Fermentation media

To obtain reproducible antibiotic production by fermentation, it is necessary to obtain a pure culture of the producing organism. Pure cultures are isolated from mixed soil sample populations by various streaking and isolation techniques on nutrient media. Once a pure culture has been found that produces a new antibiotic typically on a mg/L scale, improvement in antibiotic yield is accompHshed by modification of the fermentation medium or strain selection and mutation of the producing organism. Production of g/L quantities may take years to accomplish. 

Difficulties encountered in the separation and stabihty of the individual rifamycins led to studies to increase the yield of individual components of the complex. The addition of sodium diethylbarbitutate to the fermentation medium of N. mediterranei resulted in the formation of rifamycin B as practically the only substance formed (91,92). 

Tolypomycins. The addition of small amounts of iron salts to the fermentation medium increases the production of tolypomycin Y (48) (7,203,204), the stmcture of which was arrived at by chemical degradation (205,206) and confirmed by x-ray crystallographic analysis (207) (Fig. 5). Mild acid hydrolysis of tolypomycin Y yields tolypomycinone [22356-23-6] (49, R = H), C27H42NO23, and tolyposamine [34174-76-0] C23H23NO2, (50). Further hydrolysis of tolypomycinone using acid yields tolyponone [24317-12-2] (51), which is also formed upon mild acid hydrolysis of rifamycia S. 

Factors affecting the accumulation of ansamitocins P-2, P-3, and P-4 in JSbocardia sp. C-15003 have been studied (246) the addition of isoleucine, propionate, ptopionaldehyde, or -ptopyl alcohol to the fermentation medium resulted in the increased production of P-2 the addition of valine, isobutyrate, isobutyraldehyde, or isobutyl alcohol increased the production of P-3, reaching more than 90% of the total ansamitocins produced and the addition of leucine, isovalerate, isovaleraldehyde, or isoamyl alcohol increased the production of P-4. 

A typical fermentation medium for penicillin production contains lactose, com steep Hquot, and calcium carbonate (3,153,154). In most industrial processes the carbohydrate source, glucose, beet molasses, or lactose, is continuously added to the fermentation. The rate of glucose addition must be carefully monitored, by pH or rate of oxygen depletion, because the synthesis of penicillin is markedly reduced in the presence of excess glucose. 

In the development of new products, optimization of the fermentation medium for titer only often ignores the consequences of the medium properties on subsequent downstream processing steps such as filtration and chromatography. It is imperative, therefore, that there be effective communication and understanding between workers on the upstream and downstream phases of the produc t development if rational trade-offs are to be made to ensure overall optimahty of the process. One example is to make the conscious decision, in collaboration with those responsible for the downstream operations, whether to produce a protein in an unfolded form or in its native folded form the purification of the aggregated unfolded proteins is simpler than that of the native protein, but the refolding process itself to obtain the product in its final form may lack scalabihty. 

If the fermentation product is extracellular, the benefits come from the minimisation of product entrainment into the cake. Alternatively, if the product is intracellular or the cellular material itself, a compact cake mininiizes the contamination that would result from entrainment of the fermentation medium in the cake. 

Since the microbiological degradation is a rather specialized reaction, no experimental details are given in this chapter. The success of such a reaction depends not only on the proper choice of substrate and proper duplication of the fermentation medium, but also on obtaining the proper strain of microorganism reported to perform a particular transformation. 

A 100 ml aliquot of this medium is placed In a 500 ml Erlenmeyer flask and sterilized by autoclaving for 20 minutes under 15 psi pressure. Spores of mutant strain S. aureofaciens S1308 (ATCC No. 12,748) are washed from an agar slant Into the flask with sterile distilled water to form a suspension containing approximately 10 spores per milliliter. A 1.0 ml portion of this suspension is used to inoculate the fermentation media in the example which follows. A fermentation medium consisting of the following ingredients was prepared. 

Cycloserine may be made by a fermentation process or by direct synthesis. The fermentation process is described in U.S. Patent 2,773,878. A fermentation medium containing the following proportions of ingredients was prepared ... 

A suitable fermentation medium contains water and a source of assimilable carbon and nitrogen and essential mineral salts. A typical medium suitable for production of chlorodemethyltetracycline is as follows ... 

The clarified liquid obtained from the fermentation medium (beer) by filtration in any of the... 

In a 5-liter round flask provided with two tubes, one of which is adapted for subsequent connection to a source of sterile air, 2 liters of fermentation medium are prepared according to the following formulation ... 

The mixture of broth and mycelium thus formed was then transferred under aseptic conditions to a 3-iiter fermentor containing 2 00 ml of a sterile fermentation medium having the following composition 60 g Cerelose (dextrose hydrate), 18 g soybean meal, 5 g distillers solubles, 12 g cornmeal and tap water in a sufficient amount for a 1,000-ml total volume, adjusted to pH 7.0 to 7.2 with potassium hydroxide. 

The fermentation medium was inoculated with Bacillus polymyxa prepared as follows ... 

Germination stage 2 Asepticaliy transfer 25 ml of the fermentation medium of Germination stage 1 to a 2-Cshake flask containing 500 ml of theabovedescribed sterile germination medium. Incubate the flask and its contents for 3 days at 28°Con a rotary shaker (280 rpm, 2" stroke). 

Prior to sterilizing the abovedescribed medium, adjust the pH to 8. Aerobically ferment for 66 to 90 hours while stirring at 250 rpm with air input at 4.5 C/)2/min and 25 psi. The potency of the antibiotic produced at the end of this period reaches a peak of 150 to 225 jug/ml and remains relatively constant. The pH of the fermentation medium changes slightly during the antibiotic production, varying in the range of 6.8 to 7.3. 

The above sterilized medium was inoculated with 11 liters of seed inoculum having a bacterial count of approximately 20 billion per cc. The tank was fermented at 37°C without pH adjustment, aeration, or other modification for 14 hours at the end of which time 320 cc of 50% dextrose was added. After this the pH was adjusted to 7.0 at 15 minute intervals with 5.0 N sodium hydroxide. The volume of sodium hydroxide required for neutralization was noted and 115% of this volume of 50% dextrose solution added after each pH adjustment. At the end of about 8 hours the bacterial count had ceased to increase and the fermentation was terminated. At this time the fermentation medium contained approximately 1,000 units of streptokinase per cc. 

The flasks are then placed on a reciprocating shaker (120 one and one-half inch cycles per minute) and mechanically shaken at 25°C for 3 days. The contents of the flasks are then pooled and, after the pH of the culture is adjusted to about 4 0.2 with sulfuric acid, filtered through Seitz filter pads to separate the mycelium from the fermented medium. 

A spore sand culture containing Gibberella zeae (Gordon) NRRL-2830 was aseptically placed in a sterile tube containing 15 ml of Czapek s-Dox solution and a small amount of agar. This medium was then incubated for about 168 hours at approximately 25°C. At the end of the incubation period, the medium was washed with 5 ml of steriledeionized water and transferred to a sterile tube containing 45 ml of Czapek s-Dox solution. The contents of the tube were then incubated for about 96 hours at about 25°C after which the material was available for use in inoculation of a fermentation medium. 

To a 2-liter flask were added 300 g of finely divided corn. The flask and its contents were then sterilized and after sterilization 150 ml of sterile deionized water wereadded. To the mixture in the flask were then added 45 ml of the inoculum prepared by the process and the material was thoroughly mixed. The mixed material was then incubated for about 20 days at 25°C in a dark room in a water-saturated atmosphere. The following illustrates the recovery of the anabolic substance from the fermentation medium. 

Addition of chelating agents to the fermentation medium may help to inhibit phage multiplication by prevention of phage adsorption to the cell wall. 

Cyclic peptide from 11 amino acids. Preparation by fermentation of Tolypocladium inflatum Gams with addition of DL-a-aminobutyric acid to the fermentation medium. Isolation by homogenization of mycelium, extraction with 90 % methanol and column chromatographic purification. 

By fermentation with Streptomyces griseus, S. olivaceus, S. aureofaciens. Bacillus megatherium or Propionobacterium freudenreichii. Molasses is used generally as fermentation medium, C0CI2 and 5,6-dimethylbenzimidazole are added. Various adsorption and extraction methods are used for isolation from the fermentation liquors. 

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