DD144792A1 - METHOD FOR THE PRODUCTION OF CITRIC ACID BY YEAST - Google Patents

Abstract

The aim of the invention is to eliminate or limit bacterial infections that occur at the pH optimum of citric acid synthesis, from 5 to 6. According to the invention, yeast growth is maintained in a pH range between 5 and 6, preferably by a buffer substance such as CaCO3, while in the post-logarithmic phase, it is reduced to 2 to 3, preferably by self-acidification for a longer period, thereby inactivating the less acid-tolerant bacteria. The invention is applicable to the production of citric acid using yeast.

Description

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Title:

Process for the production of citric acid by yeasts

Area of application of the invention

The invention belongs to the field of microbial product synthesis, IPK V 12 D.

The invention can be used to prepare citrate xmd or isocitrate by yeast under aerobic submerged conditions from various carbon sources such as glucose, sucrose, n-alkanes, ethanol or acetic acid by batch or semi-continuous operation to reduce or prevent bacterial infections.

Well-known technical solutions

Citric acid has been obtained by oxidative fermentation of the fungus Aspergillus niger from sucrose-containing solutions such as molasses or from glucose solutions for over fifty years. Since it has become known that certain yeasts can also dissociate citric acid-usually together with isocitric acid-in relatively large quantities, and that it can be used as a source of carbon. Saccharides are also hydrocarbons, such as n-alkanes of a chain length C..Q to about C ₂ Q, ethanol or acetic acid are suitable (Tabuchi, T. Tanaka, Y. Abe: J. Agr. Chem. Soc ^ Jap. 43 /, 154 (1969), Abe, M. Tabuchi, Y .: J. Agr. Chem. Soc., Jap. 44, 493 (1970), are widely patented.

various \ Yege for the production of citric acid with yeasts has been described (DE-OS 2005848), (DE-OS 2028933), (DE-OS 2109094).

Particularly high product yields were obtained with strains of the yeast species Candida lipolytica (DE-OS 2050361, JA 72 / 24.152 29.10.69 / 4.7.72). But also on the recovery of powerful mutants has been reported v / obei the main goal is the suppression of Isozitratsynthese (DE-OS 2264764, DE-OS 2521469).

The fermentation can be carried out batchwise, semicontinuously or continuously (DE-OS 236009I).

Typical of the course of a discontinuous Zitronensäurefermentation is the separation of growth and Produktbiljdungsphase. Product formation only sets in when growth stops, which is generally achieved by wasting the N source.

The growth phase lasts about 18 to 24 hours, the 'production phase up to 6-to 7 days. The pH optimum for growth is generally between 4 and 5, and pHs down to 2.5 are often tolerated by yeasts, but growth does not occur (A, H, Cook: The Chemistry and Biology of Yeasts, p 304, Academic press, New York, 1958).

The pH optimum for product formation is between pH and 6.5 (Nakanishi, T. et al., J. Perm., Technol. 50 , p. 855 (1972)), although a yeast strain has been described that can be reduced to pH -WerV of 2 still remains productive (DE-OS 2134826).

If, during the production phase, the pH is reduced below 4 to about 3.5, there is a reduction in citric acid accumulation in favor of polyol precipitation.

Basically fermented under aerobic, submerged conditions at temperatures around 30 ⁰ C. The disadvantage of the discontinuous and semi-continuous processes carried out in conventional Y / eise is the risk of outgrowth of bacterial infections. The need to deplete the nitrogen source to arrest the growth stops the post-logarithmic phase of growth so that there is an opportunity for rapid multiplication of bacterial infections. Especially in semicontinuous methods, where not inoculated with newly grown cultures, but where a remnant of the ausfermentierten Produktionskujltur in the permentor as an inoculation is leave_ (usually 1/10 of the filling volume), is the suppression of the growth of PremdOrganismen - as such come only bacteria in consideration - of particular importance. In semi-continuous mode of operation, infection with premature organisms would require the termination of the fermentation

Object of the invention

It is therefore the object of the present invention to propose measures which prevent the outgrowth of bacteria in batch or semi-continuous processes.

Merkma le the invention

According to the invention, the goal is achieved by, for example, lowering the pH of the solution at the end of the logarithmic shift phase, whereby the less acid-tolerant bacteria are inhibited in growth or even damaged.

be damaged. Advantageously, the lowering of the pH is achieved by the physiological acidification and by the subsequent self-acidification due to the excretion of citric and isocitric acid. The components of the nutrient medium must therefore be chosen so that the buffering capacity in the nachlogarithmisehen phase is so low that acidification of the medium is not prevented. Appropriately, to ensure the optimum pH for the growth during, the logarithmic phase (pH 4) and for lowering the pH in the nachlogarithmiseheη phase, such compounds are selected, · are the essential components of currency and their mode of action in terms of present invention requires no additional manipulation. Such compounds are, for example, calcium salts, such as CaCO-. A certain amount of CaCO 3 prevents the pH from falling during logarithmic growth: the amount must, however, be such that in the post-logarithmic phase the pH drops slowly, but remains within a range which limits the activity of the Production yeasts not endangered, this range is between 2.0 and 3 »5» However, it can be even lower for particularly acid-tolerant yeasts. The quantity of Ca ions to be presented is determined by a number of factors, such as the amount of biomass, the degree of physiological acidification (which depends on the releasable cations of the components of the mineral salts), the desired final pH, etc. _e Determine the amount of calcium salt or other according to the invention v / irkenden salt, (eg 0.1 to 0.5 %, preferably 0.2 to 0.3 %, based on 10 g yeast dry matter). It is entirely within the meaning of the invention to set the desired pH even by adding an acid, not only mineral acids but also

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also the produced acids themselves or other organic acids can serve. The phase of the lowered pH is about 0.5 to 10 hours,

The invention is explained in more detail in the following example.

Example;

Strain: Candida lipolytica

Medium (for about 10 g / l biomass): NELCl 3.0 g; CaCO ₃ 2.5 g; KH ₂ PO ₄ 0.8 g; MgSO ₄ 0.4 g; CuSO ₄ . 5 H ₃ O 0.04 g; ZnCl ₂ 0.018 g; MnSO ₄ . 4 H ₃ O 0.03 g to 1 L was made up with tap water. To 1 liter of medium, I60 ml of n-alkanes ("Parex, Cj ₁ to C ₁ Q, preferably C ₁₃ to Cjg) were added

The medium was heated at 120 ^° C. for 30 minutes. After cooling, T mg of thiamine HCl, sterilized by filtration as an aqueous solution, was added to 1 liter of aqueous solution. The pH is 6.0 after sterilization. Cultivation: In a sterilized 200 1 Rührfermentor I60 1 medium were filled under aseptic conditions and inoculated with 10 1 of a 24 hours grown culture (medium as described above, only the paraffin content reduced to 30 ml / 1). The temperature was maintained at 30 ⁰ C (ί 1 ⁰ C). The air flow was 10 m / h. The logarithmic phase of growth was complete after 12 hours. During this time, the pH remained at 6 to 6.2 and then dropped to 2 ₅ 3 within 12 hours. At this stage, the pH was maintained for an additional 4 hours. For optimal lemon isocitric acid elimination, the pH was raised to 5.5 and automatically maintained at 5 »2 by 20% laOH solution.

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Claims (1)

claim Process for the production of citric acid by yeasts from different carbon sources, such as glucose, n-alkanes, acetate and ethanol, under submerged, aerobic conditions, by discontinuous or semi-continuous operation, characterized in that during the logarithmic growth, the pH 'by a buffer substance , preferably CaCO ₀ in a concentration of 0.1 to 0.5 %, preferably 0.2 to 0.3 % _t calculated for 10 g yeast biomass as dry matter, in the optimum range between pH 5 and pH 6 is maintained and in the nachlogarithmisehen Phase of growth, the pH is lowered to a range below pH 4, preferably between pH 2 and pH 3 for a period between 0.5 and 10 'hours » OL HK: - (Q / ti * « Λ ü-i, · '. ·