DE2313670C3 - Fermentation process for the production of microorganisms - Google Patents

Description

will.

Thermally produced phosphoric acid is often used, but it is by the wet route produced phosphoric acid is also suitable. The technical phosphoric acid must be the one in the claim have said concentration of at least 55% pure phosphoric acid. Generally have technical Phosphoric acids Concentrations of pure phosphoric acid in the range from 55 to 88% by weight.

The time during which the acid must be protected and locked against the entry of microbes from the outside is of course different depending on the acid. The values in the following table gives an indication of the relationship between the time at Raumtempeiatur (about 2O ⁰ C) and the concentration of the acid.

Concentration of Minimum time to full technical phosphorus constant mortification, acid Hours. 55% 24 60% 6th 65% 4th 70% 4th 79% 1

30th

Technical phosphoric acids with concentrations in the range from 55 to 79% therefore require a Sterilization time in the range from 1 to 24 hours.

It is convenient to keep the acid in a closed container at ambient temperature during the time that is necessary to deactivate the microorganisms already present in the acid or kill. The sterilized acid can be mixed with the sterilized aqueous medium prior to its introduction mixed into the fermenter or introduced separately into the fermenter. With separate addition the acid to the fermenter for example through pipelines, it is possible to reduce the length of the pipeline to their The bore and the flow rate of the acid should be dimensioned in such a way that the residence time of the acid in the pipeline system sufficient to deactivate the microorganisms already present in the acid or kill.

Any aqueous nutrient media, their suitability for the growth of the microorganism to be cultivated known can be used. The cultivation of the microorganism can be among the normally applied conditions. The aqueous nutrient medium can by any known methods sterilized. It is best to use heat for sterilization. The simplest and most practical sterilization is carried out by passing the nutrient medium through a heat exchanger will.

The process is of particular value in fermentations where iron salts, e.g. B. iron cations containing salts, especially iron sulfates, are required by the microorganism. If these ferruginous Salts are sterilized by heat with the aqueous nutrient medium, they form iron oxides, which settle on the inner surfaces of the heat exchanger and severely impair the heat transfer. This problem can be solved by reducing the ability of phosphoric acid to self-sterilize, is used for the separate sterilization of the iron salts. For example, you can use the iron salts in the Phosphoric acid and dissolve the solution for a time necessary to deactivate or kill the already existing microorganisms are sufficient to protect against contamination by microbes from the outside and to lock.

The method is particularly well suited for the production of yeast, especially for the production of yeast using a hydrocarbon as a carbon source. However, it can also be used for Breeding of any microorganisms that require phosphorus as a vital nutrient, applied will.

When using a hydrocarbon as a source of usable carbon, for example Kerosene (Leuchtpeiroleu.ti), η-paraffins with 10 to 30 C atoms, gas oils and lubricating oils can be used as hydrocarbons.

Yeasts, the hydrocarbons, can be used as microorganisms, e.g. B. Tribes of the genera Candida, Torulopsis, Hansenula or Neurospora were used. As examples of suitable types of Genus Candida are C. lipolytica, C. pulcherrima, C. utilis, C. utilis variati major and C. tripocalis.

example 1

Candida lipolytica, strain CBS number 6331, was fermented in a 2000-1 fermenter with a working volume from 18001 grown under aseptic conditions. An aqueous nutrient medium of those mentioned below Composition was continuously sterilized by passing it through a plate-type heat exchanger was directed.

(NH ₄ I ₂ SO ₄ 2.0 g KCI 1.15g MgSO ₄ • 7H ₂ O 0.65 g MnSO ₄ · 4H ₂ O 0.045 g FeSO ₄ • 7H ₂ O 0.068 g ZnSO ₄ · H ₂ O 0.17 g Yeast Extra U 10,000 g CuSO ₄ • 5H ₂ O 0.008 g Tap water to make up to 1 1

The medium had a residence time of 1 minute at a temperature of 140 ° C. in the heat exchanger. The sterilized medium was then continuously introduced into the fermenter. A separately sterilized mixture of C10-C _{) 4} normal paralfins in the kerosene boiling range served as the carbon source. This mixture was prepared by treating a petroleum fraction with a molecular sieve.

A thermally produced phosphoric acid with a concentration of 70% by weight as defined above was fed separately to the fermenter. The acid was stored in a container and fed to the fermenter through a pipe. The container and tubing were at ambient temperature, ie, at about 2O ⁰ C. The length and bore of the pipeline were so dimensioned that the minimum residence time of the acid that is, the time during which had been completed, the acid from the external environment, at least It was 4 hours when the acid was fed into the fermenter in an amount and at a flow rate which were sufficient to cover the phosphorus requirements of the fermentation. In the present case, the phosphorus requirement was 1 g, calculated as technical phosphoric acid, per liter of culture medium in the fermenter.

The fermenter was operated continuously in this way for a total of 2817 hours without Blockage or malfunction in the heat exchanger and operated without contamination.

For comparison, fermentation was carried out continuously carried out under exactly the same conditions and in the same manner as described above, however, the phosphoric acid was added to the aqueous nutrient medium before the mixture was taken out Nutrient medium and acid has been passed through the heat exchanger for sterilization. The continuous Operation was flawless for a period of 1820 hours, however, there was a gradual decrease in the heat transfer coefficient of the heat exchanger. After 1820 hours of continuous operation, the Pressure drop in the heat exchanger to rise sharply until after an operating time of 1970 hours of operation was seriously disturbed by the large pressure drop and the low heat transfer coefficient. After 2087 hours of continuous operation, the heat exchanger seals were leaking, so that the heat exchanger failed completely. The plates showed strong buildup and could neither with Acid can be cleaned with sodium hydroxide. Manual cleaning was only partially successful. The final cleaning was done by sandblasting which damaged the panels.

Example 2

Candida lipolytica, strain CBS number 6331, was fermented under aseptic conditions in a 2000 liter fermenter bred continuously with a working volume of 18001. An aqueous nutrient medium of the The following composition was continuously sterilized by passing it through a heat exchanger was directed by the plate type.

above definition was introduced separately into the fermenter. The acid was mixed with iron (II) sulfa (FeSO ₄ · 7H ₂ O) in such an amount that a solution with an iron (II) sulfate concentration of 2.7% by weight was obtained. The solution was stored in a container and fed to the fermenter through a pipeline system. The container and the pipeline were at ambient temperature, ie about 20 ^° C. The length and bore of the pipeline were dimensioned so that the minimum residence time of the solution in the pipeline system was at least 4 hours. The solution was fed to the fermenter in an amount corresponding to about 1 g of phosphoric acid per liter of nutrient medium in the fermenter.

The fermenter ran in this manner continuously for a total of 4270 hours without clogging or failure of the heat exchanger and operated without contamination.

Example 3

Candida lipolytica, strain CBS number 6331. was in a fermenter with a working volume of 20 l continuously bred. An aqueous nutrient medium of the following composition became continuous sterilized by passing it through a plate-type heat exchanger:

(NH ₄ SO ₄ 2.0 g KCI 1.15g MgSO ₄ · 7H ₂ O 0.65 g MnSO ₄ • 4H ₂ O 0.045 g FeSO. 7H ₂ O 0.068 g ZnSO ₄ · H ₂ O 0.17 g Yeast extract 10,000 g CuSo ₄ • 5H ₂ O 0.0008 g Tap water to make up to 1 l

(NH ₄ I ₂ SO ₄ 2.0 g KCI 1.15g MgSO ₄ • 7H ₂ O 0.65 g MnSO ₄ · 4H ₂ O 0.045 g ZnSO ₄ · H ₂ O 0.17 g Yeast extract 10,000 g CuSO ₄ • 5H ₂ O 0.0008 Tap water to make up to 1 l

40

45

The medium had a residence time of 1 minute in the heat exchanger at a temperature of 140 ^° C. The sterilized nutrient medium was then continuously introduced into the fermenter. A separately sterilized mixture of Qo-Cm - normal paraffins in the kerosene boiling range, obtained by treating a petroleum fraction with a molecular sieve, served as the carbon source.

A thermally produced phosphoric acid with a concentration of 70% according to The nutrient medium had a residence time of 1 minute in the heat exchanger at a temperature of 140 ^° C. A thermally produced phosphoric acid with a concentration of 55% according to the above definition was in a closed container stored at ambient temperature (about 20 ⁰ C) under protection against contamination from the outside Mikirobien 24 hours. The acid was then mixed with the sterilized aqueous nutrient medium in such an amount that the phosphoric acid content was 1 g / l. The mixture was then continuously fed into the fermenter. _{A separately sterilized mixture of Ci 4} -Ci7 normal paraffins in the gas oil boiling range, obtained by treating a petroleum fraction with a molecular sieve, served as the carbon source.

The fermenter ran in this manner continuously for a total of 300 hours with no clogging or failure of the heat exchanger and operated without contamination.

Claims (2)

a - ¹ Richer was therefore involved in the implementation of Bisner's _{method of the} above Claims: χΓοΤϋΞ. the phosphoric acid either separately or • Hm aqueous medium before introducing it into 1 Procedure for the production of microorganisms- sterilize with « ^m _r * _u . The sterilization takes place men by continuous cultivation of the M.kroorga- 5 denFerme ^. ^ _{D Sa of the} Ssmus in a fermenter in the presence of e.nes | ^e J ° ^hn ^ n _naltende aqueous Nahrmed.ums for eme sterilized aqueous nutrient medium and m counter ent acid ^^ ^^ _χ that was in phosphoric acid, characterized marked §enu ^ge "de _the ^ _{Sj. ure th ltenden Medmm} Te ch net. that a technical phosphoric acid ^^^ microorganisms is disabled or ververwendet having a concentration _from .ο existing _Slerilisa ^ _{(k lon} even after at least 55 wt .- "/" pure phosphoric acid. mchtet ^"he _{z B by Fl, LRATIO} "of irradiating the Gesamtgewich, technical f ^ ^e ™ £ _{sweise mi} t ultraviolet hch or Kontgen-phosphoric acid has, and thereby development without exposure £ ^S P are _jedoc h these methods kostspievon heat has been sterilized. that se during ™ ^ _s f _{r ZUV} erlässig than the H.tzester.l.sat.on. emer time that be to disable or kill -5 '"""" ^ pounds! the separated already in the acid existing microorganisms t »^ _{phosphoric acid} by the action of is sufficient against contamination by M.krob.en of ^b " ^a _{blerae has been} protected and sealed off by the corrosive nature of the outside. If phosphoric acid results. On the other hand, if the 2 method according to claim! marked thereby he Ben ^ nosP ^ ^ _{by H |. (ze s [enl, wlrd} _ characterized in that a phosphoric acid is used *> acid m _{Wärmeauslausch} it and its containing dissolved iron salt piping systems to blockage by formation of.. Storage of insoluble materials and approaches Jen B iron phosphates. Additionally result IC erosion problems. With continuous The invention relates to a method of producing 25 for long: ^ _{Korrosjon and} deposition of of microorganisms by continuous culture ^since "^ ^eir _Mater ialien be so strong that the microorganism be unusable in a fermenter 1 η counter ^ ° ^ r _ustausche, waiting a sterilized aqueous Nahrmed.ums and ^" ^ _J Presence of phosphoric acid presence of phosphoric acid. caused approaches are extremely harsh and very For the production of microorganisms, ζ B. 3 ° £ ™ _without damaging the equipment for nutritional yeast, fermentation processes are known, with difficult _chemical cleaning, ζ. Β through which a sterilized aqueous nutrient medium, which can enüerne ^^ ^^ _{or Alkaheni} contains technical grade phosphoric acid as a source of phosphorus. _{Be ERNT} Benarig _{A R} e _ini supply of is used. Technical phosphoric acid is one ^ Approaches successful. By sandblasting Mixture that removes pure phosphoric acid, water and 35 » ^an ™ _approaches , but de surface impurities, e.g. B. small amounts of Metalho- ^wer ^ _exchangers damaged, NEN. contains. Technical phosphoric acid is either de-η was _{found that} technical Phosphorsauauf thermally or wet methods ts _certain concentrations themselves manufactured. The be.den by these processes manufactured REN_ η ^ _{sje. Ambient} temperatures Manufactured technical phosphoric acids are sold in different 40 _w ™ ^e _d ^re _{a time} , the deactivation or different concentration. Killing off those already present in the acid The concentration of technical phosphoric acid weighs _against contamination by can a) as percent by weight pure phosphoric acid ™ kroorg s ^ _{hflt and} completed based on the total weight of the mixture or b) Bastenen ^^ ^ ^ _{Concentration of the} as percent by weight of phosphorus pentox.d (P ₂ O ₅ ), based on 45, υ based on the total weight of the mixture, or c) as acidic. ^ _{Invention is} accordingly a specific gravity can be expressed. A through- ^ production of microorganisms through Average technical phosphoric acid has a concentration . _{Cultivation of} the microorganism in tration of 67 parts by weight o / o, calculated as pure phosphorus ^ ™ _'F ^e _e ^r _{rmemer in} the presence of a sterilized, acid. 49% by weight. Calculated as PhosphorpentoxK ₅ o ^e J ^ ^{m Fe} ™ ^e _{nutrient medium and} in the presence of (P ₂ O ₅ ) and a specific gravity of 1.5. In de * ^a ' ^ir ' ^g _h ^en _{rsaure that is} marked by it. that present description and in the claims. t ^P hosP ^h orsa ^ r _{acid is used} , de e, ne the concentration of the technical phosphoric acid as ^ε ™ ^ ^ _{^P we} nigstens 55Gew, o / o pure by weight of the pure phosphoric acid on total ^Konz ™ _u ° _{relative re o} f the total weight of the weight of the mixture expressed. _{"Tpf, hn} i _rule phosphoric acid, and thereby has no DT-PS 4 63 333 describes a process for "" hen ς P ^. ^ _{Wor (jen} . _{$ { That} ^ the production of solutions with a strong germicidal effect. Emphasis on ^ deactivation or which is characterized by it. that for solutions * ^^ ^e ^ acids, bases or salts already present in the acid, whose concentration ^ toW "^ is smaller than 1/50 η, adding 0.85% NaCl. Generally 60 ^ kroorgamsmen g BB ^ ^ _concluded is stated in this publication as known that microbes Alkalis and acids generally been in accordance with "t ^. ^ ^ _{ADVANTAGES) haftei not} reasonable electrical their dissociation, ie the con- _STVE rstflndliche embodiment of the invention centering of the OH or Selbstverstana contained in the solution. H-ions have a disinfecting effect. For phosphoric acid is prayer 65 .. _{so ill at the best} s measured at am- However, known to _st to Orgamsmen, z.emhch ^ ^e; ^ ^S _{e y} ^e _t ^O _n ^U ™ _sterilisi e _r t, but it may act gegebewenig; it is considered unfair see for example exercise »tem £ ^ ^ ^ _outside ^^ R ö m ρ ρ, »Chemielexikon«, 6th edition, 4bös.