Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2000-10-25
2002-06-18
Tate, Christoper R. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S252310, C435S252330, C435S252700
Reexamination Certificate
active
06406895
ABSTRACT:
The present invention relates to a process for the production of 1,3-propanediol by the fermentation of a 1,3-propanediol-producing microorganism in a fermentation medium containing glucose.
In particular, the present invention relates to a process for the production of 1,3-propanediol by means of a microorganism which produces 1,3-propanediol from glucose, said process consisting in carrying out the fermentation without mechanical agitation, with the maintenance, in said fermentation medium, 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 viability of said 1,3-propanediol-producing micro-organisms.
Even more particularly, the present invention relates to a process for the production of 1,3-propanediol wherein the fermentation is carried out in a pneumatic fermenter of the bubble column type, and preferably in a pneumatic fermenter of the bubble column type without gas recirculation, so as to maintain a high gas retention, cell density and cell viability by controlling production of foam in the fermentation medium.
The preparation of 1,3-propanediol by fermentation is normally effected with the aid of microorganisms of the genera Klebsiella, Citrobacter, Clostridium, Lactobacillus, Ilyobacter, Pelobacter or Enterobacter, from glycerol as the carbon source directly assimilable by said microorganism, and under anaerobic fermentation conditions.
In these microorganisms, the metabolic path which leads to the production of 1,3-propanediol from glycerol is based mainly on two successive enzymatic activities belonging to the reductive pathways.
The first enzymatic activity is a glycerol dehydratase, which converts the glycerol to 3-hydroxypropionaldehyde. The second enzymatic activity is an NADH,H
+
oxidoreductase, which converts the 3-hydroxypropionaldehyde to 1,3-propanediol.
It is for this reason that specialists in the production of 1,3-propanediol by fermentation acknowledge that, whether it be from glycerol in the case of the natural microorganisms of the genera Kiebsiella, Citrobacter, Clostridium, Lactobacillus, Ilyobacter, Pelobacter or Enterobacter, or whether it be from glucose or other sugars in the case of recombinant microorganisms, said fermentation must preferably be performed under anaerobic conditions because the metabolic pathways for the conversion of glycerol to 1,3-propanediol are not oxidative but reductive pathways.
In fact, the oxidative pathways are only involved in equilibrating the intracellular redox potential—in this case regenerating the cofactor NADH,H
+
which is necessary for the conversion of 3-hydroxypropionaldehyde to 1,3-propanediol.
However, patent applications WO 96/35796 and WO 98/21341 describe the cloning of genes coding for these two enzymatic activities and, more particularly, the cloning and expression of at least the glycerol dehydratase activity in specific host cells, for example of the
E. coli
or
S. cerevisiae
type, which are normally fermented under aerobic conditions.
This strategy thus enables 1,3-propanediol to be produced from a less expensive carbon source than glycerol, i.e. mainly from glucose, using only one recombinant microorganism.
Nevertheless, in said patent applications, although said recombinant microorganism containing the gene coding for the dehydratase activity is capable of degrading glucose or other sugars via the pathway for the conversion of glycerol to 1,3-propanediol with a good yield and a good selectivity, the fermentation conditions used are still anaerobic.
In fact, the fermentation conditions described are batch fermentation conditions in mechanically agitated glass serum flasks, i.e. at best microaerobic. Furthermore, these fermentation conditions cannot easily be extrapolated to the industrial scale.
It is apparent from all the above that there is an unsatisfied need for a process which is simple to implement and which makes it possible both to produce said 1,3-propanediol efficiently from a less expensive carbon source than glycerol, i.e. glucose, and also to achieve this result under aerobic conditions.
The Applicant has thus overcome the technical prejudice whereby the fermentation of a 1,3-propanediol-producing microorganism can only be carried out efficiently under anaerobic conditions, by proposing a fermentation process carried out under highly aerobic conditions by using a non-mechanically agitated fermentation under specific conditions, said process furthermore making it possible to maintain a high cell viability of the 1,3-propanediol-producing microorganisms.
In terms of the invention, “highly aerobic conditions” are understood as meaning operating conditions which result in the maintenance, in the fermentation medium, of a high air retention value, i.e. a value greater than or equal to 50%, preferably greater than 70% and particularly preferably greater than or equal to 100%, this percentage being expressed as the volume of gas relative to the total volume of the liquid phase of said fermentation medium.
Also, “non-mechanically agitated fermentation” is understood as meaning a fermentation carried out in pneumatic fermenters of the bubble column type with or without recirculation of the gas used, in contrast to fermenters which contain a mechanical device for agitating the fermentation medium.
Although patent application WO 91/15590 describes a fermentation carried out in a reactor of the bubble column type with gas recirculation, making it possible to produce 1,3-propanediol microbiologically, the microorganism involved here is of the genus Clostridium and the fermentation is carried out on a glycerol basis and under anaerobic conditions.
These anaerobic conditions entail using an oxygen-depleted gas, especially of the nitrogen, argon or carbon dioxide type.
This process is therefore totally unsuitable for the fermentation of 1,3propanediol-producing microorganisms under aerobic conditions.
Anxious to develop a process which would satisfy the practical constraints better than the processes already in existence, the Applicant found that an efficient production of 1,3-propanediol from glucose by means of recombinant micro-organisms could be carried out under highly aerobic conditions in a fermenter of the bubble column type without gas recirculation.
Surprisingly and unexpectedly, the Applicant further found that the high dissolved oxygen content could be maintained in the fermentation medium by controlling production of foam in the fermentation medium, said production of foam being correlatable with the cell population.
The process according to the invention for the production of 1,3-propanediol by the fermentation of a 1,3-propanediol-producing microorganism in a fermentation medium containing glucose is characterized in that the fermentation is carried out without mechanical agitation, with the maintenance of a gas 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 production of foam in the fermentation medium.
The 1,3-propanediol-producing microorganism is selected from the group of recombinant microorganisms which are capable of producing 1,3-propanediol from glucose.
The fermentation can be carried out arbitrarily under batch conditions or continuous conditions. It will be advantageous to choose a batch fermentation and particularly preferably a fed batch fermentation, which makes it possible to feed the fermentation medium batchwise with one of the fermentation substrates, in this case glucose, as will be exemplified below.
The Applicant has noticed that 1,3-propanediol production is concomitant with cell growth and also that the total conversion of the glucose used into fermentation products, i.e. on the one hand 1,3-propanediol and on the ot
Defretin Sophie
Delelis Brigitte
Segueilha Laurent
Henderson & Sturm LLP
Roquette Freres
Srivastava Kailash C.
Tate Christoper R.
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