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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 Amount added(g/dm3)
P.chrysogenum
Glycerol
Cane molasses
Curbay BG
MgS04 ·7H2O
KH2P04
Peptone
NaCl
Agar
Molasses
KH2P04
Agar
7.5
7.5
2.5
0.05
0.06
5.0
4.0
20
300
0.5
20
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
FACTORS:
1. The amount of water added to the cereal before sterilization
2. Relative humidity of the atmosphere, which should be high
Organism Substrate Conditions,yield
Aspergillus ochraces
200 grams of 'pot‘ barley
100grams of moistened wheat
bran
5 X 10^11 conidia
After 6 days at 28
Deg.C and 98%
RH
Penicillium , cephalosporium
Cooked rice
Aspergillus, Penicillium White bread
S.aureofaciens Millet
Sporulation in submerged culture
Easy aseptic operation and Large scale application Not suited for actinomycetes Eg : Griseofulvin production
1. Organism used: P.patulum
2. For prolific sporulation the nitrogen level must be limited to between 0.05 and 0.1% wIv and good aeration must be maintained
3. The lower the degree of aeration, the lower the concentration of nitrogen needed to induce sporulation.
4. Incubation at 25° for 7 days
5. Yield : 10% inoculum for a vegetative seed stage in a stirred fermentor
Fermentation
spore inoculum vegetative inoculum - penultimate stage:penicillium - eg: clavulanic acid pdtn production - Reduces fermentation time - early stages : sagamycin - High labour cost - Reduces cost of installation and operation of seed tanks
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 offilamentous organisms in submerged culture
Two main factors that influence the morphology:
1. Concentration of spores in a spore inoculum • High spore inoculum – filamentous growth• Low conc. of spores - pellet formation
2. Inoculum development medium• Rich, complex media - varied dispersed growth• Chemically defined media – pelleted growth
Hyphal form Pelleted form
Broth becomes highly viscous Broth is less viscous and less homogenous
Difficulty in aeration Limited diffusion of O2 and nutrients to the centre
Eg: penicillin production by
P. chrysogenum Fusarium gramineatium pdtn requires hyphal length of 400 µm.
Eg: citric-acid production by Aspergillus niger
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.