Penicillins, development fermentation

The agricultural industry contributed, through its fermentation facilities and com steep liquor used for the medium of culture, to penicillin development. The production of penicillin increased by more than 10-fold. In fact, by 1944, there was sufficient penicillin to treat all of the severe battle wounds incurred on D-day at Normandy. Also, diseases like syphilis and gonorrhea could suddenly be treated more easily than with earlier treatments. 

Synthetic vitamins (Max Tishler) After synthesizing several vitamins dniing the 1930 s, Tishler and his team develop the antibiotic suUaquinoxaline to treat coc-cidiosis. He also develops fermentation processes to produce streptomycin and penicillin. 

The history of penicillin is an interesting one. Its antibacterial properties were first discovered accidentally by Alexander Fleming in 1928. Penicillin was treated as a curiosity until 1941, when research on its production coalesced through a network of laboratories in the United States. Within five years, penicillin production had developed from a crude, low-yield method to one making batches of penicillin via fermentation with industrialized mass-production techniques. Prior to 1941, laboratory scientists could only produce small quantities of crude penicillin toward the end of World War II production had mushroomed to 4 million sterile packages per month. 

AH cephalosporins found in nature (Tables 1 and 2) have the D-a-aminoadipic acid 7-acyl side chain (21). AH of these compounds can be classified as having rather low specific activity. A substantial amount of the early work in the cephalosporin area was unsuccessfiiHy directed toward replacing the aminoadipic acid side chain or modifying it appropriately by fermentation or enzymatic processes (6,22). A milestone ia the development of cephalosporins occurred in 1960 with the discovery of a practical chemical process to remove the side chain to afford 7-ACA (1) (1). Several related processes were subsequendy developed (22,23). The ready avaHabHity of 7-ACA opened the way to thousands of new semisynthetic cephalosporins. The cephalosporin stmcture offers more opportunities for chemical modification than does that of penicillins There are two side chains that especiaHy lend themselves to chemical manipulation the 7-acylamino and 3-acetoxymethyl substituents. 

Superior penicillin producing cultures ate capable of producing in excess of 30 mg/mL of penicillin G (154). Cephalosporin producing strains, however, generally grow poorly and cephalosporin C production is not as efficient as is that of penicillin. Factors such as strain maintenance, strain improvement, fermentation development, inoculum preparation, and fermentation equipment requkements ate discussed in the hterature (3,154). 

Most methods used for the production of the commercially important a-amino penicillins, such as ampicillin and amoxicillin, are based on modifications of an enamine process employing the appropriate phenylglycine and methylacetoacetate followed by coupling with 6-APA (64). Other aspects of the fermentation, strain maintenance, equipment, inoculum development, media, and procedures used in the production of penicillin are well covered in previous editions of the Enyclopedia. Developments in these areas have been reviewed (65). 

In spite of the considerable progress in developing methods for total synthesis, this route to cephalosporins cannot compete with fermentation or penicillin rearrangement (see Sections 5.10.4.1 and 2) for the industrial production of cephalosporin antibiotics. While total synthesis does provide access to nuclear analogs not readily obtainable from fermentation products, none of the totally synthetic materials have displayed sufficient advantages to Warrant their development as new drug products (b-81MI51000). 

After a strain improvement and development programme similar to, but more complicated than that of penicillin, the D-a-aminoadipyl side chain containing cephalosporin C was obtained by large scale fermentation. However, cephalosporin C could not be isolated as easily as penicillin G or V. Due to its amphoteric nature it is soluble at any pH in the fermentation broth. Several costly isolation procedures involving ion-exchange chromatography have been developed, as a result of which cephalosporin C is much more expensive than penicillin G. 

It was almost immediately recognised that the deacylated product, 7-aminocephalosporanic add (7-ACA, Figure 6.16), would be of similar importance as was 6-APA in the development of new penidllins. However, 7-ACA, the cephalosporin equivalent of 6-APA, could not be found in fermentations of Cephalosporin acremonium. In Figure 6.15 we have shown that penicillin acylase hydrolyses the acyl residue from natural cephalosporins. Up to a point this is true. These acylases will, however, only work with a limited range of acyl residues. It now seems that nature does not provide for acylases or transacylases that have the capacity to remove or change the D-a-aminoadipyl side chain of cephalosporin C efficiently in a single step. Widespread search for such an enzyme still remains unsuccessful. 

The manufacture of benzylpenicillin (penieillin G, originally just penicillin ) is chosen as a model for the antibiotic production process. It is the most renowned of antibioties and is the first to have been manufactured in bulk. It is still universally prescribed and is also in demand as input material for semisynthetic antibiotics (Chapter 5). Developments associated with the penicillin fermentation process have been a significant factor in the development of modem bioteehnology. It was a further 30 years, i.e. not until the 1970s, before there were signifieant new advances in industrial fermentations. 

From this one ancestral fungus each penicillin manufacturer has evolved a particular production strain by a series of mutagenic treatments, each followed by the selection of improved variants. These selected variants have proved capable of producing amounts of penicillin far greater than those produced by the wild strain, especially when fermented on media under particular control conditions developed in parallel with the strains. These strain selection procedures have become a fundamental feature of industrial biotechnology. 

The 1980 s and the early 1990 s have seen the blossoming development of the biotechnology field. Three-phase fluidized bed bioreactors have become an essential element in the commercialization of processes to yield products and treat wastewater via biological mechanisms. Fluidized bed bioreactors have been applied in the areas of wastewater treatment, discussed previously, fermentation, and cell culture. The large scale application of three-phase fluidized bed or slurry bubble column fermen-tors are represented by ethanol production in a 10,000 liter fermentor (Samejima et al., 1984), penicillin production in a 200 liter fermentor (Endo et al., 1986), and the production of monoclonal antibodies in a 1,000 liter slurry bubble column bioreactor (Birch et al., 1985). Fan (1989) provides a complete review of biological applications of three-phase fluidized beds up to 1989. Part II of this chapter covers the recent developments in three-phase fluidized bed bioreactor technology. 

The p-lactams, mainly penicillins and cephalosporins, are by production volume the most important class of antibiotics worldwide, enjoying wide applicability towards a range of infectious bacteria. Most of the key molecules are semi-synthetic products produced by chemical modification of fermentation products. Production of these molecules has contributed significantly to the development of large-scale microbial fermentation technology, and also of large-scale biocatalytic processing. 

Semi-synthetic penicillins are accessed from 6-aminopenicillanic acid, (6-APA), derived from fermented penicillin G. Starting materials for semi-synthetic cephalosporins are either 7-aminodesacetoxycephalosporanic acid (7-ADCA), which is also derived from penicillin G or 7-aminocephalosporanic acid (7-ACA), derived from fermented cephalosporin C (Scheme 1.10). These three key building blocks are produced in thousands of tonnes annually worldwide. The relatively labile nature of these molecules has encouraged the development of mild biocatalytic methods for selective hydrolysis and attachment of side chains. 

One of the most important developments in the history of large scale fermentations is the fed-batch process. Again, this derives from the work of Marvin Johnson at the University of Wisconsin during development of the penicillin fermentation over 50 years ago. Soltero and Johnson wrote Glucose, intermittently fed to fermentations, has given penicillin yields on synthetic medium equal to, or even better than, those obtained with lactose. Penicillin yields of twice those of lactose controls have been obtained when glucose or sucrose is continuously added to the fermentations . 

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. 

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