Production of Penicillin in Industrial Fermenters (Bioreactors)

Antibiotics such as penicillin are usually produced in large cylindrical vats, constructed of stainless steel, containing a liquid medium in which Penicillium chrysogenum is grown.

penicillin fermenter

What nutrients should the liquid medium contain?

C - carbon source: glucose/sugars

N - nitrogen source NH₄⁺ / NO₃^{- /}amino acids other mineral salts

Why is the liquid stirred?

to dissolve oxygen, mix contents (even distribution of nutrients, mould, waste, heat)

For what purpose is air pumped in?

oxygen dissolves so aerobic respiration occurs, leading to efficient growth

Why must the air be sterile?

so that no bacteria or other micro-organisms get in

Why is the temperature monitored?

to prevent overheating - inefficient / kills Penicillium

How is the temperature controlled?

by pumping cold water into jacket / varying throughput - carries away heat

Why is the pH monitored?

to prevent buildup of excess acid (e.g.), leading to adverse pH

How is the pH controlled?

additions of base / acid

Why is stainless steel considered to be an ideal material for fermenter construction?

strong - strain of stirring, general knocks / does not tarnish - inert - no rust / withstands heat / allows heat to pass through

Before use, fermenters must be sterilised, usually with superheated steam.

Usually these fermenters are operated in a batch process.
After a certain amount of time for fungal growth, followed by gradual production of antibiotic, the contents are removed and processed to extract the antibiotics, then the fermenter is cleaned, sterilised and the process is repeated.

penicillin production graph

Penicillin extraction

After 6-8 days of batch culture, the liquid medium is pumped out, filtered and concentrated. The basic antibiotic - benzyl penicillin - is precipitated as crystals when potassium compounds are added.

This antibiotic may then be modified by the action of other micro-organisms or by chemical means, before being mixed with inert substances and pressed into tablets or converted into syrup or injectable form.

Although the molecular structure of penicillin is known, and it may be synthesised by chemical methods, it is not economic to do so. The production process still relies on fungal fermentation based on biological principles, although modern strains are much more productive than the early strains. This has been achieved through screening programmes involving isolates from different sources, and treatment to encourage mutations.

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 source
How might mutations be encouraged?
radiation (ultra-violet, X-rays etc) or chemicals
What is meant by a screening programme?
Many experiments, testing lots of strains to see if they work or not

Antibiotics - mode of action

These are defined as substances produced by some micro-organisms which are in some way lethal to other micro-organisms. It is thought that these substances give the organisms that produce them (usually moulds or actinomycetes - which grow slowly) some sort of advantage in competition with other micro-organisms (usually bacteria - which grow fast) in the same habitat.

However, their great medical advantage in healing infections is that the purified forms of antibiotics are more or less harmless to most humans. This means that they must act on some aspect of of the growth of micro-organisms which differs from ordinary mammalian cells.

There are in fact several versions of Penicillin, variations on a common formula, produced by different strains of Penicillium, or using different culture media and methods.

These other types of penicillin differ in the efficacy and ease of use in different applications, e.g. some are better in injectable form, or less likely to be broken down by enzymes of host or bacterial origin.

All penicillin type antibiotics operate by inhibiting the production of cell walls by bacteria, which therefore prevents growth. It is now known that penicillin has a bactericidal rather than a bacteriostatic action, i.e. it kills the bacteria, rather than merely inhibiting their growth, as some antibiotics do. The bacteria may die due to osmotic damage because they are not protected by their outer wall.
Various other antibiotics have also been developed with different modes of action, e.g. interfering with bacterial protein synthesis.

Why do antibiotics kill bacteria and not (usually!) humans?
Bacteria have cell walls, & die without them. Human cells do not!

Antibiotic Resistance

Some strains of bacteria produce enzymes which break down and thus inactivate antibiotics such as penicillin, so they can still grow in its presence. These are said to be resistant strains to these antibiotics.

What problem would this cause to an individual person infected with a resistant strain of bacteria ( or that person's G.P.)?
This means that the infection could not be controlled by the usual methods, so their condition would probably deteriorate.

It is thought that in a large population of bacteria there are always a few bacteria with resistance to some antibiotics. The few bacteria with this ability can grow and multiply greatly when the other bacteria - "sensitive" to the antibiotic - have been killed. Bacteria can also pass on the antibiotic resistance to other unrelated bacteria. In effect, these resistant strains of micro-organisms are showing rapid evolution based on the currently accepted principles of evolution - natural selection favouring some sections within populations which normally show variation, most of which is due to mutation, i.e. chance alteration of DNA.

In some cases, other ("second line") antibiotics are available which work in a different way, and may therefore bring infections under control. This is an area of constant application by microbiologists.

Nowadays there are, unfortunately, frequent instances of multiple resistance to antibiotics. Bacteria with this multiple resistance are extremely hazardous and threaten to put the clock back to times before the introduction of antibiotics.

Cause of antibiotic resistance

In the past, antibiotics have been added to animal feed, because they cause increased growth rates (by modifying the balance of bacteria in the animals' gut) and hence gave greater profitability.

However, this practice has been banned because it led to the development of resistance which could be passed to other bacteria.

When taking antibiotics for medical reasons, it is also important to complete the course of antibiotics prescribed, in order to maximise the benefit of the treatment, and not to allow survival of some bacteria exposed to a sub-lethal concentration.

Other infections which cannot be controlled by antibiotics

It is said that infections due to viruses cannot be cured by the use of antibiotics, unless there is a secondary infection by bacteria.
Explain why viral diseases are not controlled by antibiotics.
Most antibiotics damage cell walls of bacteria, which viruses do not have. Bacteria are metabolically active, so they can be "hit" by antibiotics, whereas viruses are not active unless inside the host's cells. Once a virus is inside a cell, the cell is doomed anyway.Viral activities inside cells can only be stopped by killing cells!

Testing bacteria for sensitivity to antibiotics

Individual bacterial strains can be tested against a variety of antibiotics (or vice versa) by growing the bacteria as "lawns" on agar in the presence of different concentrations of a single antibiotic, or several different antibiotics may be tested at the same time.

Under what circumstances might individual bacterial strains be tested against a variety of antibiotics?
to see if a bacterium causing an infection can be controlled by a specific antibiotic/find the best one (for a particular patient)
Under what circumstances might individual antibiotics be tested against a variety of bacterial strains?
to see if an antibiotic under development by a drug company is likely to work in a given circumstance - i.e. control a certain disease

This testing may be achieved either by either of 2 methods:
- placing antibiotic liquid into wells or ditches which have been cut into the agar, or
- applying discs containing measured amounts of antibiotics, which will diffuse out.

bacterial sensitivity testing

What is meant by the term bacterial lawn?
an even layer of bacteria growing on the surface of /within an agar plate

The size of the zone of inhibition - in which bacteria will not grow - gives an indication of the sensitivity of the strain involved, i.e. how easily the bacterial strain will be controlled by the particular antibiotics.

In a medical context, the prescription by a doctor of an appropriate antibiotic or dose rate for a particular patient may thus be confirmed from these laboratory tests.

How long would these laboratory tests take? Explain why.
about 24 hours (or at least overnight) - to give the culture a chance to grow (or be killed!)

The history of antibiotics

The early work done by Fleming, who discovered the effect of penicillin, and Florey and Chain, who developed it further, are interesting examples of recent scientific history. Development of this and other antibiotics such as actinomycin and aureomycin and other tetracyclines solved many problems during the second world war.