Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2000-05-09
2001-07-31
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S171000, C435S813000, C435S911000, C435S921000, C435S930000, C435S940000
Reexamination Certificate
active
06268190
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
Not Applicable
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not Applicable
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
The present invention relates to a method of producing arabitol by continuous fermentation of at least one sugar by micro-organisms producing arabitol.
It relates more particularly to a method of producing arabitol by continuous fermentation of at least one sugar by micro-organisms producing arabitol, employing at least two interconnected fermentation areas.
To be more precise, in the method according to the invention, arabitol is produced in a first fermentation area in such a manner that some of the sugar introduced into the fermentation medium that it contains is consumed, some of said fermentation medium is transferred into a second fermentation area, the volume of said first area being maintained constant by adding sugar, and production of arabitol continues in the second a fermentation area in such a manner as to consume the residual sugar.
Thereafter, in the second fermentation area, the fermentation medium with a low residual sugar content obtained in this way is separated into a fraction concentrated in micro-organisms and another, soluble fraction rich in arabitol, possibly with recycling of said micro-organisms to the entry of the first fermentation area.
The industrial preparation of arabitol is based mainly on fermentation processes.
Generally speaking, micro-biological methods of producing arabitol can employ two different fermentation modes: a batch mode and a continuous mode.
In the batch mode, two techniques are conventionally used: the batch fermentation technique and the fed batch fermentation technique.
In the batch fermentation technique, all the substrates necessary for feeding the micro-organisms are introduced at the beginning of the fermentation and the arabitol produced is extracted at the end of fermentation.
The term “substrates” refers to all the nutrients that are introduced into the fermentation medium. In the context of the invention, the substrates are mainly carbonaceous sources (including sugar) and nitrogenous sources which can be assimilated directly by the arabitol-producing micro-organisms.
However, this continuous fermentation technique has the drawbacks of requiring long fermentation times to enable total consumption of the sugar introduced into the fermentation medium and of necessitating the use of large volumes of fermentation medium, and therefore large fermenters, to obtain satisfactory conversion yields. This leads to a mediocre productivity per unit volume.
Moreover, variation of the concentration of one of the ingredients of the fermentation medium can affect the arabitol yield or productivity.
One solution is to use the second batch fermentation technique, known as fed batch fermentation.
In particular, this technique introduces the substrates progressively into the fermentation medium.
The advantage of this technique is that the substrate concentration in the fermentation medium can be controlled and all the sugar can be consumed.
Thus the fed batch fermentation technique solves problems associated with the potential inhibiting effects of some ingredients of the fermentation substrates on the growth of the micro-organisms or on the production of arabitol or, for example provides finer adjustment of the aeration that conditions arabitol production.
However, to obtain satisfactory yield and productivity, it is necessary to use high substrate concentrations. Most importantly, the fermentation times are long and the various fermentation steps require complex and painstaking control.
The method described by ESCALANTE et al. (in Journal of Fermentation and Bioengineering, 70-4, 228-231, 1990) for the production of arabitol by Hansenula using fed batch fermentation cannot achieve industrially viable yield and productivity.
This is because, although arabitol is selectively produced, and although the fed batch fermentation process ensures that all of the glucose introduced into the fermentation medium is consumed, arabitol is obtained with a yield of the order of only 14%.
The second fermentation mode conventionally used for the biological production of arabitol is the continuous mode, as described by JAKOBUS van ZYL and PRIOR in Appl. Microbiol. Biotechnol. (1990), 33, 12-17, for example.
In continuous mode, all the fermentation substrates are added continuously to the fermenter and fractions of the fermentation medium are extracted at the same flowrate as the introduction of substrates, to maintain a constant working volume.
This fermentation mode significantly increases yield and productivity but does not produce a product of high purity because the arabitol drawn off is necessarily contaminated by the substrates re-introduced during fermentation.
Consequently, the major drawback of continuous mode fermentation processes is the need to purify the arabitol. Also, available purification techniques can separate the arabitol from the residual sugar not assimilated by the micro-organisms only with difficulty.
For this reason, complex, large and costly purification installations are conventionally associated with these continuous fermentation processes.
Fermentation experts therefore devote all their efforts to seeking operating conditions which can reconcile improved arabitol yield and productivity with a low residual sugar content enabling easy and low-cost purification of the arabitol produced.
Thus the aim of the present invention is to solve the problem of producing arabitol of satisfactory purity with excellent yield and productivity by employing a particular continuous fermentation technique.
The method of producing arabitol developed by the Applicant company consists of fermenting at least one sugar using arabitol-producing micro-organisms in two fermentation areas.
The invention relates more particularly to a method of producing arabitol by continuous fermentation of at least one sugar by micro-organisms producing arabitol, characterised in that:
a) arabitol is produced in a first fermentation area including at least one fermenter in such a manner that some of the sugar introduced into the fermentation medium is consumed by said micro-organisms,
b) some of the fermentation medium obtained in this way is transferred into a second fermentation area including at least one fermenter, the volume being maintained constant in the first fermentation area by adding sugar,
c) production of arabitol continues in said second fermentation area in such a manner as to consume the residual sugar of the fermentation medium,
d) the fermentation medium thus obtained from said second fermentation area is separated continuously into a fraction concentrated in micro-organisms and another, soluble fraction enriched in arabitol, and
e) the arabitol thus obtained is collected.
The first step of the process according to the invention consists of producing arabitol in a first fermentation area comprising at least one fermenter in such a manner that part of the sugar introduced into the fermentation medium is consumed by said micro-organisms.
The micro-organisms are advantageously selected from natural or modified osmosis-tolerant yeasts producing arabitol from sugar as a directly assimilable source of carbon.
The term “osmosis-tolerant yeasts” refers to yeasts which can withstand high osmotic pressures and are of the genus Yamadazyma, Debaromyces, Hansenula, Candida, Zygosaccharomyces or Saccharomyces. The above list is not limiting on the invention.
The term “natural osmosis-tolerant yeasts” refers to osmosis-tolerant yeasts which are isolated from their environment for their natural capacity for producing a given metabolite, here arabitol.
The term “modified osmosis-tolerant yeasts” refers to osmosis-tolerant yeasts whose natural capacity for producing a given metabolite, here arabitol, has been optimised by the use of random or controlled mutagenesis techniques or the techniques of molecular biology.
The term “sugar” refers in the context of the present invention to all
Duflot Pierrick
Lanos Pierre
Machu Fabrice
Segueilha Laurent
Henderson & Sturm LLP
Lilling Herbert J.
Roquette Freres
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