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.