Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1999-06-22
2002-04-02
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S155000, C435S157000, C435S169000, C435S170000, C435S171000
Reexamination Certificate
active
06365383
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method of producing erythritol by fed-batch and repeated fermentation of sugars by microorganisms which produce erythritol.
BACKGROUND OF THE INVENTION
By fed-batch fermentation is meant a fermentation in which microorganisms are fed by the successive addition of substrates, and in which the product and the co-products of the fermentation remain in the medium until the end of fermentation.
By repeated fed-batch fermentation is meant a fed-batch fermentation in which the fraction of the fermentation medium containing the microorganisms is removed in order to use it as an inoculum for a following fed-batch fermentation.
The term substrates is defined as being all the nutritive elements which are introduced into the fermentation media. In the sense of the invention, the substrates are principally carbonaceous and nitrogenous sources which may be directly assimilated by the microorganisms producing erythritol.
The industrial preparation of erythritol is based principally on fermentation methods which use yeasts or unicellular fungi, and this from hydrocarbons or from sugars as directly assimilable sources of carbon.
By sugars is meant in the present invention all the carbonaceous sources which may be directly assimilated by the microorganisms which produce erythritol. Such sugars are chosen for example from the group consisting of glucose, saccharose, fructose, maltose, xylulose and maltulose, on their own or in a mixture. By extension, sugars also means certain sugar alcohols (or polyols) such as mannitol or sorbitol which, being assimilated by said microorganisms, will also lead to the production of erythritol.
The microorganisms producing erythritol from sugars are principally yeasts capable of withstanding significant osmotic pressures and belonging to the types Moniliella, Aureobasidium, Torulopsis, Candida, Trigonopsis, Trichosporon, and Yallowia . . . without this list being restrictive. These yeasts are preferably chosen from the family of microorganisms called “osmotolerant” and are for example isolated from honey.
In a general manner, the microbiological methods of producing erythritol can implement two different methods of fermentation: the batch method and the continuous method.
In the batch methods, all the substrates necessary for feeding microorganisms are introduced at the start of the fermentation, and the product and the co-products of the fermentation are extracted at the end of fermentation.
Such batch methods of fermentation have been carried out for the production of erythritol, and are described for example in patents EP 136 803 and JP 61-31 091.
However, the drawbacks of the batch methods for the production of erythritol which are found in these two patents are, on the one hand, the long fermentation time, of the order of one to two weeks, and on the other hand, the necessity of managing large volumes of fermentation media, for a productivity which does not exceed 1.5 grams per liter and per hour (g/l/hour) and a yield of the order of 10 to 30%, whence raised production costs.
In the continuous methods, all the fermentation substrates are added in a continuous fashion to the reactor, and fractions of the fermentation medium are extracted at the same rate as the supply of substrates, so as to work at a constant volume.
A continuous method has been used for the production of erythritol and is described in patent U.S. Pat. No. 4,923,812. This method presents the advantage of resolving the majority of the drawbacks of the batch method, and thus permits a gain in productivity and yield which is not inconsiderable.
However, these continuous methods do not make it possible to obtain extremely pure products, since the erythritol extracted is of necessity contaminated by the substrates re-introduced during fermentation.
Thus, for the fermentation of glucose by a colony of Aureobasidium, the values presented in the examples of these two patents illustrate this increase in yield and in productivity since the yields of erythritol are between 45 and 50% and the productivities are of the order of 4 to 4.5 g/l/hour. But with the erythritol produced is also found approximately 10% of non-consumed residual glucose, and, in addition, the high osmotic pressures borne by the microorganism necessarily result in inducing the synthesis of glycerol, ribitol and other polyols with that of the erythritol.
Consequently, the main disadvantage of the continuous methods of fermentation is the necessity of purifying the erythritol. Furthermore, the purification techniques only make it possible to separate with difficulty the erythritol from the co-products of the fermentation, and not any better from the residual sugars not assimilated by the microorganisms.
For this reason, one always finds associated with these methods of continuous fermentation complex, heavy and expensive purification installations.
Moreover, all the specialists agree in recognising that the methods of producing erythritol by fermentation remove certain difficulties which neither the batch method nor the continuous method has totally resolved.
For example, in order to reduce the costs of implementation, all the methods of fermentation are based on the use of fermentation media whose sugar content is maximum.
In fact, the microorganisms producing erythritol can withstand high sugar contents in fermentation substrates. The resultant high osmotic pressures then lead said microorganisms to synthesise polyols including erythritol, but also glycerol, arabitol and/or ribitol.
The problem is that with too high a dose of assimilable sugars, the saturation of the metabolic paths induces the instability of the microbial cultures and a greater mortality of the microorganisms. Furthermore, in these operating conditions, all the microorganisms producing erythritol also produce other polyols in an increased proportion (up to 20% or more ribitol and/or glycerol). In certain cases, when the deviation of the metabolic paths is too significant, the erythritol becomes itself a co-product of the fermentation.
All the efforts of the fermentation experts thus focus on the search for operating conditions which lead to reconciling the best yield and productivity of erythritol of the microorganisms with their level of tolerance to maximum concentrations of sugars.
Besides these standard batch or continuous methods of fermentation, there exists a third method of fermentation called a method of fed-batch fermentation, which is chosen specifically when it is noted that the variation of the concentration in one of the fermentation substrates affects the yield or productivity, which is the case with glucose for the production of erythritol by the fermentation path. This fed-batch system thus makes it possible to resolve the problems linked to inhibition by one of the fermentation substrates.
This method consists in introducing the sugars gradually into the fermentation medium of the microorganisms, not carrying out any decanting, and thus leaving the product in the fermenter, in this case the erythritol, until the end of the fermentation.
Patents EP 136 802 and EP 136 804 describe a method of producing a mixture of polyols (erythritol, glycerol, ribitol), by the fermentation of a sugar, preferably glucose, saccharose, fructose or maltose, by
Moniliella tomentosa
var
pollinis,
using the fed-batch and repeated system. The pre-culture serving to culture the fermenter is possibly removed during a preceding fermentation.
However, patent EP 136 802 gives the fermentation conditions which favour the production of erythritol, glycerol and ribitol from concentrations of sugars, preferably glucose, greater than 30% by weight/volume, and the conditions which favour in particular the production of ribitol by controlling the degree of aeration in the fermentation medium.
Patent EP 136 804 describes, for the production of this same mixture of polyols, the addition of 40 to 80% by weight/volume of sugars to the fermentation medium, the initial concentration of sugars being fixed at between 20 and 35%
Henderson & Sturm LLP
Prats Francisco
Roquette Freres
LandOfFree
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