Penicilin Production

Microorganism selection

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

Process description

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 After that, the product is withdrawn by solvent extraction in the downstream.

Inoculum development

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 37oC .

Fermentation

Fed-batch reactors are used to avoid oxygen limitation. Preventing contamination stainless steel 316 can be used for tank material.

Fermentation media:

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, 20-25% for growth and 10-12% 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 6.4-6.8 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 20-40% 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 phase. When the microorganism has grown

enough to reduce the substrate concentration, the penicillin production phase (idiophase)

starts.

Penicillin is excreted into the medium and is recovered at the end of the fermentation. Whole broth extraction is usually performed at acidic pH by most manufacturers and has resulted in a 2-5% improvement in overall extraction efficiency by the elimination of the rotary vacuum filtration step. Solvent extraction of chilled acidified broth is carried out with amyl, butyl or isobutyl acetate. Multiple back-extractions into buffer and solvent at varying pH using countercurrent contactors has led to considerable penicillin concentration in the early recovery

stages of the purification process. Pigments and other broth impurities are removed by the use of activated charcoal. The penicillin is crystallized upon the addition of potassium acetate and is isolated as a crystalline potassium salt. Additional carbon treatments and solvent washes results in a highly purified final product.

Variables

* Input Variables

Feed Temperature

Feed Rate

Inflow Air Rate

Agitator Power Input

Coolant Flow Rate

Coolant Temperature

* Process Variables

Culture Volume

Fermentor Temperature

Generated Heat

Concentrations of

Substrate