FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
PROVISIONAL SPECIFICATION
(See Section 10; rule 13)
"AEROBIC PRODUCTION OF 1,3-PROPANEDIOL FROM CRUDE GLYCEROL FROM BIODIESEL PROCESS"
RELIANCE LIFE SCIENCES PVT.LTD
an Indian Company having its Registered Office at Dhirubhai Ambani Life Sciences Centre,
R-282, TTC Area of MIDC,
Thane Belapur Road, Rabale,
Navi Mumbai - 400 701
Maharashtra India.
The following specification particularly describes and ascertains the nature of this invention and the manner in which it is performed:-


FIELD OF THE INVENTION:
The present invention relates to an efficient fermentation process for the production of propanediol under aerobic conditions. The present invention in particular employs pure glycerol as well as glycerol obtained from biodiesel process without any purification to obtain propanediol with competitive yields.
BACKGROUND OF THE INVENTION
1,3-Propanediol (1,3-PD) or Trimethylene glycol (TMG) is a three-carbon diol. For a long time, 1,3-propanediol has been considered a speciality chemical. However, the recent development of new polyester called polypropylene terephthalate, with unique properties for the fiber industry necessitates a drastic increase in the production of this chemical.
There are currently two processes for the chemical synthesis of 1,3-propanediol. Both of these processes produce toxic intermediates and require a reduction step under high hydrogen pressure. The biological production of 1,3-propanediol from glycerol was demonstrated for several bacterial species, e.g., Lactobacillus brevis, Lactobacillus buchnerii, Bacillus welchii, Citrobacter freundii, Klebsiellaa pneumoniae, Clostridium pasteurianum, and Clostridium butyricum. (1)
The production of 1,3-propanediol from glycerol is generally performed under anaerobic conditions using glycerol as the sole carbon source and in the absence of other exogenous reducing equivalent acceptors. In some strains of Citrobacter, Clostridium, and Klebsiella, a parallel pathway for glycerol operates under aerobic conditions, which first involves oxidation of glycerol to dihydroxyacetone (DHA) by a NAD+-(or NADP+-) linked glycerol dehydrogenase (Equation l)followed by DHA phosphorylation to dihydroxyacetone phosphate (DHAP) by a DHA kinase (Equation 2), and would be helpful for biosynthesis and supporting ATP generation via, for example, glycolysis. In contrast to the 1,3-propanediol pathway under anaerobic conditions, this pathway may provide carbon and energy to the cell and produce NADH rather than consuming it.


The above equations under aerobic and anaerobic conditions is summarised below::
Glycerol NAD+®DHA+NADH+H+ (Equation 1) DHA+ ATP®DHAP+ADP (Equation 2)
Under anaerobic/micro aerobic conditions
Glycerol®3-hydroxypropionaldehyde +H20 (Equation 3) 3-hydroxypropionaldehyde + NADH®l,3-PDO+NAD+ (Equation 4)
The 1,3-PDO pathway has been studied primarily in K. pneumoniae. Glycerol is transported into the cell through the glycerol facilitator. The glycerol is then converted into 3-hydroxypropionaldehyde by vitamin B12-dependent dehydratase (Equation 3). The 3-hydroxypropionaldehyde is reduced to 1,3-PDO by an NADH-dependent 1,3-PD oxidoreductase (Equation 4). 1,3-PDO is then excreted into the medium. The 1,3-PDO pathway in K. pneumoniae is part of the dha regulon. The dha regulon is induced by dihydroxyacetone (DHA) in the absence of an exogenous electron acceptor, such as oxygen, fumarate, or nitrate. The enzymes of the dha regulon that are not directly involved in 1,3-PDO production convert glycerol to DHA by an NAD+ dependent glycerol dehydrogenase and then to dihydroxyacetone phosphate by an ATP-dependent DHA kinase; the dihydroxyacetone phosphate is further metabolized to provide carbon and energy for growth. The physiological reason for 1,3-PDO formation is most likely to regenerate NAD+ needed by the DHA branch of the dha regulon (2)
Among the organisms capable of producing propanediol, K. pneumoniae ferments glycerol to PDO with a high yield and productivity. However, due to the product inhibition (3), it is difficult to obtain a high PDO concentration in the fermentation broth. In view of the high cost of diol recovery from aqueous solution, an economical production of PDO from glycerol requires the improvement of both product concentration and productivity. According to previous reports, production of PDO is mainly done under anaerobic conditions (4), although PDO can still be obtained under micro aerobic or low aerobic conditions (5, 6). The effect of aeration conditions on the fermentation process has not yet been fully explored. (7). The maximum yield of 1,3-PD to glycerol obtained under hitherto


anaerobic conditions is maximum of 0.72 mol mol- and 0.85 mol mol-1 under micro aerobic conditions.
The US patent number 7,169,588 and US patent number 7,074,608 provides a process of obtaining 1,3-propanediol by recombinant E. coli from glucose.
The US patent number 6,603,048 provides a process by either recombinant E. coli, Bacillus, Pseudomonas or Streptomyces from glucose and separate 1,3-propanediol, glycerol, or a mixture of 1,3-propanediol and glycerol from a biological mixture using a molecular sieve.
The US patent number 6,406,895 provides a process for the production of 1,3-propanediol by either recombinant E. coli, Bacillus, Pseudomonas or Streptomyces from glucose by a fermentation that is carried out without mechanical agitation, with the maintenance of an air retention greater than or equal to 50%, expressed as the volume of gas relative to the total volume of the liquid phase of the fermentation medium, and with the maintenance of a high cell density and a microorganism viability value, determined by a test A, greater than or equal to 95%, preferably of between 95 and 99%, by controlling frothing in the fermentation medium.
The US patent number 5,254,467 provides a process for the transformation of glycerol into 1,3-propanediol by microorganisms comprising fermenting the microorganisms in media having a glycerol content of from about 5% to about 20% by weight under standard anaerobic fermentation conditions and recovering the 1,3-propanediol
Production of 1,3-propanediol under aerobic conditions by K. pneumoniae was studied, and the effects of aeration, initial glycerol concentration, pH and 3-hydroxypropionaldehyde were investigated by Chen et al. (Study on 1,3-propanediol production by Klebsiella pneumoniae under aerobic conditions Xiandai Huagong/Modern Chemical Industry; Volume 26, Issue SUPPL. 2, October 2006, Pages 297-300) The process describes the accumulation of the intermediate 3-hydroxypropionaldehyde.


Further the aerobic fermentation process of 1,3-propanediol by K. pneumoniae with glycerol as substrate was studied by Cheng in batch system. (Kinetic analysis of aerobic batch fermentation of 1,3-propanediol by K. pneumoniae; Xiandai Huagong/Modern Chemical Industry Volume 25, Issue SUPPL., July 2005, Pages 185-188)
1,3-propanediol production by Klebsiella pneumoniae was studied by Cheng (1,3-propanediol production by Klebsiella pneumoniae under different aeration strategies Biotechnology Letters Volume 26, Issue 11, June 2004, Pages 911-915 ) in batch cultures under N2 flow and four airflow systems. Different byproducts were formed under different aeration conditions. An anaerobic/aerobic combined fed-batch culture was developed giving 70 g 1,3-propanediol per litre and 16 g 2, 3-butanediol per litre with total diol yield of 0.6 per mol glycerol.
PCT publication WO/2007/099161 relates to a process for the preparation of 1 ,2-propanediol, in which a glycerol-containing stream, in particular a stream obtained on an industrial scale in the preparation of biodiesel, is subjected to a hydrogenation.
PCT publication WO/2004/041421 provides a process for hydrogenating 3-hydroxypropionic acid, or esters thereof, or mixtures of the acid and the ester, in a liquid phase, in the presence of a ruthenium catalyst, alone, or in combination with at least one or more additional metal catalyst wherein the metal is molybdenum, tungsten, titanium, zirconium, niobium, vanadium, chromium, or mixtures of the metals.
The practical yield of 1,3-PD obtained by conventional techniques is usually much lower compared with the theoretical maximum because some part of glycerol is taken up to produce biomass and byproducts, e.g. ethanol and acetate. To enhance the yield of 1,3-PDO and decrease the cost of production, strategies involving the utilization of raw materials (e. g. industrial-crude glycerol, directly issued from bio-diesel production units) by cells capable of resisting the impurities of the medium have been utilized. Raw, unpurified glycerol, has been used in this type of fermentation. Likewise, a cheap carbon source, such as glucose, is often used as a H-donor substrate instead of the fraction of glycerol to provide both reducing equivalents for 1,3-PDO formation and ATP for biomass. A higher


yield of 1,3-propanediol to glycerol was obtained by using glucose as co-substrate, but a high concentration of glucose in the culture medium appears to have a strong inhibition to the enzymes involved in the production of 1,3-PDO. The ratio of glucose to glycerol hence seems to be an important parameter for 1,3-PDO production. However, it is still not investigated experimentally or theoretically. K. pneumoniae is a facultatively aerobic bacterium. Recent experimental results and theoretical analysis show that the micro aerobic conditions are favorable for cell growth, 1,3-PD formation and its productivity in K. pneumoniae (8).
The present invention has thus focused on the production and partial purification of 1,3-propanediol by Klebsiella pneumoniae using glycerol under aerobic conditions. The inventors of the present invention have successfully developed an efficient fermentation process for the production of propanediol under aerobic conditions using pure glycerol as well as glycerol obtained from biodiesel process without any purification. The fermentation process is cost effective and commercially viable as the glycerol used is in crude form without undergoing the tedious process of purification, resulting in high and comparative yields of propanediol. The present fermentation process was scaled up to 10L level with competitive and reproducible yields. The optimized culture conditions of the present conditions are responsible for such competitive and reproducible yields.
OBJECT OF THE INVENTION
It is the object of the present invention to provide an efficient process for production of 1,3-propanediol by Klebsiella pneumoniae using glycerol.
It is the object of the present invention to provide a process for partial purification of 1,3-propanediol.
It is the object of the present invention to provide a fermentation process under aerobic conditions.


It is the object of the present invention to provide a fermentation process using pure glycerol as well as glycerol obtained from biodiesel process without any purification.
It is the object of the present invention to provide a process, which is cost effective, commercially viable and feasible on a large scale.
SUMMARY OF THE INVENTION
The present disclosure provides an efficient process for the aerobic production of 1,3-propanediol by Klebsiella pneumoniae using glycerol.
In one embodiment the present invention provides a process without the need for using genetically modified organisms. In particular the present invention aims to provide a process using Klebsiella pneumoniae.
In one embodiment the present invention provides the effects of pH, temperature, glycerol concentration, yeast extract concentration on the yields of propanediol.
In one embodiment the present invention provides the process using batch and fed batch fermentation.
In one embodiment the present invention provides the process for producing 1,3-propanediol using crude glycerol from the biodiesel process.
In one embodiment the present invention provides the process on large scale fermenter of upto 10 L scale.
In one embodiment the present invention provides the partially purified PDO from
glycerol.
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.
Figure 1: shows the growth curve of Klebsiella pneumoniae ATCC 15830. 1,3-propanediol production commenced during the mid-log phase and continued until the stationary phase.
Figure 2: shows the effect of initial pH on the production of PDO wherein an intiai pH of 8.0 was favorable for 1,3-propanediol production.
Figure 3: shows the effect of temperature on the production of PDO wherein incubation at 25°C was optimum for 1,3-propanediol production.
Figure 4: shows the effect of glycerol concentration on the production of PDO. A initial concentration of 5 % glycerol was favorable for production of 1,3-propanediol.
Figure 5: shows the effect of yeast extract concentration on the production of PDO. Yeast extract at 0.5 % concentration was optimum.
Figure 6: shows growth and 1,3-propanediol production profiles of Klebsiella pneumoniae ATCC 15830 in optimized medium in 1.5 L fermentor.
Figure 7: shows the PDO and glycerol profiles in 1.5L fermenter during the fed batch fermentation using glycerol.
DETAILED DESCRIPTION OF THE INVENTION
Definitions:
The term "PDO", "propanediol" refers to the 1,3-propanediol of molecular weight 76.10
g/mol, boiling point of 214°C and with a molecular formula of C3H8O2
The term" aerobic" refers to the fermentation conditions of at least 1VVM aeration.


The term "bacteria" refers to wild type Klebsiella or strains that are not genetically modified.
The present invention provides a process of aerobic fermentation of glycerol using Klebsiella pneumoniae to yield PDO.
The present invention thus provides an efficient fermentation process for the production of propanediol under aerobic conditions using pure glycerol as well as glycerol obtained from biodiesel process without any purification. The fermentation process is cost effective and commercially viable as the glycerol used is in pure form without undergoing the tedious process of purification, resulting in high and comparative yields of propanediol. The present fermentation process was scaled up to 10L level with competitive and reproducible yields. The optimized culture conditions of the present conditions are responsible for such competitive and reproducible yields.
The present invention has provided the effect of culture conditions on the aerobic fermentation The culture conditions which were studied in detail for aerobic fermentation in order to increase the yields were pH of the media, glycerol and yeast extract concentrations, temperature and the rate of agitation. The effect of these parameters is illustrated below supported with examples.
The growth and 1,3-Propanediol production patterns of Klebsiella pneumoniae culture in PDO media is shown in Fig.l. Production of 1,3-Propanediol was monitored concomitantly with growth and was found to start increasing during the early exponential phase and extending well into the stationary phase before reaching a plateau (Fig.l).
Growth media adjusted to different initial pH values viz., 4, 5, 6, 7, 8, 9 and 10 were inoculated with K. pneumoniae culture. Sampling was done to monitor the growth of Kpneumoniae and production of 1,3-Propanediol. Propanediol production was found to be increasing with increase in pH from 4.0 to 8.0 and then decreased with increase in pH (Fig.2).


Growth media adjusted to the optimized pH was inoculated with K. pneumoniae culture and incubated at different temperatures viz., 25°C, 30°C, and 35°C. Sampling was done to monitor the growth of K. pneumoniae and production of 1,3-Propanediol. Propanediol production was optimum at 25°C and was found to be decreasing with increase in temperature (Fig. 3).
Growth media with different glycerol concentrations viz., 0.5%, 1%, 1.5%, 2%, 5%, 7.5% and 10% were prepared and inoculated with K. pneumoniae culture and were incubated at the optimum pH and temperature. Sampling was done to monitor the growth of K. pneumoniae and production of 1,3-Propanediol. The propanediol yield was found to be increasing with increase in glycerol concentration (Fig. 4). However, beyond 5 % glycerol, there was no significant increase in 1,3-propanediol.
Growth media with different yeast extract concentrations viz., 1 to 6g/L, were prepared and inoculated with K. pneumoniae culture and were incubated at the optimum pH and temperature. Sampling was done to monitor the growth of K. pneumoniae and production of 1,3-Propanediol. The propanediol concentration increased with increase in yeast extract concentration up to 5g/L and thereby decreased with increase in yeast extract concentration (Fig. 5).
Optimized production media for 1.5L fermenter was sterilized and inoculated with 100ml of 24h old K. pneumoniae culture and incubated at 25°C, 200 rpm agitation. Aeration was set to 3VVM. Sampling was done to monitor the growth of K. pneumoniae and production of 1,3-Propanediol. The log phase commenced after initial 2 h of lag phase and extended up to 13 h of incubation followed by stationary phase till 34 h. Maximum propanediol concentration was obtained at late stationary phase (Fig. 6)
The present invention provides for a fermentation process for the production of propanediol under aerobic conditions using pure glycerol as well as glycerol obtained from biodiesel process is devoid of purification steps. This has resulted in cutting down on operational


inputs for addition of different media and accordingly thus provides a simple and cost effective with high and comparative yields of propanediol.
The below paragraphs list the process in batch and fed batch citing the important parameters.
In the present invention, the conversion rate of 0.8 - 0.9 mole 1,3-PDO/ mole of glycerol obtained is much higher than any other reported in the literature. This is advantageous as there is maximum utilization of glycerol. Besides, the present process is completely aerobic using a wild type strain of Klebsiella pneumoniae.
Batch fermentation of 1,3-Propanediol in 10L
A yield of 27 to 32g/L propanediol was obtained after 24h of incubation from 50g/L of glycerol. The yield was less compared to that obtained in 1.5L fermenter but the incubation time was reduced to 24 h compared to that of 34 h in 1.5L fermenter.
Fed batch fermentation of 1,3-Propanediol in 10L fermenter
Optimised production media for 10L fermenter containing 5 % glycerol was sterilized and inoculated with 1000ml of 24h old K. pneumoniae culture and incubated at 25°C, 200 rpm agitation at an aeration of 18L/min. After the complete consumption of glycerol, additional autoclaved media containing 3% glycerol was added as feed. Growth and 1,3-Propanediol production was monitored continuously until complete glycerol consumption occurred. A yield of 56g/L of propanediol was obtained after 48h of incubation. The glycerol added initially was totally consumed within 24h of incubation and additional supplementation of glycerol added after 24 h was also consumed within 24h. Thus, 8 % glycerol was totally consumed in 48 h and propanediol production reached a maximum of 56g/L. It took 200h in 1.5L fermenter for getting a similar yield of 1,3-PDO.
Production of 1,3-propanediol from crude glycerol
A yield of 62 g/L of propanediol was obtained after 120 h of incubation from 8% glycerol. 5% glycerol was added initially to the media before inoculation and remaining 3% was added after 36 h of incubation along with all other media components after the depletion of


glycerol added initially. After 120 h of incubation, glycerol was totally consumed and propanediol production reached a maximum of 62g/L as compared to 70g/L PDO with pure glycerol.
Purification of 1,3-Propanediol
The broth obtained from fermentation was treated with IP A for precipitation of proteins. The supernatant after removal of proteins was subjected to charcoal treatment for colour removal. The colourless broth was then distilled to get pure PDO. The purity obtained at this step ranged from 90-95%. The purification process has to be developed further to obtain polymer grade 1,3-propanediol (99%).
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLE 1: STUDY OF VARIOUS PARAMETERS FOR AEROBIC FERMENTATION: Media and Culture
Klebsiella pneumoniae ATCC 15830 was obtained from ATCC. The strain was cultured and maintained on nutrient agar (NA) slants. The media used for production of 1,3-Propanediol was:
Glycerol : 50 g/1
Glucose : 5 g/1
Yeast extract : 5 g/1
KH2PO4 : 3g/l


Na2HPO4 : 6g/l
MgSO4 : 0.5g/l
NaCl : 0.5g/l
NH4Cl : 2g/l
Growth of Klebsiella pneumoniae
Klebsiella pneumoniae culture was inoculated from the NA slants into 250 ml of media and incubated at 30°C for 48 h on a shaking incubator at 250 rpm. Cell growth was monitored by collecting 1ml of culture from fermented broth every 24h and centrifuged at 13200 rpm for l0 min. The supernatant was used for PDO estimation and the pellet washed thrice with saline was used for determining cell growth
Analytical procedures
Glycerol and PDO were analyzed by HPLC with a refractive index detector and organic acid (Rezex) column with 0.01N H2SO4 as mobile phase at 0.5 ml min-1 flow rate. 1,3-propanediol elutes at retention time of 24 min under these conditions whereas glycerol elutes at 18 min (9).
Growth curve of Klebsiella pneumoniae
The growth and 1,3-Propanediol production patterns of K. pneumoniae culture in PDO media is shown in Fig.l. After an initial lag of 14h, there was an exponential increase in growth. This log phase continued up to 28h before reaching a plateau indicating the commencement of stationary phase. Production of 1,3-Propanediol was monitored concomitantly with growth and was found to start increasing during the early exponential phase and extending well into the stationary phase before reaching a plateau (Fig.l).
Effect of pH
Growth media adjusted to different initial pH values viz., 4,5,6,7,8,9 and 10 were inoculated with K.pneumoniae culture. Sampling was done to monitor the growth of Kpneumoniae and production of 1,3-Propanediol and the pH permitting high 1,3-Propanediol production was chosen for further studies. Propanediol production was found


to be optimum at initial pH of 8 with a yield of 12.2 g/L. The growth of Klebsiella pneumoniae was found to be optimum at initial pH of 9. Propanediol production was found to be increasing with increase in pH from 4.0 to 8.0 and then remained constant with increase in pH (Figure 2).
Effect of Temperature
Growth media adjusted to the optimized pH was inoculated with Kpneumoniae culture and incubated at different temperatures viz., 25°C, 30°C, and 35°C. Growth and 1,3-Propanediol production was monitored and the temperature permitting high 1,3-Propanediol production was chosen for further studies. Propanediol production was found to be optimum at 25°C with a yield of 11.2g/L. Although the growth of K. pneumoniae was found to be optimum at 35°C, propanediol production was found to decrease with increase in temperature (Figure 3).
Effect of Glycerol concentration
Growth media with different glycerol concentrations viz., 5g/l, 10g/l, 15g/l, 20g/l, 50g/l, 75g/l and 100g/l were prepared and inoculated with K. pneumoniae culture and were incubated at the optimized pH and temperature. Growth and 1,3-Propanediol production were monitored and the glycerol concentration permitting high 1,3-Propanediol production was chosen for further studies. Propanediol production improved with increasing initial concentration of glycerol with a yield of 32 g/L at 5% glycerol concentration and 37 g/L at 10% glycerol concentration (Figure 4). All further experiments were carried out at 50 g/L glycerol concentration since there was no significant effect on the yield of the product at higher glycerol concentration..
Effect of yeast extract concentration
Growth media with different yeast extract concentrations viz. 1 to 6 g/L were prepared and inoculated with Kpneumoniae culture and were incubated at optimized pH and temperature. Growth and 1,3-Propanediol productions were monitored and the yeast extract concentration permitting high 1,3-Propanediol production was chosen for further


studies. Propanediol production was found to be optimum at 5 g/L yeast extract with a yield of 32 g/L. (Figure: 5).
Optimized parameters for production of 1,3-Propanediol in batch mode includes an initial pH of 8.0, agitation of 200rpm, aeration of 3VVM for fermentation at 1.5L scale and 18 L/min for 10L scale, initial glycerol concentration of 5 % and yeast extract at 5 g/L concentration. During fed-batch, after the complete consumption of glycerol, an additional feed of 3 % glycerol along with other media components is added. Sampling was done to monitor the growth of K.pneumoniae and production of 1,3-Propanediol.
EXAMPLE 2: Batch fermentation of 1,3-Propanediol in optimized medium at 1.5L scale
Production media with optimized parameters at 1.5L scale was prepared, autoclaved and was inoculated with 100ml of 24h old K.pneumoniae culture. The log phase commenced after initial 2h of lag phase and extended up to 13h of incubation followed by stationary phase till 34h. Maximum propanediol concentration was obtained at late stationary phase (Figure: 6). A yield of 34g/L propanediol was obtained after 34h of incubation , which is stoichiometrically equivalent to 0.83 mol/mol of glycerol. This value is closer to that of 0.85mol/mol propanediol obtained from micro aerobic conditions.
EXAMPLE 3: Fed-batch fermentation of 1,3-Propanediol at 1.5L scale
Production media with optimized parameters at 1.5L scale was prepared , autoclaved and inoculated with 100ml of 24h old K.pneumoniae culture. After the complete consumption of glycerol, which occurred in 50 h, additional autoclaved media containing 3% glycerol was added as feed. The rate of glycerol consumption ceased completely at 120 h with an yield of 63g/L of propanediol(Figure: 7). The stoichiometry of 1,3-PDO produced is 0.94 mol/mol glycerol which is the highest reported so far.
EXAMPLE 4: Batch fermentation of 1,3-Propanediol at 10L scale
Production media with optimized parameters at 10L scale was prepared, autoclaved and was inoculated with 1000ml of 24h old Kpneumoniae culture. A total of 27g/L


propanediol was obtained after 24h of incubation from 50g/L of glycerol. Although the yield was less compared to that obtained atl.5L scale, the incubation time was reduced to 24h instead of 34h at 1.5L scale.
EXAMPLE 5: Fed batch fermentation of 1,3-Propanediol at 10L scale
Production media with optimized parameters at 10L scale was prepared ,autoclaved and was inoculated with 1000ml of 24h old Kpneumoniae culture. After the complete consumption of glycerol which occurred in 24 h, additional autoclaved media containing 3% glycerol was added as feed. The rate of glycerol consumption was almost complete at 48 h with only traces of glycerol remaining. A yield of 56g/L of propanediol was obtained after 48 h of incubation from a feed of 8% glycerol.
EXAMPLE 6: Production of crude glycerol from Jatropha oil
To 1000ml of jatropha oil, 8% (80mL) of methanol was added and mixed thoroughly. Then 0.1 %V/V (1ml) of cone, sulphuric acid was added at a constant temperature of 35°C and at a constant stirring for 1 hour and was incubated for overnight. After the incubation, 7.5g of NaOH was dissolved in 120ml of methanol and was added drop wise to the oil at a constant temperature of 65°C and with constant shaking for lhr. The mixture was kept aside for phase separation. The yield of crude glycerol which separates out as bottom layer was 10-12%.
EXAMPLE 7: Purification
2000ml of broth obtained from fermentation was centrifuged to remove the cells. The supernatant obtained was concentrated to 50ml. 300ml of IP A was added to the concentrate and the salts were allowed to precipitate. The supernatant obtained was again centrifuged and concentrated to 50ml. 150ml of water was again added and the pH was adjusted to 3.0 with concentrated HC1.. The solution was then treated with charcoal at concentration of 3% and kept agitating overnight. The broth was centrifuged and was distilled at 130°C and 10 mbar vacuum. The distillate obtained was analyzed by HPLC for 1,3-propanediol purity. The purity obtained varied from 90-95%. After IPA


precipitation, the salts from the media precipitate out and the charcoal treatment removes the colour.
References
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Thus, while we have described fundamental novel features of the invention, it will be understood that various omissions and substitutions and changes in the form and details may be possible without departing from the spirit of the invention. For example, it is expressly intended that all combinations of those elements and/or method steps, which perform substantially the same function in substantially the same way to achieve the same results, be within the scope of the invention.







ABSTRACT
The present invention relates to aerobic production of 1,3-Propanediol by Klebsiella pneumoniae ATCC 15380. The process in particular provides an efficient conversion of glycerol to 1,3-Propanediol which is stoichiometrically over 0.9 mol of propanediol/mol of glycerol compared to 0.85 mole/mole theoretical yield under microaerobic conditions. The invention also describes production of 1,3-PDO from crude glycerol obtained from biodiesel process at a competitive and reproducible yield as compared to pure glycerol.