23395987 penicillin-fermentation (1)

Title : Production of Penicillin by Fermentation

Maulik Suthar

• Alexander Fleming, 1952

Chains of conidia (spores) produced by hyphal branch from mycelium

Mechanism : Penicillium fungi blocks the growth of gram positive bacteria in culture

The strain

Mold conidiophores, fruiting structures, sporangia, conidia, and asexual spores of Penicillium notatum, also known as Penicillium chrysogenum. The mold is commonly found in homes, is used in the production of green- and blue-mold cheese, and is used to produce the antibiotic penicillin. Penicillin was the first antibiotic to be discovered by Alexander Fleming. Magnification of X600

Penicillium notatum Penicillium chrysogenum

• More than fifty years have passed since penicillin was first produced in volume

Mechanism of antibiotics

Inhibition of Bacterial Cell Wall Synthesis

• large fermenting vats which can hold 10,000 gallons of liquid. The network of pipes keep the temperatures constant during the process of making Penicillin

Outline of Presentation

1. Media

2. Inocula preparation

3. Process and control parameters (ph, Aeration , Agitation , Temp , D.O. etc)

4. Downstream processing (Recovery and Purification)

Media

Component Concentration(gm/ltr)

Cornsteep liquor 21.88

(NH4)2SO4 10.70

KH2PO4 2.74

Sodium phenyl acetate 15.90

Glucose 81.90

MgSO4 7H2O,CaCO3, antifoam oil

qs

• Total Composition of Typical Media:

• Solids (40-60%), lactic acid (12-27%),total nitrogen(7.4-7.8%) , amino nitrogen(2.6-3.3%),reducing sugars as glucose (1.5-14%)magnmasum(18-20%).

• Carbon source: Lactose in concentration of 6% satisfactory. Cornsteep (greatly enhances the yeield of penicillin ). And/or one of the protein rich oil cakes like cotton seed and groundnut.

• One or more sugars like lactose, sucrose and glucose and glucose along with a vegetable oil like soybean oil, groundnut oil

• Nitrogen source : Sodium nitrate,ammonium sulphate,ammonium acetate, ammonium lactate,cornsteep liquor etc.serve as.

• Amino acids such as L-cystine,L-cysteine and valine are important in the synthesis of the b-lactum thiazolidine ring system of the penicillins.

• Mineral salts including sources of sulphate and phosphate• Precursors are used to increase the yield of penicillin by the

fermentation The requisite precursor, eg. phenylacetic acid,phenoxy acetic acid and phenylacetamide are commonly used as precursors.

Inoculation methods

• Various media employed in the manufacture of penicillin can be inoculated by several methods, like ..

surface culture : surface of the medium is inoculated with dry spores. the spore material is applied in such a way as to cover the surface as uniformly as possible.

submerged culture : production medium is inoculated with dry spores,by pellet inocula or by ungerminated spores can be prepared in sterile 0.1% soap solutions, in sterile water containing 100 ppm of sodium lauryl sulphonate.pellet inoculation saves time in the production stage. pellet inocula are prepared by growing mycelium from mold spores under submerged conditions.

Fermenter

Fed-Batch Penicillin Production

Conditions of fermentation

• Optimum Temperature – 25 OC.• Optimum ph Range: 5-7.5, lower ph range yield penicillin substantially

lower• Buffering agent : Calcium carbonate , however it is not suitable in

surface culture production as it decreases the growth of the molds and the yield of penicillin.P.chrysogenum being strictly aerobic,

• Rate of Aeration: adequate aeration of the fermenter is essential, rate vary from around 0.5 volume of air/volume of liquid/minute., Effectiveness may be enhanced by increase in pressure of abt 20lb/sq inch.

• Aeration rate is also attained by the use of proper type of stirrer and at correct speed.

• Antifoam agents such as tributyl citrate, octadecanol, and lard oil, prevent excessive foam formation during the production of penicillin by submerged culture method.

• Prevention of contamination during the production of penicillin is essential because contamination usually causes rapid destruction of penicillin.

• Sterilization of facilities and media are easily achieved through steam.

• lag phase: when a suspension of bacteria is inoculated into a fresh medium growth may not occur immediately. The length of the delay, or lag, in growth depends on the organism, the growth phase of the inoculum, the composition of the new medium and other factors. During this period the viable bacteria in the inoculum undergo physiological readjustment enabling them to utilise substrates in the new medium.

• exponential phase: bacteria enter a period of balanced growth, during which all constituents of cells increase by the same proportion over the same time interval and the population doubles in a definite and constant time (the generation time).

• stationary phase: eventually the bacteria stop growing, because some nutrient becomes exhausted or because a metabolic end product reaches toxic concentrations. Balanced growth is not possible and cells no longer have a constant composition. Also, cells in the stationary phase are smaller than those in exponential phase because cell division continues after the synthesis of most macromolecules has slowed down.

• What does the term productive mean?producing large quantities of something useful (antibiotic)What is meant by an isolate?a single strain (pure culture) of organism from a particular sourceHow might mutations be encouraged?radiation (ultra-violet, X-rays etc) or chemicalsWhat is meant by a screening programme?Many experiments, testing lots of strains to see if they work or not

Isolation and Purification

The first step is the recovery process is the removal of mycelium or cells by filtration or centrifuging.

Second step is to remove the antibiotic from the spent production medium by solvent extraction, adsorption or precipitation.

Additional solvent extraction,distillation,sublimation, column chromatography or other methods accomplish purification.

Semi-synthetic penicillins.

Semi synthetic such as penicillin such as Ampicillin, Methicillin, Oxocillin, Propicllin are prepared by chemical acylation of 6-aminopenicillanic acid.

• The instability of the penicillin molecule under acidic conditions and its low concentration in the fermentation broth required the development of extraction equipment that could efficiently contact the aqueous penicillin-containing broth with the water-immiscible extraction solvent, and then rapidly separate the two phases.

• This permitted rapid extraction of the penicillin from the acidified aqueous phase and rapid neutralization of the penicillin-rich solvent so as to minimize acid degradation of the penicillin. The multi-stage counter-current contactor developed by Walter Podbielniak for this purpose enjoyed wide usage for penicillin production, and for other systems as well.

Assay for standardization

Assay for standardization