Fermentation

All technologies developed for industrial scale have the same aims: maximal production of desired ergot alkaloids, minimum amount of accompanying contaminants (other alkaloids, toxins, etc.), the shortest possible time of fermentation, minimized cost of medium, energy, equipments, and labor. For the production of ergot alkaloids, different fermentation technologies have been employed (a) stationary cultivation on liquid or solid medium (Kybal and Vlcek 1976; Trejo-Hernandez and Lonsane 1993), (b) submerged cultivations (Kobel and Sanglier 1986) also adapted to semicontinuous or continuous processes (Kopp and Rehm 1984). In some cases the immobilized microorganisms were used for production of ergot alkaloids under condition of submerged fermentation (Komel et al. 1985; Kopp and Rehm 1983; Kren 1991).

5.3.1 Stationary Surface Cultivation

In the beginning of sixties, the development of processes for production of ergot alkaloids under conditions of stationary cultivation on liquid media started (Adams 1962; Kybal et al. 1960; Molnar et al. 1964; Rochelmeyer 1965). The stationary surface cultivation on agar slants was commonly used for preparation of starting cultures. When transformed to industrial scale this technology showed some limitations mostly due to difficulties with control of aseptic conditions of large surfaces. Vlcek and Kybal (1974) developed technology for stationary cultivation of C. purpurea in plastic bags partially filled with inoculated liquid medium. This procedure was used for production of ergotoxins and later adapted to production of asexual spores of C. purpurea for field production.

5.3.2 Submerged Cultivation

Submerged fermentation in laboratory scale, i.e., shaker cultivation, is a primary step in getting knowledge of production microorganism physiology, biosynthesis, sporulation, stability, and influence of medium composition on production of alkaloids. In industrial scale, submerged fermentation in shaker culture is mostly used for preparation of sufficient amount of inoculum for further cultivation step. During the inoculum preparation, an optimal state of the culture for biosynthesis of ergot alkaloids in the production step can be established (Socic et al. 1985; 1986). Usually, the whole industrial process consist of four steps i.e., (a) shaker culture, (b) preinoculating fermentation, (c) seed fermentation, and (d) production fermentation followed by downstream processing (Malinka 1999). Industrial production of clavine alkaloids, agroclavine, and elymoclavine by submerged fermentation of different strains of Claviceps sp. was reported in a number of patents (Kren et al. 1988; Rehacek et al. 1986; Trinn et al. 1990), reaching the maximum production of alkaloidal mixture about 6 g/l (Pazoutova and Tudzynski 1999; Pazoutova et al. 1987). In contrast to high production and well elaborated procedure of submerged cultivation the industrial production of clavine alkaloids does not receive much attention due to limited amount of procedures leading to preparation of desirable final products (cf. Table 1). Production of lysergic acid and its simple derivatives is of high technological and industrial interest due to relatively simple procedure of chemical modifications to semisynthetic ergot preparations. The main producers of these alkaloids are strains of C. paspali. The basic studies on biosynthesis, physiology, and production of simple derivatives of lysergic acid were done on relatively limited number of strains including C. paspali 31 (Rosazza et al. 1967), C. paspali ATTC 13892 (Socic et al. 1986), and C. paspali MG-6 (Bumbova-Linhartova et al. 1991). The concentration of produced alkaloids reached nearly 3 g/l of fermentation broth (Pertot et al. 1990) with the strain C. paspali L-52. One of the very interesting features of C. paspali strains is their ability to convert clavine alkaloids added to the cultivation medium to the simple derivatives of lysergic acid (Mothes et al. 1962). Flieger et al. (1989a, b) and Harazim and Malinka (1989) used this capability of C. paspali CCM 8061 and developed technology of aggressive bioconversion of clavine alkaloids to simple derivatives of lysergic acid with total production of nearly 6 g/l in batch cultivation and about 3 g/l in industrial fermentor.

Different strains of C. purpurea, as the only producers of ergopeptines, were described for their submerged production. Ergotamine producing strains and their cultivation are the best-studied processes due to direct therapeutical use of ergotamine. Industrial technologies were developed for the following strains: (a) F.I. 32/17 producing 2g/l of ergotamine and a-ergokryptine mixture (Amici et al. 1966), (b) IBP 47, IMET PA135 producing 1.5 g/l [mixture of alkaloids containing 75% of ergotamine (Baumert et al. 1979)], (c) L-4 (ATTC 20103) producing 1.5 g/l of ergotamine (Komel et al. 1985). Another type of ergopeptines produced by submerged cultivation belongs to group of ergotoxines, i.e., ergocristine, ergocornine, and a-ergokryptine. Between many published procedures and industrial technologies (Malinka 1999) the process developed for strain C. purpurea L-17 resulted in relatively high production of ergotoxines (2.4 g/l). This strain was further studied and intermediary metabolism and production of secondary metabolites were correlated (Gaberc-Porekar et al. 1992).

5.3.3 Solid Substrate Fermentation

Robinson et al. (2001) has recently proposed solid substrate fermentation (SSF) for the production of enzymes and secondary metabolites. The production of ergot alkaloids by C. fusiformis using SSF procedure was found to be 3.9 times higher than that obtained by submerged liquid fermentations (SLF) (Hernandez et al. 1993). One of the reasons could be the necessity of use of antifoam chemicals and the shear stresses caused by stirring in SLF. Also, better air circulation can be achieved in SSF thus further increasing the ergot alkaloid yields (Balakrishnan and Pandey 1996). The SSF has been shown to produce a more stable, requiring less energy in smaller fermentors with easier downstream processing measures. Also, by removal, the cost and trouble associated with antifoaming chemicals and by maximizing yield production, SSF may be seen as a viable option for industrial scale production of ergot alkaloids.

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