept at their optimum level. Ail the medium components were steam sterilized at 121° C for 15 minutes in situ.
Example 13: Effect of different vvm of airflow were carried out in 10 L fermentor (New Brunswick Scientific, USA) with 7.5L of the medium by Citrobacter sp. for the production of 1,3-propanediol and the results are shown in Table 4.
Table 4: Different experiments yield based on the carbon dioxide air-flow in a 10 L bioreactor.

Air flow (wm) 1,3-propanediol (g/L)
0.4 . 9.3
0.5 10.5
0.6 13.0
0.7 12.8
0.8 10.7
Example 14: Effect of different vvm of airflow as standardized in 30 L fermentor (Scigenics) with 15L of the medium by Citrobacter sp. for the production of 1,3-propanediol are shown in Table 5. Table 5:

Air flow (wm) 1,3-PD (g/L)
0.4 9.5
. 0.5 11.6
0.6 13.0
0.7 12.3
0.8 11.2
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The results showed that a maximum of 13.0g/L of 1,3-propanediol was produced in 24h. The sugar concentration decreased linearly with the increase in the growth and propanediol production.
While the present invention has been described with respect to certain preferred embodiments, it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the scope of the invention as defined in the following claims.
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We claim:
1. A process for efficient production of 1,3-Propanediol comprising incubating a natural microorganism, Citrobacter in a reaction medium comprising of high glycerol substrate concentration, a nitrogen source, and additives including a second carbon source, and metal ions; at pH 6.0 to 7.5, maintained at 25°C to 35°C temperature, with lOOrpm to 200rpm agitation, 3.0% to 5.0 % inoculum density, for an incubation period of 24 hrs, and optionally providing an air flow at the rate of 0.4 to 0.8 vvm.
2. The process for efficient production of 1,3-Propanediol as claimed in claim 1 wherein glycerol is in 0.5 - 7.0 % concentration.
3. The process for efficient production of 1,3-Propanediol as claimed in claim 2 wherein said glycerol concentration is 2.0 %.
4. The process for efficient production of 1,3-Propanediol as claimed in claim 1 wherein said nitrogen source comprises organic nitrogen sources including peptone, tryptone, beef extract, yeast extract and inorganic nitrogen sources including ammonium sulphate, sodium nitrate and urea
5. The process for efficient production of 1,3-Propanediol as claimed in claim 4 wherein said nitrogen source is tryptone.
6. The process for efficient production of 1,3-Propanediol as claimed in claim 5 wherein said tryptone concentration is 1 %- 5%.
7. The process for efficient production of 1,3-Propanediol as claimed in claim 7 wherein said tryptone concentration is 2%.
8. The process for efficient production of 1,3-Propanediol as claimed in claim 1 wherein said second carbon source includes glucose, fructose, lactose and sucrose.
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9. The process for efficient production of 1,3-Propanediol as claimed in claim 9 wherein said second carbon is glucose in a concentration 0.05% to 0.3%.
10. The process for efficient production of 1,3-Propanediol as claimed in claim 10 wherein said glucose concentration's 0.15%.
11. The process for efficient production of 1,3-Propanediol as claimed in claim 1 wherein said metal ion is selected from Sodium Na, Magnesium Mg, Calcium Ca, Iron Fe, Manganese Mn, Potassium K, Zinc Zn and Cobalt Co.
12. The process for efficient production of 1,3-Propanediol as claimed in claim 12 wherein said metal ion is Magnesium.
13. The process for .efficient production of 1,3-Propanediol as claimed in claim 12 wherein said magnesium ion concentration is 0.5 to 3.0% .
14. The process for efficient production of 1,3-Propanediol as claimed in claim 13 wherein said magnesium ion concentration is 1% .
15. The process for efficient production of 1,3-Propanediol as claimed in claim 12-14 wherein said magnesium is in the form of Magnesium carbonate. ■
16. The process for efficient production of 1,3-Propanediol as claimed in claim 1 wherein said reaction medium comprises 0.5 to 7% of glycerol in the presence of 1 - 5% tryptone and 1.0 % of Mg.
17. The process for efficient production of 1,3-Propanediol as claimed in claim 1 wherein said reaction medium comprises 2% glycerol; 2% of tryptone and 1.0 % Mg.
18. The process for efficient production of 1,3-Propanediol as claimed in claims 1 wherein said incubation is carried out at pH 7.0.
19. The process for efficient production of 1,3-Propanediol as claimed in claim "1 wherein said temperature is maintained at 30°C.
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20. The process for efficient production of 1,3-Propanediol as claimed in claim 1 wherein said agitation is at 150 rpm.
21. The process for efficient production of 1,3-Propanediol as claimed in claim 1 wherein said inoculum density is 4%.
22. The process for efficient production of 1,3-Propanediol as claimed in claim 1 wherein said reaction medium comprises 1-5% glycerol, 1 - 2% tryptone, 3% inoculum density, a second carbon source and magnesium salt, the agitation rate being 150 rpm, pH being 6.0-7.5, temperature being 25°C-35°C and the incubation period being 24 hrs.
23. The process for efficient production of 1,3-Propanediol as claimed in claim 22 wherein said reaction medium comprises 2.0% glycerol, 1.5% tryptone, glucose as second carbon source, and magnesium salt, the agitation rate being 150 rpm, pH being 7, temperature being 30°C and the incubation period being 24 hrs.
24. The process for efficient production of 1,3-Propanediol as claimed in any preceding claim wherein the process is carried out in a volume of more than 10 litre and said air flow rate is 0.6 vvm of carbon dioxide attained by cascading.
25. The process for efficient production of 1,3-Propanediol substantially as herein described with respect to accompanying drawings and examples.
Dated this 16th day of March 2006

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ABSTRACT
Process for efficient production of 1,3-Propanediol
The production of 1,3-propanediol from glycerol using anaerobic fermentation in presence of Citrobacter by using a novel medium composition which allows optimum production in less incubation time. The process allows intake of high concentration of glycerol as raw material by the natural bacteria for efficient 1,3-propanediol production.
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