DE1517852A1 - Process for the production of vitamin B12 - Google Patents

Description

US ELSACE fHJU.'JG
OR. VCLK'if? VCS ^ IUS

PATcMTAi ivVÄLTE
8Münchcn23-5iegosstr. 26
. iBteion; 345Ö6Z

u.z. » G 4Ö4

JRICHTüfi GüDxäOli VEGYESZiiTI GYJU IT Budapest X., Cserkeaz utca 63 (Hungary)

fOEBSAHRSN TO HEHSTELLÖIIG FROM "VHEAJHH

Priority »December 8, 1965 / Hungary Aninelde-Ho. BI-2yO

01 · Invention relates to a new process for the production of Vitonin B ₁₂ and other similarly acting factors.

It is known that those in the mud of urban Wastewater living microorganisms produce hereditary amounts τ of vitamin R, 2. There are also methods of promotion the growth of these microorganisms and to increase their vitamin production by adding various substances to the sewage or to the sludge known by the such The increase in the vitamin Byj ^ content achieved was the rational operational processing of the waste water

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Extraction of vitamin B, _o fero oil light. When working up, the sludge is used on the one hand as an inoculum and on the other, supplemented with various additives, as a nutrient medium for cultivating the microorganisms that produce vitamin B ^ p. In the further fermentation, the sludge serving as inoculum can then be partially or completely replaced by an inoculum obtained from earlier fermentations, but sludge is always used as the main nutrient medium in known processes.

These methods, in which the natural sewage sludge is used as inoculum and as a nutrient medium, at least in part, have the advantage that the fermentation is not carried out under sterile conditions; as a result, the operational vitamin B-yo production can be carried out in a relatively simple and cheap way in large fermentors of, for example, 500 or 1000% . Volume to be carried out; Constant stirring and ventilation is also not necessary, and the vitamin B ^ production achieved under such circumstances is sufficiently high to ensure the economic viability of the process.

However, the septic fermentation processes carried out using sewage sludge also show fairly serious drawbacks, including the following in particular to mention are:

a) For the fermentation continuously large amounts are needed of sewage sludge (eg 100 mr for each batch), the continued provision In general difficulties offers and neistens not by a single wastewater treatment plant

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is possible; but the Schiarm of nehr «<rfcn which is Abwassernnl * pfn caught up, then often businesses difficulties arise because of the difference in the quality and Mikroorganisuenflora of data originating from different places and treated in various ^one Aeise Schlämmt-,

b) The staujner.de urban household, mainly from households Wastewater shows different compositions in the different seasons and changes accordingly also the composition and the microorganism populrites of the sewage sludge. The urban sewage is astonishing wise also from industrial companies from which harmful substances that interfere with fermentation get into the Schlaoia can.

c) from the sludge in the Fernentation reach the wild ^fl "Mikroorganisnenstämue not allow kterienpcpulation the formation of a permanent, stable, B ^, which, however, the precondition of the reproducibility of the fermentation process and the uniform, stable operation would sführung.

d) The dirty ones. - Form sediments of the sludge an uncomfortable ballast in the processing of the fermentation product; the clever one holds a lot of water in which makes it more difficult to filter the fermentation product and there are also losses during work-up.

The addition of fresh sewage lamb inoculum.

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endeavors, since the experience has always been made that in aolchen Cases when only a Tnoculun taken from previous fermentations. without the addition of fresh sewage sludge - with otherwise the same fan base - was always used there has been a considerable reduction in vitamin B - ^ production is,

Over UNCERT-r, on removing de "obenerwähn« «th disadvantage" d- r ₅ septic fermentation-related research, we have found, surprisingly, that the aforementioned sharp decline in vitamin B ^ -JPjrocluktion in without fresh Abwasserschlaciii-Zusati , only with inoculum removed from earlier fetuses, with the application of err ^ pro ¹ .other, methanol-containing nutrient medium through the O ^ ührtni r ^ nr.L / ^ - AonRt "is only a temporary phenomenon. . W'.rd ofLn? Series of such fermentations

carried out in such a way that always a portion of the inoculum earlier fermentation product is added to the nutrient medium, then after a temporary lowering of the vitamin B ^ g production in the first 1-2 fermentations an increase in the vitamin B_p content was observed again,

■ further / ^λ which after 1-2 / fermentations not only reaches the vitamin B, p ~ Gtehftlt of the usual fermentation products obtained with fresh sludge inoculum, but also significantly exceeds it. This experience shows that the vitamin-producing mixed bacterial population of the waste sludge can not only be adapted to the successive fermentations carried out without fresh sludge addition, always with the previous fermentation product as inoculum,

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Äondem also f-i ™. *. ^ b similar- increase in • is capable of such an adaptation. We also have 'at The microbiological study of the microorganism population found that the original population of the sewage sludge shows a gradual change in the course of this adaptation, and that is what was originally predominantly from The population of bacilli-like organisms is gradually replaced by a coccus species and bacilli in approximately the same proportion containing gbinical population replaced.

The vitamin B - ^ production of the to those without fresh Mud inoculum, exclusively with the mature Fennen-

inoculated successive tation broth of the previous fermentation Fermentations gradually adopting and accordingly gradually changing microorganism population becomes: -xi of the following table with the production data in the usual way, always with fresh mud inoculum performed remote entation series compared. The numbers given in the table show the date on fifth day of fermentation determined by paper chronographs Vitanin B-j ^ -Ctenalt of the fermentation broth

Number of rows of
Fermentations Vitamin: B _{12 {} r / M.teT 1 with fresh
Sludge additive without fresh
mud J-. 4244 3544 3 3560 4200 4th 5400 6120 5 6000 7360 5420
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Similar results are not obtained during the lent product! On , in remote operating gates of 500 units, in the course of a fermentation series continued for one year. The on without Schlainmzusatz continued fermentations adopted ^one Mikroorganisiaenpopulation has its higher Poduktionafähigkeit kept unchanged and the average] cozy vitamin B ^ -JProduktion was un 23% higher than that of in the usual Weis ·, otherwise unttr same conditions with fresh Abwasserschlamn-Inoculira company fermentations have taken place.

The object of the present invention is therefore a process for the production of vitamin Bj ^ and other EjO ^-011 ^ iS ⁶¹¹ factors by fermentation carried out in a nutrient medium containing ethanol, organic nitrogen source and inorganic salts under septic conditions, which is characterized in that the mixed microorganism population obtained from the sewage sludge without further addition of sewage sludge

in

Fresh nutrient medium containing methanol at least five times in succession re-piqued and in this way adapted to the further development without the addition of fresh sludge and then the vitarain-producing Fermentations of these adopted microorganisms], population, also in nethanolhalfeigen, organic Culture medium containing a nitrogen source and inorganic salts, by inoculating ndfc the mature fermentation product of a previous fermentation, with complete closure des AbwassersciilaniLies continues.

The series of these, without the addition of fresh lamb, by inoculating the mature far-end product into the

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next 3Tfcrr:. 'irx-br.-t; i erfclstndfen nritoiainppodusi ^ iiendfcii Fermentations can be practically limitless without a decrease in the production capacity continued; the production capability who already adopted the schlar.Lj.ose nutrient medium Microorganism population remains in an appropriate ethanol The nutrients are constantly maintained.

As a nbthanolhaltiges nutrient medium according to the invention Process an otherwise common, nc-bt-n den as the main carbon source methanol and other organic nutrients that provide carbon and nitrogen, such as yeast extract, Corn soaking water, glycine, succinic acid, and more Aluminum hydroxide, inorganic salts and 5,6-Benzimidaeol as Nälirboden containing precursor can be applied. One too

which can be used with good success in the method according to the invention Nethanolhaltigfcs Näliriiediun is e.g. in the Hungarian patent specification Kr, 151,065; this medium contains' calculated on a 1 liter volume, the following constituents ·: 1 g molasses

1 g yeast extract

2 ml liver extract (10%)

5 g luise spring water concentrate 0.1 g K ₂ HPO ₄

0.1 g MgCl ₂

3 g NH ₄ HCO ₃

10 mg Co CHO ₃ ) ₂ .6H ₂ O

6 mg glycine

6 mg succinic acid
20 ml protein hydrolyzate

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3 ng DiuethylV / nTinidozole

10 ug KeECO,

0.4 ml NH ₂ , OH cone.

5 ial of methanol

It τ '. : · 1 * also found that the vitamin B ^ g production of the inventive fermentation, carried out without the addition of sewage sludge, in ethanol-containing mnediun can be further increased considerably if the nutrient medium provides calcium-ioiien-supplying substances, etc. adding water-soluble or at least partially water-soluble calcium salts. All such additives can be, for example, calcium sulfate, advantageously calcium sulfate freshly prepared from calcium hydroxide and sulfuric acid; The use of superphosphate has proven to be particularly advantageous for this purpose, since with this additive, besides the calcium ions, phosphate ions are also introduced into the nutrient medium, which, according to our experience, have an advantageous influence on the production of BJ-like factors exercise. If, however, superphosphate is used as such an additive, then, according to our experience, the need of the microorganisms for organic nitrogen increases, so in such cases, in order to be able to fully utilize the beneficial effect of the superphosphate additive2, the organic content of the nutrient medium should also be increased Nitrogen source, e.g. of yeast extract, can be increased.

The beneficial effect of the additive on the Calciun Vitamin B ^ g-production v / ith by the following Vereuehsergebnisse illustrates t

α) For the near axis specified in Example 1 below

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dium in an amount of 20% by volume of a hydroxide solution from Cal "iunhydrc and lu / ilRer sulfuric t smelling Calciumeulfnticsunp prepared of 0.13 s / liter concentration is added on the fourth day of the nit aniiil, adap- to sch! eyelets nutrient medium" In the fermentation carried out on the microorganism population, the following vitamin B ^ contents (Γ / liter) in the fermentation product were determined.
Experiment no. With addition of CaSO ^ without CaSO ^

1 7000 4900

2 8600 7800 *

3 6800 6480

4 8200 6480

b) In a series of experiments carried out under similar conditions, various amounts (0.15 to 0.5 %) of commercially available gypsum were added to the nutrient medium instead of the freshly prepared OaSO ^ solution mentioned above. The following results were obtained on the fourth day of the fermentations, compared with the fermentations carried out without the addition of gypsum, otherwise under the same conditions:

Attempt no. Gipszuso- ^ Increase in
Vitamin B ^ product clone. PI 0.1 % 19 % 2 0.2 % 22 % 3 0.3 % 45 % 4th 0.5% 25 %

c) Furthermore, under the same test conditions , 0.1 % superphosphate as a calcium compound was found in the nutrient medium

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^ ct tinr! gl γ-i? el M ₁ -.-. .be the amount of yeast hydrolyzate9 in the Mhiracdi'vn before * 5 g / litar increased fivefold, 25 g / lt * r: On the fii.-: ': / n day of fermentation v / urde the vitamin Bjg content of the Medium determined by lap chromatography. In parallel test series of 30 fermentations a higher average of 29% vitamin B ^ 1b media obtained in ohno Superphoephat additive was found 2 ~ ^K oncentration in the results obtained with superphosphates additional Fennentationsprodukten, *.

As a further advantage of the process according to the invention, we have found that "in the fermentation carried out with the microorganism population adopted with the sludge-free methanol-containing medium according to the invention, the 5,6-rilethylbenzimidazcl usually used as a precursor to increase the vitamin B production can partially be replaced by the considerably cheaper o-xylidine, since these microorganisms can also use this compound to build up the vitamin B-yo molecule, if one part of the fermentation carried out according to the invention, e.g. VT.-80 % of the usual amount (e.g. 3.3 g / liter) of the 5,6-dimeth ^ ^1% >: ^ 7i "iilc £"durc> ~. - " ⁷ ^ i -J-3in replaced, neither the amount of the vitamin produced "-"% - and the Bj ^ -like factors. Still, the mutual proportions of this product = -changed et ..

Basically appropriate procedures can therefore be advantageous be carried out in such a way that the otherwise usual components (Lfecn & noi, other organic carbon and Nutrients containing nitrogen sources and inorganic salts)

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medium, a calcium-containing compound, advantageously calcium sulphate or superphosphate, expediently added in amounts of 0.5 to 1.0 g / liter and about 50 to 30 % of the usual amount of the 5 used as precursor | 6-DiLiGthyrbenziiaidazols replaced by the same amount of o-xylidine.

The method according to the invention shows compared to & <«r previously known methods using sewage sludge as inoculum have the following important advantages:

1, The operational implementation of the fermentation is much safer and "better controllable, since the" wild "microorganisms originating from the sewage sludge, superfluous or even harmful from the point of view of vitamin B ^ production, and the toxic substances that may get into the fermentation medium from the industrial waste water are eliminated and through the adaptation of the microorganisms to the sludge-free medium, a stable microorganism population of very good productivity is formed.

2, The quality of the fermentation broth becomes essential improved by the changes in the sewage sludge Any fluctuations in quality caused are eliminated and the fermentation product can be processed becomes uniform, which is particularly extreme in the case of semi-continuous or continuous operating operations important is.

3, It will be high in a short time, in 4-5 days, 6000 to 7000 .j '/ liter of vitamin B ^ concentrations in the fermentation product achieves the production of vitamins

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becomes * · compared to under similar condition · »from · ** 1Λ the usual way, carried out with Abwaeserßohlaiam-Inoculuffi Fermentation - increased by about 20-2555 on average.

4. Especially the farms that can be used as feed additives cationic products of high (15000 to 2000 ^ '/ liter) LLI> activity can advantageously be carried out in a short time and in a «· c compartments are made,

5. The fermentation to produce vitamin B ^ can be used both periodically and semi-continuously or continuously with equally good success executed v / earth.

The ancestor according to the invention is given by the following Examples illustrated in more detail.

Example 1: Periodic fermentation

2 liters of urban sewage sludge were inoculum old inoculum in a laboratory fermentor of 10 liters capacity entered and diluted with 8 liters of a nutrient medium with the following composition:

20 β molasses

50 g hay hydrolyzate

20 ml liver extract (10%) 50 g corn extract concentrate 30 g NH ₄ HOO ₃

10 ml HH OH cone.
100 mg

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100 mg glycine

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100 mg succinic acid 200 mg NeHSO ₃

10 mg 5,6-dimethylbenzimidazole 40 mg o-xylidine 10 g superphosphate 60 ml methanol • -8000 nX tap water

The content of d ^ r Fermectore was 5 days "cubed at 28 ⁰ C ^in-1, and during this time an additional 60 ml MethanM were added daily, then 2 liters were dee taken medium vice inoculated into 8 liters frischec nutrient medium and then for 6 days at 28 ⁰ C ₁ with daily addition of 60 ml of methanol, and this procedure was noth repeated four times. Before the reinoculation of the vitamin B was 2-Gekalt ^d © e by paper medium, as determined spectrophotometrically & r measurement. Were identified in the 5 successive fermentations the following B ^ -Go & lte t

^ / Liter> / liter> / litor f / liter .i / liter

Reaching the B ^ p concentration of 7000 ^ y / liter showed that the microorganismpopttlation already adapts to the has adapted sludge-free fermentation} Now the fermentation product can already be used as an inoculum to produce vitamins Fermentation can be used. This was done in the same apparatus, under the same conditions, with the same

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1. 3500 2. 4200 3. 6100 4th 7300 5. 7000

Nutrient medium - "TP ^ noLrifcr.- c'Ow-fnllE ur-tK" r ta £ lj.ehfci "60 ml of methanol. The fermentation was ended on the fifth day and 4/5 of the medium was replaced with vitamin B ^ g in the usual way and factors worked up. The medium showed a vitamin content of 6000 f / L ± ter vitamin B ₁₂ and 18000 ^ / liter B ₁₂ -like factors,

1/5 of the iiedium (2 liters) was used as an inoculum used for further fermentation under the same conditions. There were consecutive at randomly continued obtain similar results in further fermentations,

Example 2

Halbkontinuierlic hey Fe rmentation The Ad & ptieruiig the microorganism population of the ex-

wasserschlani.ies cn the slippery fermentation and the first

will be / producing fermentation until the fifth day / exactly in the in 3 example 1 described manner carried out. On the fifth day, 1 liter of liquid was removed from the fermentor and by 1 liter of fresh nutrient ingredients of the following composition replaces:

2 g LIlasse

5 g of yeast hydrolyzate

2 ml liver extract (10% is)

5 g corn swell concentrate

3 g of 23H ₄ HCO ₃

1 ml MOH cone.

0.1 s MgCl ₂

0.001 g of 5 _f 6-diinfethylbenzimidazole

O ₁ Ol g Co (NO ^) ₂ .6H ₂ O

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O ₁ oil g glycine

0 * 01 g of sooted acid

0.02 g KeH ₂ SO ₃

0.00% g of o-xylidine

0.001 e tkiperphosphete

48 ml

The fermentation was continued at 26 ⁰ C and the decrease of 1 liter of liquid and di · addition of 1 liter of fresh ilkhmodjuui the above combination se t * ing was repeated daily. The 1 liter of pernentation products taken every day were processed in the usual way, called Titanin B, and B, g-like factors. In the thus obtained oriented "day fermentation product a Vitoaingehalt from an average of 6000 J ^ / liter vitamin B ^ g and 16,500> 7L B was ₁₂ -like factors found. ·

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Claims (3)

PATiiiTAVAUTIES 1. Procedure for the production of vitamin B ^ and - of other B - ^ - like factors by fermentation carried out under septic conditions in nutrient media containing methanol, organic nitrogen source and inorganic salts, characterized by ? ... <& denotes that the from the The mixed population of microorganisms obtained from wastewater sludge is inoculated at least five times in succession into fresh nutrient medium containing nethanol without further addition of wastewater sludge and in this way adapted to the development of pus without the addition of fresh sludge and then the fermentations that produce vitamins, also in this adopted vitamin-containing organic nitrogen population nutrient medium containing source and inorganic salts, by inoculating with the mature fermentation product of a previous fermentation, with complete exclusion of the waste water sludge. 2. Process according to Claim I _9, characterized in that a nutrient medium is used which, in addition to the methanol serving as the main carbon source, also contains organic acids, yeast extract, corn steep liquor, inorganic Aimoniufirvsrbindungen, growth factors such as liver extract and besides the usual inorganic salts also calcium salts, advantageously calcium sulfate or Contains superphosphate. 3. The method according to claim 1, characterized in that nan as a precursor in addition to a smaller one Amount of 5,6-diethylbenzinidazole predominantly o-xylidine your sewing tr ^ 'Y.'JJ?' 'zusstzt, BAD cr ^ ilNAL -16- 909aR5 / ruii