Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1994-03-08
2001-10-16
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S135000, C435S142000
Reexamination Certificate
active
06303351
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a process for the continuous production of citric acid by fermentation with the aid of yeast.
BACKGROUND OF THE INVENTION
citric acid, an aliphatic hydroxy acid, has been known as a natural fruit acid since the last century.
In nature, it is widely present as the acid and in the form of its salts and can be found in fruit, vegetables, milk and meat.
It is widely used in foods and beverages, in the pharmaceutical industry and for purification process and as an antioxidant so that commercial preparation independent of natural sources is necessary.
Citric acid has been long made from sugar or molasses by a fermentation process utilizing molds of the genus Aspergillus by the surface process and since the middle of this century by a submerged process.
Both processes are batch operations which can achieve yields between 57 to 77% within 1 to 2 weeks.
Since 1960, predominantly in Japan, citric acid has been produced utilizing other microorganisms, especially of the genus Candida, on other substrates of which n-alkanes are the most prominant.
In the Japanese Patent Publication J-58-138387, a process for producing citric acid from n-alkanes is described in which yeast strains of Candida or Saccharomycopsis are used to carry out fermentation in an agitated oxygen-aerated two-phase system where the optimum conversion is achieved with oxygen saturation values of 10 to 45%. The citric acid which is formed is isolated after interruption of the agitation by a phase separation from the aqueous phase which can be replenished a number of times. In this manner, citric acid yields up to 44 g/l can be obtained. Particulars with respect to the formation of isocitric acid are not available. A commercial production by this process is unknown.
In recent years, investigations into citric acid production has concentrated on the use of sugar containing media.
In the East German Patent Document 248,376, for example, citric acid is recovered in a batch process in which initially yeast (
Yarrowinia lipolytica
) is cultured upon a nutrient medium containing n-alkanes or substances with long CH
2
chains with addition of the customary mineral and trace element salt solutions until the N source has been consumed, sugar (125 g/l glucose or invert sugar) being Added and being transformed 90% to citric acid with 5% to isocitric avid. In this batch process significant problems arise with respect to the handling of the residual solution.
A continuous citrate production which would be favorable on economic grounds has not been industrially performed heretofore; on the contrary doubts about the utility of such a process have been repeatedly expressed (H. J. Peppler et. al. “Microbial Technology” Vol. I, Page 363, Acad. Press., London (1979); P. E. Milsom, “Food Biotechnology” Vol. I (1987) Page 291).
T. K. Klasson et al. (Appl. Biochem. Biotechnol. Vol. 20/21 (1989) Pages 491-509) have reported on comparative tests with continuous and bathwise production of citric acid by fermentation with yeast (
Candida lipolytica
). The continuous fermentation is carried in a single fermentor with a maximum residence time of 30 hours and >50% air saturation with glucose content (in the feed) of 30 to 150 g/l with variable ammonium concentrations from 0.3 to 1.3 g/l NH
4
Cl. With the continuous fermentation, fairly specific production rates from 0 to 0.11 g/gh are obtained with significant isocitric acid formation (3 to 6.5g citric acid/g isocitric acid). In the batch process up to 40 g/l citric acid with isocitric acid is formed while in continuous operation the acid concentration runs up to 14 g/l.
The results given by Klasson et al are not promising for a commercial production since both the total acid concentration and the citrate/isocitrate proportions are unsatisfactory.
OBJECTS OF THE INVENTION
It is therefore an object of the present invention to provide an improved process for the production of citric acid or citrate which can be readily converted to citric acid whereby drawbacks of the earlier processes are avoided.
Another object of this invention is to provide a process for the production of citric acid in the form of citrate which satisfies the need for the commercial requirements.
It is also an object of this invention to provide a commercially acceptable process for the production of citric acid in the form of citrate which gives rise to high citrate concentrations in the outflow of a continuously operated fermenter with high ratios of citrate to isocitrate.
It is a further object of the invention to provide an economical improved process for the production of citric acid as citrate.
SUMMARY OF THE INVENTION
These objects and others which will become apparent hereinafter are attained, in accordance with the invention by providing in the fermentation feed a C/N-ratio corresponding to a ratio of 12 to 22 moles of glucose to moles of ammonium compounds.
Preferably the molar ratio of glucose to ammonium compounds in the feed is maintained at 15 to 18 and the glucose concentration in the feed between 100 and 400 g/l, especially 200 to 300 g/l, with the residence time being between 60 and 120 hours(h).
With this relationship of carbon and nitrogen sources in the feed, surprisingly high concentrations with relatively low isocitrate proportions and high conversion of glucose are obtained with space-time yields in excess of 1.5 g citrate/l h. The invention thus provides in a continuous process a limited formation of biomass which is sufficient however to compensate for washout losses and to renew the biocatalyst.
By contrast with hitherto favored intense aeration and high oxygen concentrations in the fermenter, it is preferred with the present invention to operate in the fermentation with 15 to 30% of air saturation (with reference to the maximum oxygen saturation levels with air under standard conditions, which is lower by a factor of 5as when oxygen is used for the saturation instead of air).
It is especially preferred to maintain 20% air oxygen saturation in the fermenter.
The N limitation dependent upon the residence time is preferably held above the nitrogen minimum concentration required to maintain the viability of the living and producing microorganism. This corresponds to a preferred N concentration in the feed of 50 to 150 mM. The favorable operating conditions had been found to utilize a N concentration of about 0.1M, a residence time in the range of 70 to 90 h and a C concentration corresponding to 300 g/l glucose. The pH value in the fermenter is preferably between 4 and 5.5 (especially between 4.5 and 5.0 and most preferably around 5.0. The presence of traces of iron (in the &mgr;M range) in the fermentation medium shifts the pH optimum to a lower value of about 4.6).
Temperatures betwen 29 and 31° C. are preferred for the fermentation, with 30° C. being most desirable.
It has been found, surprisingly, that the addition of iron ions to the fermenter medium have a significant negative effect, i.e. the best results are obtained by excluding iron from the food.
Zinc and Manganese ions have a positive effect and it has been found that the minimum zinc ion concentration should be 0.05 mM especially about 0.15 mM (for Zn
++
)and about 0.75 mM (for, Mn
++
) which apply for nitrogen contents in the fermenter feed of 3 g/l NH
4
Cl and are proportionately higher with increased nitrogen concentrations.
The copper ion concentration is preferably that which corresponds to approximately 2 mg/l copper sulfate while the magnesium ion concentration is in the range of 3 to 5 mM each at N concentrations corresponding to 3 g/l NH
4
Cl and proportionately higher with increasing N concentrations in the feed.
Preferably the invention is practiced with yeast without a carrier and without biomass hold back or retention to yield optimum productivity parameters and enable a steady state to be maintained in a continuous manner over long periods of time since the biomass which is washed from the reactor is constantly resupplied by the growth (tough limited). Wit
Aivasidis Alexander
Anastassiadis Savas
Wandrey Christian
Anastassiadis Savas
Dubno Herbert
Lankford , Jr. Leon B.
Myers Jonathan
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