Fermentation, solvent-extractive

Figure 2.5 Possible technological solutions to bioprocess problems a) Fed-batch culture b) Continuous product removal (eg dialysis, vacuum fermentation, solvent extraction, ion exchange etc) c) Two-phase system combined with extractive fermentation (liquid-impelled loop reactor) d) Continuous culture, internal multi-stage reactor e) Continuous culture, dual-stream multi-stage reactor f) Continuous culture with biomass feedback (cell recycling). (See text for further details).

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 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. 

Isolation. Isolation procedures rely primarily on solubiHty, adsorption, and ionic characteristics of the P-lactam antibiotic to separate it from the large number of other components present in the fermentation mixture. The penicillins ate monobasic catboxyHc acids which lend themselves to solvent extraction techniques (154). Pencillin V, because of its improved acid stabiHty over other penicillins, can be precipitated dkecdy from broth filtrates by addition of dilute sulfuric acid (154,156). The separation process for cephalosporin C is more complex because the amphoteric nature of cephalosporin C precludes dkect extraction into organic solvents. This antibiotic is isolated through the use of a combination of ion-exchange and precipitation procedures (157). The use of neutral, macroporous resins such as XAD-2 or XAD-4, allows for a more rapid elimination of impurities in the initial steps of the isolation (158). The isolation procedure for cephamycin C also involves a series of ion exchange treatments (103). 

Polyethers are usually found in both the filtrate and the mycelial fraction, but in high yielding fermentations they are mosdy in the mycelium because of their low water-solubiUty (162). The high lipophilicity of both the free acid and the salt forms of the polyether antibiotics lends these compounds to efficient organic solvent extraction and chromatography (qv) on adsorbents such as siUca gel and alumina. Many of the production procedures utilize the separation of the mycelium followed by extraction using solvents such as methanol or acetone. A number of the polyethers can be readily crystallized, either as the free acid or as the sodium or potassium salt, after only minimal purification. 

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). 

The concentration of enzyme is veiy low, about several hundred milligrams per litre in the fermentation broth. Solvent extraction is a suitable process to recover a small amount of enzyme. The chance of some enzyme being intracellular is high, therefore cells are ruptured to liberate enzyme, which can then interact with organic solvents. Figure 7.1 shows a simple diagram for a jacketed fermentation vessel for operation at constant temperature. 

The microorganism Streptomyces halstedii elaborates an antibiotic substance, called carbomycin32 or Magnamycin,33 which has been isolated from the fermentation broth by simple, solvent extraction, and then purified by recrystallization. (Examination of the resulting mother liquors has led to the isolation of a closely similar compound designated as Magnamycin B. 34) Carbomycin has low toxicity,85 and appears to be an effective and satis-... 

Hotchkiss and Dubos have utilized solvent extraction to separate gramicidin and tyrocidine105. Several patents have been issued on procedures for isolating gramicidin from fermentation broth106, 107, 108. 

Recently an isotope dilution method has been reported O for assaying neomycin sulphate. However, it is first necessary to prepare 14C-labelled neomycin sulphate. This is accomplished by adding l4C-labelled glucose to a small-scale fermentation of 5. nad-iat. l4C-labelled neomycin can then be extracted by solvent-extraction or by ion-exchange chromatography. 

Recently however, it has been recognized that liquid extraction is a potential method in the primary recovery of fermentation cell culture products, such as proteins and amino acids. The separation problem, however, is difficult because the product mixtures are often complex, including cell debris and enzymes. Proteins are not suitable for conventional solvent extraction because of incompatibility with organic solvents, but can be handled in aqueous two-phase systems or by extraction in reverse micellar systems (Chapter 15). 

Acetic acid is an important intermediate organic tonnage chemical that may be produced by the petroleum industry and fermentation. The latter process requires the recovery of acetic acid from water solutions, and several techniques have been applied to this separation, including solvent extraction, azeotropic distillation, and extractive distillation. A comparison of economics between azeotropic distillation and solvent extraction combined with azeotropic distillation (Table 10.3) shows that the introduction of... 

The fermentation broth typically contains 20-30 mg/L of antibiotics, which is to say 30 parts per billion, and must be extracted into concentrated form using solvent extraction. The solvent extraction method was developed by Shell Oil and by Podbielniack and is based on the principle that penicillin is hydrolyzed in aqueous medium to H+ and RCOO ions. Thus, equilibrium in an acidic medium (i.e., one with low pH or high H+ concentration) is favored by the neutral RCOOH form, whereas equilibrium in an alkaline medium (i.e., one with high pH or low H+ concentration) is favored by the RCOO ionic form. The neutral form is more soluble in an organic medium, and the ionic form is more soluble in an aqueous medium. Thus, with amyl acetate as the organic solvent the partition coefficient of penicillin between solvent and water is about 100 at pH 3 and about 1 at pH 6. In the industrial process, the aqueous broth was acidified to pH 3 for the extraction into the organic solvent, and alkalized to a pH 6 for reverse extraction back into an aqueous medium. 

Rates of gas-liquid, liquid-liquid, and solid-liquid mass transfer are important and often control the overall rates in bioprocesses. For example, the rates of oxygen absorption into fermentation broth often control the overall rates of aerobic fermentation. Ihe extraction of some products from a fermentation broth, using an immiscible solvent, represents a case of liquid-liquid mass transfer. Solid-liquid mass transfer is important in some bioreactors using immobilized enzymes. 

Traditional methods of pharmaceutical analysis involve a series of multiple steps. For example, the identification of natural products traditionally involves the scale-up of fermentation broths, solvent extraction, liquid/liquid or column fractionation, chromatographic fraction collection, and spectroscopic analysis (usually NMR) of the individual components. Figure 5.2 illustrates the integration of these bench-scale steps into a dedicated LC/MS/MS system (Lee et al., 1997). Integration provides unique and powerful advantages for the on-line identification of natural products (Kerns et al., 1994 Ackermann et al., 1996a). Experiments that once required 2 weeks to perform with traditional approaches are now performed in half a... 

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