Broth, fermentation

All these highly complex compounds are produced on the industrial scale from microorganisms. Only the S-lactams are modified chemically after the isolation from the fermentation broth. We shall describe these partial syntheses as well as some typical synthetic sequences of academic total syntheses. 

The separation of cells from the culture media or fermentation broth is the first step in a bioproduct recovery sequence. Whereas centrifugation is common for recombinant bacterial cells (see Centrifugal separation), the final removal of CHO cells utilizes sterile-filtration techniques. Safety concerns with respect to contamination of the product with CHO cells were addressed by confirming the absence of cells in the product, and their relative noninfectivity with respect to immune competent rodents injected with a large number of CHO cells. 

The recovery of recombinant chymosin from a yeast fermentation broth showed that large-scale hydrophobic interaction chromatography could... 

Citric Acid Separation. Citric acid [77-92-9] and other organic acids can be recovered from fermentation broths usiag the UOP Sorbex technology (90—92). The conventional means of recovering citric acid is by a lime and sulfuric acid process ia which the citric acid is first precipitated as a calcium salt and then reacidulated with sulfuric acid. However, this process generates significant by-products and thus can become iaefficient. 

UOP has developed a UOP Sorbex process for the recovery and purification of citric acid from fermentation broths. The process provides technical-grade citric acid, C HgOy, which can be further recrystaUized to obtain food-grade citric acid (qv). 

Each equation is independent of impeller type. As pointed out eadier, the absolute kpi values vary considerably from Hquid to Hquid. However, similar relationships have been found for other fluids, including fermentation broths, and also for hold-up, 8. Therefore, loss of power reduces the abiHty of the Rushton turbines to transfer oxygen from the air to the broth. 

Antibiotics. Solvent extraction is an important step in the recovery of many antibiotics (qv) such as penicillin [1406-05-9] streptomycin [57-92-17, novobiocin [303-81-1J, bacitracin [1405-87-4] erythromycin, and the cephalosporins. A good example is in the manufacture of penicillin (242) by a batchwise fermentation. Amyl acetate [628-63-7] or -butyl acetate [123-86-4] is used as the extraction solvent for the filtered fermentation broth. The penicillin is first extracted into the solvent from the broth at pH 2.0 to 2.5 and the extract treated with a buffet solution (pH 6) to obtain a penicillin-rich solution. Then the pH is again lowered and the penicillin is re-extracted into the solvent to yield a pure concentrated solution. Because penicillin degrades rapidly at low pH, it is necessary to perform the initial extraction as rapidly as possible for this reason centrifugal extractors are generally used. 

Biopolymer Extraction. Research interests involving new techniques for separation of biochemicals from fermentation broth and cell culture media have increased as biotechnology has grown. Most separation methods are limited to small-scale appHcations but recendy solvent extraction has been studied as a potential technique for continuous and large-scale production and the use of two-phase aqueous systems has received increasing attention (259). A range of enzymes have favorable partition properties in a system based on a PGE—dextran—salt solution (97) ... 

The demonstration unit was later transported to the CECOS faciHty at Niagara Falls, New York. In tests performed in 1985, approximately 3400 L of a mixed waste containing 2-chlorophenol [95-57-8] nitrobenzene [98-95-3] and 1,1,2-trichloroethane [79-00-5] were processed over 145 operating hours 2-propanol was used as a supplemental fuel the temperature was maintained at 615 to 635°C. Another 95-h test was conducted on a PCB containing transformer waste. Very high destmction efficiencies were achieved for all compounds studied (17). A later bench-scale study, conducted at Smith Kline and French Laboratories in conjunction with Modar (18), showed that simulated chemical and biological wastes, a fermentation broth, and extreme thermophilic bacteria were all completely destroyed within detection limits. 

Some of the economic hurdles and process cost centers of this conventional carbohydrate fermentation process, schematically shown in Eigure 1, are in the complex separation steps which are needed to recover and purify the product from the cmde fermentation broths. Eurthermore, approximately a ton of gypsum, CaSO, by-product is produced and needs to be disposed of for every ton of lactic acid produced by the conventional fermentation and recovery process (30). These factors have made large-scale production by this conventional route economically and ecologically unattractive. 

A number of fungal immunosuppressives have been isolated from fermentation broths and demonstrated to have immunotherapeutic efficacy. Other than cyclosporin (35), two fungal metaboHtes, sirolimus (36), previously known as rapamycin (80), and FK-506 (37) (81) are in various stages of development (see Antibiotics, macrolides). 

The batch and fed-batch procedures are used for most commercial antibiotic fermentations. A typical batch fermentor may hold over 150,000 Hters. When a maximum yield of antibiotic is obtained, the fermentation broth is processed by purification procedures tailored for the specific antibiotic being produced. Nonpolar antibiotics are usually purified by solvent extraction procedures water-soluble compounds are commonly purified by ion-exchange methods. Chromatography procedures can readily provide high quaHty material, but for economic reasons chromatography steps are avoided if possible. 

Kanglemycin. Kanglemycia (74) (Fig. 8) is isolated from the fermentation broth filtrate of Nocardia mediterranei var kanglensis (1747-64) and its stmcture determiaed by x-ray crystallographic studies. The antibiotic is active against gram-positive bacteria (28). 

Recovery and Purification. The dalbaheptides are present in both the fermentation broth and the mycelial mass, from which they can be extracted with acetone or methanol, or by raising the pH of the harvested material, eg, to a pH of 10.5—11 for A47934 (16) (44) and A41030 (41) and actaplanin (Table 2) (28). A detailed review on the isolation of dalbaheptides has been written (14). Recovery from aqueous solution is made by ion pair (avoparcin) or butanol (teicoplanin) extraction. The described isolation schemes use ion-exchange matrices such as Dowex and Amberlite IR, acidic alumina, cross-linked polymeric adsorbents such as Diaion HP and Amberlite XAD, cation-exchange dextran gel (Sephadex), and polyamides in various sequences. Reverse-phase hplc, ion-exchange, or affinity resins may be used for further purification (14,89). 

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). 

Recovery. Citric acid fermentation broth is generally separated from the biomass using filtration or centrifugation. The citric acid is usually... 

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. 

Product Requirements. When an enzyme is recovered from fermentation broth, it is usually present in an aqueous solution or processed to a dried state. Both types of preparation have to be formulated to comply with requirements appropriate to their final appHcation. 

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. 

Formulations. Any formulation is a compromise between the previously mentioned requirements. For example, the fermentation broth may contain enzyme-stabilizing substances, but the appHcation of the enzyme or precipitation problems in the formulation may demand a high degree of purification that eliminates the stabilizers. Alternatively, the pH necessary for good microbial or physical stabiHty may differ from the pH that gives optimum enzyme stabiHty, or a preservative that is effective at the optimum pH for enzyme stabiHty may have a denaturing effect on the enzyme. 

Regardless of the location of the protein and its state, cell separation needs to be inexpensive, simple, and reliable, as large amounts of fermentation-broth dilute in the desired product may be nandled. The objectives are to obtain a well-clarified supernatant and solids of maximum diyness, avoiding contamination by using a contained operation. Mechanical methods, almost exclusively centrifugation and... 

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