The highest antibacterial activity and highest productivity was found in P. chrysogenum. And also P. chrysogenum transforms substrates rich in carbohydrates into penicillin. Because of these reasons P. chrysogenum strain is chosen for penicillin G fermentation.
Microorganism can be kept lyophilized or stored as a spore suspension 20% glycerol at -20 0C
The classical penicillin production process is an aerobic fermentation in fed batch fermentors made with some Penicillium strains, usually Penicillium chrysogenum The incubation of the culture strains provides the seed that grows in seed fermentors until a stage of maturity is reached. Then, the seed is transferred to fermentor. These fermentors are operated in fed-batch mode under standard conditions in order to optimize the synthesis of penicillin. After that, the product is withdrawn by solvent extraction in the downstream.
The first step in the fermentation process is inoculation of cultures. The main purpose of this and subsequent inoculum development steps is to increase the concentration of fungal mycelium (biomass) to give a population which can be added to the next step to assure that each step will be reasonably short and the large-scale equipment is used efficiently.
Inoculum development stages are typically conducted at around 25 0C in shake cultures and agitated vessels.
The media had the following constituents:
NaNO3, g : 3.00
KCl, g : 0.50
MgSO4.7H2O, g : 0.50
FeSO4.7H2O, g : 0.01
KH2PO4, g : 1.00
Glucose, g : 40.00
Distilled water, l : 1.00
All components was autoclaved. The final pH was adjusted to 6. In seven 250 ml flasks; 100 ml of the above media were taken and the flasks were re-autoclaved at 1.0546 kgf/cm2 (15 psi). Then 10 ml of spore suspension was added to each flask and they were kept in a rotary shaker moving at 244 rpm at 37°C .
Fed-batch reactors are used to avoid oxygen limitation. Preventing contamination stainless steel 316 can be used for tank material.
Glucose or molasses (by cont. Feed) %10 of total
Corn-steep liquor %4-5 of total
Phenylaceticacid(by cont. Feed) 0.5-0.8% of total
(Lard oil or vegetable oil) antifoam by cont. Addition 0.5% of total pH to 6.5-7.5 by acid or alkali addition
Trace elements Fe, Zn, Cu
Phenylaceticacid(PAA) is the precursor of penicillin G. Residual PAA concentrations below 0.2 for inoculation to first 20 h., then 0.45-0.60 until the end of the fermentation. This was done to avoid toxic effect of PAA on biomass growth. The addition of vegetable oils or corn steep liquor significantly improved the penicillin production by P.chrysogenum. The replacement of lactose with the continuous addition of glucose or sucrose did not change antibiotic production, but resulted in a financial advantage. Corn steep liquor represent major nitrogen source.
About 65% of the carbon is metabolized for cellular maintenance, 2025% for growth and 1012% for penicillin production. Sugar and precursor are fed continuously and the sugar is also used to help regulate the pH of the fermentation to between 6.46.8 during the active penicillin production phase. In this process, pH 6.5 can be chosen.
Mini-harvest protocols are often used in penicillin fermentations. This "batch-fill and withdraw" system involves the removal of 2040% of the fermentor contents with replacement with fresh sterile medium. This procedure can be repeated several times during the fermentation.
At the beginning, initial substrates (carbon, phosphorus and nitrogen sources) and penicillin-G reaction precursor (phenyl acetic acid) are introduced in the reactor to start the fermentation. The process follows with an exponential growth phase (tropophase), in which the substrate concentration in the fermentor is high. Since penicillin production is a substrate repression process there is almost no production in this...