Penicillin production-biosynthesis, media preparation, fermentation

§  The basic structure of penicillin consists of a thiazolidine ring condensed with a B-lactam ring.

§  6-amino-penicillanic acid (6-APA) is penicillin nucleus

                                        

Penicillin Biosynthesis 

Penicillin is a modified tripeptide made from three amino acids aminoadipic acid, cysteine and valine. The principle enzyme involved in penicillin biosynthesis is ACV-synthetase. The β-lactam ring is formed  by isopenicillin-N synthetase; finally, side chains are added

 

By fermentation technology penicillin is produced from Penicillium chrysogenum.

 

ü  If penicillin fermentation is carried out without addition of side chain precursor, the natural Penicillins are produced.

ü  But fermentation can be better controlled by adding a side chain precursor to obtain derived Penicillin.

ü  The synthetic Penicillins are produced by enzymatic hydrolysis of 6 APA by penicillin acylase enzyme and then addition of desired side chain by chemical means

Penicillin Fermentation- Medium preparation

Raw materials are primary requirement to design the fermentation broth. Fermentation broth contains all the necessary elements required for the proliferation of the microorganisms.  Generally, it contains a carbon source, nitrogen source, mineral source, precursors and antifoam agents if necessary.

Basic composition of inoculation/fermentation media

Corn steep liquor (3.5%)

Lactose (3.5%)

Glucose (1%)

CaCO₃ (1%)

KH₂PO₄ (0.4%)

Edible Oil (0.25%)

and Penicillin precursor in fermentation media (absent during inoculum preparation)

Inoculum preparation

Aim is to develop a pure inoculum in an adequate amounts. Inoculum is built up sequentially from small flasks to small fermenters. All the parameters like temperature, pH, aeration, agitation etc. should be properly maintained.  

pH: near 5.5 to 6.5

 Temperature: 26°C to 28°C

Aeration: a continuous stream of sterilized air is pumped into it.

Agitation: have baffles which allow constant agitation. 

Carbon Source

Lactose acts as a very satisfactory carbon compound for fermentation. Other carbohydrates like glucose is used during inoculum preparation. Lactose is preferred in fermentation media to provide slow feeding rate and starvation conditions to induce antibiotic formation. Sucrose may also be used. Complex as well as cheap sources like molasses, or soy meal can also be used.

Nitrogen Source

Another essential compound for metabolism of organisms is nitrogen. Ammonium salts such as ammonium sulfate, ammonium acetate, ammonium lactate or ammonia gas are used. Corn steep liquor also may also be used.

Mineral Source

Additionally, some minerals are necessary for the proper growth of these organisms like phosphorus, sulfur, magnesium, zinc, iron, and copper which are generally added in the form of water soluble salts. 

Precursors

Various types of precursors are added into production medium to produce specific type of penicillin. The most important naturally occurring penicillin is Penicillin-G. Depending upon the precursors added, the type of penicillin going to produced can be changed.

For example, if phenyl acetic acid is provided then  penicillin-G will be produced but if hydroxy phenyl acetic acid is provided then penicillin-X will be produced. Phenoxy acetic acid is provided as precursor for penicillin-V production. 

 When corn steep liquor is provided as nitrogen source, it also provides phenyl acetic acid derivatives; therefore, it is widely used in the production of penicillin-G.

Anti-foam agents

Anti-foaming agents such as lard oil, octadecanol and silicones are used to prevent foaming during fermentation. 

Following three points should be kept in mind before choosing raw materials for manufacture of penicillin,

1. An abundant growth of mycelium 

2. Maximum accumulation of penicillin 

3. Ease of extraction and purification of antibiotics.

Penicillin Production 

Penicillin production was done previously by surface process ie. Solid state fermentation and surface liquid fermentation. Nowadays a commercial production is carried out by fed batch submerged process

Microorganism 

Initially Penicillium notatum strain was used for fermentation of penicillin. Later, Penicillium chrysogenum was discovered and improved by using strain improvement program by genetic mutation This strain should be maintained properly by lyophilization technique or storage in liquid nitrogen in frozen state.

 

 

Penicillin Production

Steps

1)      Inoculum preparation

2)      Production fermentation

3)      Product recovery

1) Inoculum preparation:

In the process of inoculum preparation the pure culture of Penicillium chrysogenum is prepared in adequate amount for production of penicillin.

 

ü  The primary working stock is added in special sporulating agar which allow maximum sporulation of spores. The sporulated spores are suspended in water or non-toxic wetting agents like SLS - sodium lauryl sulphonate solution in a proportion of 1:10,000.

ü  These spores are added in a nutrient medium of wheat bran plus nutrient for heavy sporulation and the flask are incubated for 5 to 7 days at 24°C. This is done in small tanks with aeration and agitation for sufficient mycelial growth

ü  These mycelia from inoculum medium are used as a inoculum in fermentation tank.

can be used for production fermenter at 10% levels

2) Production fermentation:

§  Method: fed-batch or batch

• Substrate: glucose, lactose, phenyl acetic acid (precursor), Corn steep liquor, Additional nitrogen source ie, soya meal, yeast extract, lactic acid, inorganic ions, growth factors 

ü  Precursors for penicillin G – phenylacetic acid.

ü  Precursors for penicillin X – Hydroxy phenylacetic acid.

ü  Precursors for penicillin V – Phenoxy acetic acid.

Fermentation media used in penicillin fermentation should provide a sufficient amount of carbon source, nitrogen source, Nutrients, amino-acids, salts and precursors. The fermentation media should provide growth of mycelium, accumulation, extraction, purification of penicillin. Exact composition of media used in industries are not know as it’s a matter of trade secrets.

§  Fermenter: stirred tank or air lift tank

§  pH:  5.5 - 6.0 

     (which increase up to 7-7.5 (optimum) due to consumption of lactic acid and liberation of NH₃ gas. If pH is 8 or more, CaCO₃ or MgCO₃ or phosphate buffer is added)

§  temperature: 25-28°C

§  aeration

§  agitation: 120-150 rpm

§  time: 3-5 days

§  antifoam: edible oil (0.25%)

 

Fermentation of penicillin is carried out by surface fermentation in trays or by submerged culture method. The 10 % of inoculum is added in the fermenter aseptically. The temperature of about 25°C to 28°C is maintained. The sterile air supply is provided continuously as the fermentation is aerobic fermentation. The fermentation is carried out for about 3 to 5 days.

During this fermentation process the samples are withdrawn aseptically and checked for yield of penicillin, pH and contamination. The checking for contamination is very important if the fermentation media gets contaminated by organism producing penicillinase enzyme then it can result in a great economic loss to industry. The fermentation process is monitored for foam formation also and if foam is produced, it is controlled by antifoaming agent.

Initially the pH of fermentation media remain constant as the inoculum initially utilizes carbon as a source of energy but further when concentration of carbon is reduced the micro-organism starts utilization of nitrogen as a source of energy so at this point the pH of the fermentation media increases to 7.0 to 7.5 due to deamination and release of ammonia. Now at this point the micro-organism starts product synthesis by utilization of lactose and production of penicillin. After product formation the concentration of lactose is decreased and pH rises to 8 or even more which results in autolysis of mycelium. Here at this point the fermentation is stopped and recovery and harvesting of product is started.

Initially during 20 to 30 hours the fungal spores utilizes carbohydrates and corn steep liquor and fungal spores develop as mycelium and further in the duration of 48 to 96 hours the mycelium starts production of penicillin product.

The yield obtained is 3 % to 5 % and 1500 unit per milliliter of fermentation medium.

(contd..)