Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2000-11-14
2002-08-06
Tate, Christopher R. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S106000, C435S115000, C435S116000, C562S513000
Reexamination Certificate
active
06428992
ABSTRACT:
FIELD AND OBJECTS OF THE INVENTION
The present invention relates to a process for the purification of 1,3-propanediol from a fermentation medium comprising 1,3-propanediol and fermentation coproducts.
The present invention relates more particularly to a process for the purification of 1,3-propanediol by subjecting a clarified aqueous solution originating from said fermentation medium to chromatography on a cationic resin to give at least one fraction comprising purified 1,3-propanediol and at least one fraction comprising the fermentation coproducts.
In the invention, “clarified aqueous solution” is understood as meaning the aqueous solution obtained after removal of the 1,3-propanediol-producing microorganisms from the fermentation medium.
Also, “fermentation coproducts” are understood as meaning the products resulting from a secondary metabolism, such as glycerol in particular, or the unconverted substrate, for example glucose or glycerol, or various residues originating from the nutrients in the fermentation medium or from the bacterial metabolism, such as acetic acid, ethanol or 2,3-butanediol.
The present invention relates even more particularly to a process for the purification of 1,3-propanediol by chromatography using a cationic resin whose cation is advantageously selected from the group consisting of lanthanum, lead, zinc, iron and aluminum.
Finally, the present invention relates to a treatment by microorganisms which specifically convert the glycerol to 1,3-propanediol, of said clarified aqueous solution and/or the fractions respectively containing glycerol and/or a mixture of glycerol and 1,3-propanediol, obtained by the chromatographic separation.
BACKGROUND OF THE INVENTION
The methods of purifying 1,3-propanediol from a fermentation medium which are known in the state of the art are principally based on a series of steps consisting in extracting the 1,3-propanediol from said fermentation medium with organic solvents and/or distilling it.
The conventional distillation techniques normally used in these purification processes require a large input of energy, which reflects on the cost of the final product purified in this way. This distillation point of 1,3-propanediol is in fact 214° C.
MALINOWSKI (for example in his article in
Biotechnology Techniques,
1999, 13, pp 127-130) proposed a liquid-liquid extraction process with organic solvents to extract the 1,3-propanediol from dilute aqueous solutions containing it.
However, this purification process requires the handling of large quantities of solvents of the pentanol type (up to nonanol) or hexanal type (up to decanal) and, in particular, its 1,3-propanediol extraction and separation efficiency is too low.
MALINOWSKI moreover recognizes the need to turn to an alternative, more efficient method as 1,3-propanediol is too hydrophilic.
Another alternative, proposed by BROEKHUIS et al. in
Ind. Eng. Chim. Res.,
1994, 33, pp. 3230-3237, was to use chemicals of the formaldehyde or acetaldehyde type to form a dioxolane derivative of the 1,3-propanediol, which then facilitates its solvent extraction.
However, although the process consumes less energy, the cost of the chemicals used makes the extraction and purification process prohibitive because of the additional need to regenerate the 1,3-propanediol from its dioxolane derivative.
Likewise, the processes for complexation of the 1,3-propandiol with organoborates, proposed by BROEKHUIS et al. in
Ind. Eng. Chim. Res.,
1996, 35, pp. 1206-1214, demand substantial means to regenerate the 1,3-propandiol, making these extraction processes economically non-viable.
It is therefore apparent from all the above that there is an unsatisfied need for a process which is simple to carry out and makes it possible to purify 1,3-propanediol from the fermentation medium of a 1,3-propandiol-producing microorganism.
Anxious to develop a process which satisfies the practical constraints better than the process already in existence, the inventors have found that it is possible efficiently to purify 1,3-propanediol from said fermentation medium by a means which consists in performing a specific chromatographic separation of the components of the fermentation medium.
A process for the separation of 1,3-propanediol from a fermentation medium by chromatography has in fact been proposed by BÜNZEL et al. (for example in
DECHEMA BIOTECHNOLOGY Conferences,
1990, 4 pt. B, pp. 713-716) and consists in employing adsorption techniques, especially adsorption on hydrophobic zeolites such as silicalite-1 or non-aluminous NaY zeolites, or even active charcoal.
However, the capacity of 1,3-propanediol to adsorb onto these supports is very mediocre; furthermore, some of the fermentation coproducts regrettably bind to said supports.
MORE DETAILED DESCRIPTION
The process according to the invention for the purification of 1,3-propanediol from a fermentation medium of a 1,3-propanediol-producing microorganism, comprising 1,3-propanediol and fermentation coproducts consisting at least of glycerol, is thus characterized in that:
a) the 1,3-propanediol-producing microorganism is separated from the other components of the fermentation medium to give a clarified aqueous solution;
b) said clarified aqueous solution is passed over a cation exchange resin to give at least one fraction comprising purified 1,3-propanediol and at least one fraction comprising the fermentation coproducts; and
c) the 1,3-propanediol is recovered.
The 1,3-propanediol-producing microorganism is selected from the group of recombinant microorganisms which are capable e.g. of producing 1,3-propanediol from glucose, i.e. recombinant microorganisms of the
E. coli
or
S. cerevisiae
type, such as those described in patent applications WO 96/35796 and WO 98/21341.
The inventors have noticed that these microorganisms predominantly produce on the one hand 1,3-propanediol and on the other hand glycerol in particular as the fermentation coproduct. The fermentation medium may also contain some glucose which as not been completely consumed and a small amount of acetic acid.
The first step of the process according to the invention consists in separating the 1,3-propanediol-producing microorganisms from the other components of the fermentation medium to give a clarified aqueous solution.
This separation is effected by any technique otherwise known to those skilled in the art and can consist of a microfiltration or centrifugation of said microorganisms or a precipitation thereof with flocculants. These techniques can also be combined.
The preferred technique in the process according to the invention is that of microfiltration using a microfiltration membrane whose porosity is adapted to the size of the microorganisms in question, for example TECHSEP membranes of porosity 0.1 &mgr;m for 1,3-propanediol-producing recombinant microorganisms of the genus
E. coli.
It is advantageously chosen to pass the resulting clarified aqueous solution through a column of charcoal and demineralize it on a strong cationic resin and then on a weakly and/or strongly basic anionic resin, for example of the acrylic type, employing the techniques widely known to those skilled in the art.
These steps make it possible to remove the cellular debris and the proteins resulting from any lysis of the 1,3-propanediol-producing microorganisms, and the effect of desalination is to remove the mineral load from the fermentation medium.
It can then be chosen to treat said clarified aqueous solution with a microorganism which converts the residual glycerol to 1,3-propanediol.
In such a case this microorganism is selected from the group consisting of microorganisms of the genera Klebsiella, Citrobacter, Clostridium, Lactobacillus, Ilyobacter, Pelobacter or Enterobacter, and is preferably a Citrobacter or a Clostridium.
In one particularly preferred embodiment of the process according to the invention, it is chosen to use this microorganism capable of converting the glycerol to 1,3-propanediol, in the free or immobilized form and preferably in the immobilized form.
It is possible to effect t
Berghmans Elie
Fouache Catherine
Roturier Jean-Michel
Henderson & Sturm LLP
Roquette Freres
Srivastava Kailash C.
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