Fermentation medium preparation

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 ... 

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 ... 

After 55 hours of fermentation, the contents of the round flask is transferred under aseptic conditions into a metal reactor of about 100 liters capacity containing 60 liters of sterile medium prepared as follows ... 

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

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. 

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. 

The fermenter was assembled and filled with 900 mL of the medium prepared above (see step 1). This setup was then sterilized by autoclaving (121 °C 20 min) and allowed to cool to room temperature. Then, the fermenter was properly connected to air supply, pH titrants (ammonia solution and phosphoric acid) and anti-foam PP-G200, which was sterilized prior to usage. 

Substrate and Pretreatment. Sweet corn (hybrid Lingodor) of W.H. Perron Laval, Quebec was grown in well prepared soil in a plot of 3 x 2 meters. Corn stalks were ground to 20 mesh to be used as a substrate. It was pretreated with 1.5% sodium hydroxide (NaOH) wt/vol with substraterwater ratio of 1 10 at 121 C for 60 minutes. The substrate was not washed after the pretreatment, and all the solubilized polymers (hemicelluloses and lignin) were retained along with the insoluble polymer (cellulose) in the fermentation medium. The composition of corn stalk is presented in Table 1. 

These defects have spurred attempts to prepare analogs. The techniques used have been (1) natural fermentation (in which the penicillin-producing fungus is allowed to grow on a variety of complex natural nutrients from which it selects acids for incorporation into the side chain), (2) biosynthetic production (in which the fermentation medium is deliberately supplemented with unnatural precursors from which the fungus selects components for the synthesis of "unnatural" penicillins), (3) semisynthetic production (in which 6-aminopenicillanic acid (2) is obtained by a process involving fermentation, and suitably activated acids are subsequently reacted chemically with 6-APA to form penicillins with new side chains) and (4) total synthesis (potentially the most powerful method for making deep-seated structural modifications but which is at present unable to compete economically with the other methods). 

Unit operations for biological products obtained from fermentation or cell culture can largely be subdivided into four parts medium preparation, inoculums expansion, bioreactor, and harvest operations. 

The fermentation medium was inoculated with Bacillus polymyxa prepared as follows A culture of Bacillus polymyxa in a tube with Trypticase soybean broth was incubated overnight at 25°C. 5 ml of this culture was transferred to 100 ml of the tank medium in a 500 ml Erlenmeyer flask which was incubated for 48 hours at room temperature. This 100 ml culture served as inoculum for one tank. During the course of fermentation the medium was aerated at the rate of 0.3 volume of air per volume of mash per minute. The temperature was maintained at about 27°C. Samples of mash were taken every 8 hours in order to determine pH and the presence of contaminants and spores. After 88 hours of fermentation the pH was about 6.3 and an assay using Escherichia coli showed the presence of 1,200 units of polymyxin per cubic centimeter. 

For liquid substrates the most significant equipment cost is medium preparation/fermentation/harvesting/drying, ranging from X to X% of the total running costs. 

The commercial production of antibiotics for medicinal use follows a general pattern, differing in detail for each antibiotic. The general scheme may be divided into six steps (a) preparation of a pure culture of the desired organism for use in inoculation of the fermentation medium (b) fermentation. 

The product was finally identified as 9a-OH-PS by NMR, after isolation by semi-preparative chromatography. A two-step semi-preparative chromatographic separation was run in order to achieve high purity of the sample. First NPLC was performed to get rid of yellow strongly nonpolar compounds from the fermentation medium. The fraction from the NPLC eluate containing the product was further purified using a high resolution RPLC column. 

Enzyme Preparation. A crude enzyme mixture was prepared from the fermentate of Candida rugosa (American Type Culture Collection, ATCC No. 14830. The rugosa was revived and cultivated in YM Agar slants at 24°C for one week. The growth on one YM Agar slant was transferred aseptically to a two-liter flask containing 400 ml sterilized fermentation medium consisting of 2.0% defatted soyflour,... 

The seeds for solid-state fermentation are prepared either by submerged culture or solid-state culture. Bran is an excellent medium for solid seed preparation. After the spores have matured, the solid seeds are harvested and mixed with the pre-sterilized medium for solid-state fermentation. The ratio between solid seeds and medium is around 1 20-50. 

Preparation of fermentation medium — Starch, sugar, soybean powder,... 

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