Inoculum Development For Fermentation Of Fungi

30°C for 4-5 days aeration: 500 liters/min agitation: 150 rpm

60 liters of fermentation medium in 1000 liters fermentation tank

Fig. 6.6. The inoculum development programme for the production of sagamicin by Micromonospora sagamiensis (Podojil, 1984).

be seen that in the clavulanic acid process the spore inoculum is used to inoculate the final seed stage, in the Chlortetracycline process a vegetative stage is interspersed between the spore inoculated batch and the production fermentation, and in the sagamicin process the spore inoculum is used at a very early stage followed by vegetative growth.

When considering the production of gluconic acid by Aspergillus niger, Lockwood (1975) discussed the merits of inoculating the final fermentation directly with a spore suspension as compared with germinating the spores in a seed tank to give a vegetative inoculum. Direct spore inoculation would avoid the cost of installation and operation of the seed tanks whereas the use of germinated spores would reduce the fermentation time of the final stage, thus allowing a greater number of fermentations to be carried out per year. However, labour costs for the production of the vegetative inoculum could be almost as high as for the final fermentation although some of these costs may be recovered, in that gluconic acid produced in the penultimate stage would be recoverable from the final fermentation broth and would contribute to the buffering capacity throughout the fermentation. Thus, Lockwood claimed that the choice of inoculum for the production stage depends on the length of the cycle of the fermentation process, plant size and the availability and cost of labour.

Inoculum development for vegetative fungi

Some fungi will not produce asexual spores and, therefore, an inoculum of vegetative mycelium must be used. Gibberella fujikuroi is such a fungus and is used for the commercial production of gibberellin (Borrow et al., 1961). Hansen (1967) described an inoculum development programme for the gibberellin fermentation. Cultures were grown on long slants (25 X 10 mm test tubes) of potato dextrose agar for 1 week at 24°. Growth from three slants was scraped off and transferred to a 9-dm3 carboy containing 4 dm3 of a liquid medium composed of 2% glucose, 0.3% MgS04 • 7HzO, 0.3% NH4C1 and 0.3% KH2P04. The medium was aerated for 75 hours at 28° before transfer to a 100-dm3 seed fermenter containing the same medium.

The major problem in using vegetative mycelium as initial seed is the difficulty of obtaining a uniform, standard inoculum. The procedure may be improved by fragmenting the mycelium in an homogenizer, such as a Waring blender, prior to use as inoculum. This method provides a large number of mycelial particles and therefore a large number of growing points. Wor-gan (1968) has given a detailed account of the use of this technique in the preparation of inocula for the submerged culture of the higher fungi.

The effect of the inoculum on the morphology of filamentous organisms in submerged culture

When filamentous fungi are grown in submerged culture the type of growth varies from the 'pellet' form, consisting of compact discrete masses of hyphae, to the filamentous form in which the hyphae form a homogeneous suspension dispersed through the medium (Whitaker and Long, 1973). The filamentous type of habit gives rise to an extremely viscous broth which may be very difficult to aerate adequately, whereas the pellet type of habit gives rise to a far less viscous, but also less homogeneous, broth (see Chapter 9). In a pelleted culture there is a danger that the mycelium at the centre of the pellet may be starved of nutrients and oxygen due to diffusion limitations. Also, there is considerable evidence that the morphological form of the organism influences the productivity of the culture, but whether this is due to the phenomena already mentioned or to some form of metabolic control is far from clear. Thus, some fermentations are carried out with the fungus in a filamentous habit, whereas others are carried out with the organism growing as pellets. For example, filamentous growth has been claimed to be optimum for penicillin production by P. chrysogenum (Smith and Calam, 1980), whereas pelleted growth has been claimed to be optimum for citric-acid production from Aspergillus niger (Al Obaidi and Berry, 1980) and lovastatin from Aspergillus teireus (Gbewonyo et al, 1992; see also Chapter 9). The necessity for filamentous growth is taken to the extreme in the I CI Rank Hovis McDougal mycoprotein process where Fusarium graminearium is produced for human consumption. A highly filamentous morphology is required to produce the desired texture in the product which resembles the strength and eating texture of white and soft, red meats (Trinci, 1992). Thus, in this process a median hyphal length of 400 pm is required.

The relevance of this consideration of mycelial morphology to inoculum development is that the morphology may be influenced considerably by both the concentration of spores in a spore inoculum and the inoculum development medium. Usually, a high spore inoculum will tend to produce a dispersed form of growth whilst a low one will favour pellet formation (Foster, 1949). The effect of the concentration of a spore inoculum on the morphology of P. chrysogenum is given in Table 6.6. Thus, in the commercial production of fungal products it is critical to grow the organism in the desired morphological form which necessitates the use of an inoculum which achieves this end. If the production fermentation is to be inoculated with a spore suspension then the spore concentration must be such as to produce the production culture in the desired morphological form; if a vegetative inoculum is to be used for the production fermentation then, again, the concentration of its spore inoculum must be such as to produce the vegetative inoculum in the desired morphological form. Although the effects of media on morphological form can be extremely varied dispersed growth is more likely in rich, complex media and pelleted growth tends to occur in chemically defined media (Whitaker and Long, 1973). Thus, the medium used in the spore germination stage must be optimized in terms of the morphology of the inoculum.

An interesting series of experiments on the effects of inoculum conditions on the morphology of Pénicillium citrinum were reported by Hosobuchi et al. (1993). This Pénicillium species synthesizes compound ML-236B, a precursor of pravastatin which is a cholesterol-lowering drug. Optimum productivity was achieved when the organism grew as compact pellets in the production fermentation. The vegetative inoculum for the production fermentation had to contain an optimum number of short, filamentous propagules in order to initiate pellet formation in the final culture. This was achieved by using a four-stage inoculum development programme (initiated by a spore-inoculated shake flask) with very rich media in the third and fourth cultures. Thus, this system required a dispersed vegetative inoculum to generate a pelleted production fermentation.

The information available on the morphology of actinomycetes in submerged culture is very limited compared with that on fungi. However, Whitaker (1992) has reviewed the area and it is obvious that actinomycetes are capable of producing a wide range of morphological types. Also, it appears to be accepted that a dispersed mycelial morphology is desirable for most industrial actinomycete fermentations. Mycelial forms have been shown to be desirable for the production of streptomycin by Streptomyces griseus and turimycin by S. hygroscopicus, whereas the pelleted form of S. nigrificans was better for glucose isomerase production (Whitaker, 1992). As already discussed for the fungi, the concentration of spores in the inoculum has also been shown to influence the morphology of certain streptomycetes (Lawton et al., 1989). These workers also demonstrated that medium composition and the shear forces operating during culture also affect morphological form. Thus, the principles applied to the optimization of fungal inoculum development regimes are also relevant to actinomycete processes. Hunt and Stieber (1986) described the optimization of the inoculum regime of a small-scale streptomycete cephamycin C fermentation. Pellet formation was observed to be detrimental to product formation and

Table 6.6. The effect of spore concentration and medium on the morphology and penicillin productivity of Penicillium chrysogenum

Medium Spore concentration Morphology in the medium

Table 6.6. The effect of spore concentration and medium on the morphology and penicillin productivity of Penicillium chrysogenum

Medium Spore concentration Morphology in the medium

Camici et al. (1952):

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  • melody
    How to prepare fungal innoculum for fermentation?
    2 years ago
    What is innoculum development?
    10 months ago

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