Fermentation broth impurities

Some fermenter broth impurities have been listed by Holbrook, Bailey and Bailey (13) and repeated in a description... 

Pharmaceuticals. Pharmaceuticals account for 6% of the Hquid-phase activated carbon consumption (74). Many antibiotics, vitarnins, and steroids are isolated from fermentation broths by adsorption onto carbon foUowed by solvent extraction and distillation (82). Other uses in pharmaceutical production include process water purification and removal of impurities from intravenous solutions prior to packaging (83). 

Lime-Sulfuric. Recovery of citric acid by calcium salt precipitation is shown in Figure 3. Although the chemistry is straightforward, the engineering principles, separation techniques, and unit operations employed result in a complex commercial process. The fermentation broth, which has been separated from the insoluble biomass, is treated with a calcium hydroxide (lime) slurry to precipitate calcium citrate. After sufficient reaction time, the calcium citrate slurry is filtered and the filter cake washed free of soluble impurities. The clean calcium citrate cake is reslurried and acidified with sulfuric acid, converting the calcium citrate to soluble citric acid and insoluble calcium sulfate. Both the calcium citrate and calcium sulfate reactions are generally performed in agitated reaction vessels made of 316 stainless steel and filtered on commercially available filtration equipment. 

Purification. The objective of crystallization also can be purification of a chemical species. For example, L-isoleucine (an essential amino acid) is separated by crystallization from a fermentation broth that has been filtered and subjected to ion exchange. The recovered crystals contain impurities deleterious to use of the product, and these crystals are, therefore, redissolved and recrystalHzed to enhance purity. 

Physica.1 Sta.bihty, Physical stabiHty depends primarily on the purity of the enzyme. Impurities remaining from the fermentation broth may precipitate or form a hazy solution. Unwanted sedimentation is often related to Ca " or acidic polysaccharides. The solubiHty of some enzymes can be increased by optimizing the ionic strength or changing the dielectric constant of the solution by a dding low molecular-weight polyols. 

This step is almost always performed to get rid of the coloured impurities in the fermentation broth. The method is based on the fact that amino adds (esperially the non-aromatic amino acids) do not adsorb onto activated charcoal. Although the treatment is very effective, some of the amino acid is lost during this step. 

The fermentation step to produce penicillin GA is the major cost element in the overall process to produce 6-APA. This is substantially due to the high cost of sterile engineering (Table 4.6 and 4.7). Clarification, extraction and solvent recovery steps are also significant, a reflection of the dilute and impure composition of fermentation broths. The concentration of 6-APA in the final broth has a big effect on total process costs. Thus increasing final 6-APA concentrations from 1.2-6.0% have been calculated to reduce production costs by over 50% (Table 4.8). By contrast the 6-APA production step cost is quite small, and is less that half the cost of the solvent recovery process (Table 4.6). The costs of the immobilized enzyme is not insignificant in a recent calculation it was estimated at 2.5 /kg 6-APA (Rasor and Tischer, 1998). 

Once mycelia have been separated via continuous filtration from exhausted production media, citric acid may be recovered by using three different methods, such as direct crystallization upon concentration of the filtered liquor, precipitation as calcium citrate tetrahydrate, or liquid extraction. Since molasses are extremely rich in impurities, direct crystallization cannot be applied unless very refined raw materials, such as sucrose syrups or crystals, are used. The precipitation process (that is based on subsequent addition of sulfuric acid and lime to clarified fermentation broths) is used by the great majority of world citric acid manufacturers, including Archer Daniels Midland Co. (ADM) in the United States. Liquid extraction with mixtures of trilaurylamine, n-octanol, and Cio or Cn isoparaffin was used by Pfizer Inc. in Europe and Bayer Co. (formerly Haarmann Reimer Co., subsidiary of Miles) in the Dayton (OH, USA) and Eikhart (IN, USA) plants only (Moresi and Parente, 1999), even if such plants might have been shut down in 1998. 

Following the initial stages of product recovery from a fermentation broth, a number of purification stages will be required in all but the simplest industrial processes. In the case of high-purity pharmaceutical products, a large number of separation stages are usually required to remove all impurities from the desired final product. By identifying some difference between the product and its impurities, either physical or chemical, the desired bioseparation can be achieved. 

Specifically, a new composition of matter (NCM) or new chemical compound prepared by synthetic methods is the primary area of interest. They may be made using synthetic methods or are isolated from natural sources such as plant or oceanic material or from fermentation broths. At times, a compound is known in an impure state that is unusable as a pharmaceutical product. If it is obtained in a purified state and meets the requirements of patentability, it can be claimed as a compound of a defined purity. 

Immiscible-liquid solvent extraction is a well-established practice for recovery, concentration, and purification of organophilic solutes (e.g., antibiotics, amino acids, vitamins) present in aqueous process streams such as fermentation broths or plant or animal tissue extracts [88]. The process is, however, frequently rendered difficult or impossible by problems of emulsification, loss of entrained solvent, and contamination by particulate impurities in the feed. Integrated membrane separation with liquid/liquid extraction is iUustrated in Fig. 9.7. 

When separation by distillation is ineffective or very difficult, liquid extraction is one of the main alternatives to consider. Qose-boiling mixtures or substances that caimot withstand the temperature of distillation, even under a vacuiun, may often be separated from impurities by extraction, which utilizes chemical differences instead of vapor-pressure differences. For example, penidliin is recovered from the fermentation broth by extraction with a solvent such as butyl acetate, after lowering the pH to get a favorable partition coefficient. The solvent is then treated with a buffered phosphate solution to extract the penicillin from the solvent and give a purified aqueous solution, from which penicillin is eventually produced by drying. Extraction is also used to recover acetic acid from dilute aqueous solutions distillation would be possible in this case, but the extraction step considerably reduces the amount of water to be distilled. 

Since one of the objectives of scale-up is to increase yield, i.e., proportion of available product isolated, knowledge of the compound s stability is a useful asset. Procedures that employ conditions in which the compound is less stable can then be avoided or operated to minimize loss. If the data have not already been determined, it is useful to determine stability under a range of pH and temperature conditions. It is important to appreciate that a compound in an impure mixture may be more or less stable than in its pure state. Enzymes present m crude extracts or fermentation broth may transform the product the rate is likely to be highly dependent on pH and temperature. Some compounds that are relatively unstable in the pure state may be stabilized by conjugation with other components. 

Reverse phase silica (e.g., octadecyl silica) is widely used for analytical purposes and is very effective when used preparatively. However, its capacity is relatively low. It is resistant to fouling by the majority of impurities found in fermentation broths but certain long-chain aliphatics such as may be found in fermentation antifoams can cause problems. Once fouled, these stationary phases are less readily cleaned than the polymenc resins. 

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