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This document discusses methods for developing inocula for mycelial processes, including sporulation techniques and factors that influence morphology in submerged culture. Sporulation can occur on solidified media like agar slants or directly on surfaces like cereal grains. Inocula for vegetative fungi are often developed in liquid media under aeration. The concentration of spores and composition of the inoculum development medium impact whether growth is filamentous or pelleted in submerged culture. Both forms are useful depending on the organism and fermentation goals.
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0% found this document useful (0 votes)
409 views

Tech Seminar

This document discusses methods for developing inocula for mycelial processes, including sporulation techniques and factors that influence morphology in submerged culture. Sporulation can occur on solidified media like agar slants or directly on surfaces like cereal grains. Inocula for vegetative fungi are often developed in liquid media under aeration. The concentration of spores and composition of the inoculum development medium impact whether growth is filamentous or pelleted in submerged culture. Both forms are useful depending on the organism and fermentation goals.
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPT, PDF, TXT or read online on Scribd
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THE DEVELOPMENT OF INOCULA FOR MYCELIAL PROCESSES

Sporulation on solidified media Sporulation on solid media Sporulation in submerged culture

Sporulation on solidified media


ROLL BOTTLE METHOD: Large surface area for cultivation of spores 300c.c of medium with 3% agar is sterilized in 1dm3 bottles. Cooled to 45deg.C

On rotation in roller mill, agar sets as a cylindrical shell inside the bottle
Inoculation with a spore suspension of P.chrysogenum and incubation at 24 for 6 to 7 days. Roux bottle is a similar method used in production of clavulanic acid from S.clavuligerus

Organism

Media contents
Glycerol Cane molasses Curbay BG MgS04 7H2O KH2P04 Peptone NaCl Agar Molasses KH2P04 Agar

Amount added(g/dm3)
7.5 7.5 2.5 0.05 0.06 5.0 4.0 20 300 0.5 20

P.chrysogenum

Sporulation on solid media

Substrates used are barley, hard wheat bran, ground maize and rice. Filamentous organisms will sporulate profusely on the surface of cereal grains

1.

FACTORS:
The amount of water added to the cereal before sterilization Relative humidity of the atmosphere, which should be high

2.

Organism

Substrate
200 grams of 'pot barley 100grams of moistened wheat bran

Conditions,yield
5 X 10^11 conidia After 6 days at 28 Deg.C and 98% RH

Aspergillus ochraces

Penicillium , cephalosporium
Aspergillus, Penicillium S.aureofaciens

Cooked rice
White bread Millet

Sporulation in submerged culture


Easy aseptic operation and Large scale application Not suited for actinomycetes Eg : Griseofulvin production
1.

Organism used: P.patulum For prolific sporulation the nitrogen level must be limited to between 0.05 and 0.1% wIv and good aeration must be maintained The lower the degree of aeration, the lower the concentration of nitrogen needed to induce sporulation. Incubation at 25 for 7 days Yield : 10% inoculum for a vegetative seed stage in a stirred fermentor

2.

3.

4. 5.

Fermentation spore inoculum - penultimate stage:penicillium production - early stages : sagamycin - Reduces cost of installation and operation of seed tanks vegetative inoculum - eg: clavulanic acid pdtn - Reduces fermentation time - High labour cost

Choice of inoculum depends on: Length of fermentation process, plant size, cost of labour.

Inoculum development for vegetative fungi

Gibberellin production using Gibberella fujikuroi: - Long slants of potato dextrose agar (1 week at 24 deg.) - Growth scraped off and transferred to a liquid medium: 2% glucose,0.3% MgS04 7H2O,0.3% NH4CI and 0.3% KH2P04. - The medium was aerated(75 hours at 28) - Transfer to a seed fermenter Difficult to get uniform, standard inoculum Mycelium fragmented in homogeniser prior to use

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

Two main factors that influence the morphology: Concentration of spores in a spore inoculum High spore inoculum filamentous growth Low conc. of spores - pellet formation Inoculum development medium Rich, complex media - varied dispersed growth Chemically defined media pelleted growth

1.

2.

Hyphal form

Pelleted form
Broth

Broth becomes highly viscous

is less viscous and less homogenous


Limited

Difficulty in aeration

diffusion of O2 and nutrients to the centre

Eg: penicillin production by Eg: citric-acid production by Aspergillus niger P. chrysogenum Fusarium gramineatium pdtn requires hyphal length of 400 m.

Actinomycetes

Mycelial form : streptomycin by S. griseus Pelleted form : glucose isomerase pdtn by S. nigrificans Inoculum development for cephamycin C pdtn : Key factor : concentration of iron in the seed medium Pellet formation was observed to be detrimental to product formation The principles applied to the optimization of fungal inoculum development regimes are also relevant to actinomycete processes.

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