Antibiotic Production

Although Fleming discovers penicillin in 1929, large

scale processes for its production were not realised
until the early 1940s.

The treatment of wartime casualties became a priority

and the work of Florey realised the potential of this
drug in the battlefield.

Initially penicillin was produced in milk bottles

because the technology existed for the handling and
filling of these vessels.

Over time it became apparent that deep tank

fermenters be used as they resulted in a great leap
forward in process efficiency and productivity.

Valuable lesson:
Scientific discovery is not the singular catalyst to bring
about significant change. It is also essential that
suitable technology be proposed to exploit the
potential to its full commercial impact.

The early success in isolating a medically useful

compound from a microorganism lead to the massive
ongoing hunt for antibiotics that continues to the
present day.

Today, over 5000 compounds have been isolated, by

far the largest proportion have been isolated from
streptomyces cultures.
Of this only a relatively small number of compounds
have been commercially successful in therapy, with
hundreds of tonnes of compound being produced
each year by fermentation.

Penicillins and cephalosporins account for

approximately 70% of market share. These are used
either in their natural form or as semi synthetic
derivatives.

Antibiotics are produced generally as a result of some

environmental stress that triggers a metabolic
response.

Antibiotics are produced as a defense mechanism to

prevent the proliferation of other organisms when
environmental conditions are challenging.

The organism will incur significant metabolic overhead

systhesising these compounds, so only does so under
environmental stress.

This is an important consideration when considering

process design.
Production Processes
Almost all antibiotics are produced in mechanically
agitated and aerated vessels, the design of which is of
crucial importance to the process.

Media used for production are extremely varied and

most contain both complex nitrogen and carbohydrate
sources.

Each production process is generally divided into the

following key stages.

• Culture Preservation
• Innoculum Preparation
• Seed Stage
• Production Stage
• Harvest, Extraction and Purification

Culture Preservation
The preserved culture is a valuable asset and as little
should be used as possible to initiate the process.

Generally this preserved stock is in the form of inert

spores.

Spores can be stored in dry soil, lyophilised or frozen

in a preservation solution.
Production Strain
A high yielding strain is a prerequisite of any good
antibiotic producing process. Continuous modification
of process conditions will result in steady increases in
yield however, large steps forward generally result
from the introduction of a superior strain.

Such strains are generally obtained through genetic

manipulation. Random mutagenesis and subsequent
screening are key strategies in strain yield
improvement.

Modern techniques of genetic engineering has seen

the isolation and cloning of gene cartridges
associated with the metabolic pathway for
biosynthesis of antibiotics into non producing
organisms with more desirable growth attributes.

Remember if this approach is to be successful, then

the means of inferring resistance to the antibiotic also
has to be provided to the host microorganism.

Scale Up
Scale up is an important part of many cell driven
processes and no more so than in antibiotic
fermentations.

Starting off with a small volume of a preserved

culture, it is not feasible to inoculate your production
fermenter immediately with this material.

It is essential to amplify the starting innoculum

through a series of ever increasing volumes until
enough material exists to innoculate the production
system.

In bacterial and yeast systems where vigorous growth

is anticipated, 1:20 to 1:100 pitches are common. A
common chain would be

1ml – 100ml – 10L – 1m3 - <1000m3

Innoculum resuspension – shake flask – pilot plant

fermenter – seed fermenter – production fermenter.
For antibiotics the production vessel is generally in the
range of 10-300m3 depending on the value of the final
product.

While the overall aim of the process is to have the

microorganism producing antibiotic for as long a time
as possible, it is always essential to consider that the
catalyst has to be grown prior to its use. Low biomass
concentrations in the production reactor may have
deleterious impact on process timescale.

Also consider our earlier statement, that antibiotic

production is not growth associated and is as a result
classified as a secondary metabolite. Biocatalyst
growth and and antibiotic production are mutually
exclusive.

The solution to this issue is to have a growth medium

which is used throughout the scale up process and
have a production medium which is used in the final
stage of the process.

The growth medium is formulated to encourage

biomass growth to the detriment of antibiotic
production and the production medium (in conjunction
with process conditions) simulates the required
environmental stress required for the synthesis of
antibiotics.

Production process
Process economics key, one of the major process
costs associated with the sterilisation of large volumes
of fermentation medium.

Continuous sterilisers often used due to superior

efficiencies.

The production reactor must be designed with the

following considerations in mind (these considerations
also apply to a lot of other processes):

• High gas liquid mass transfer coeffients must be

obtained.
• High interphasial nutrient transfer to ensure that
nutrients are rapidly distributed and supplied to the
organism. (organism growth morphology is a
significant consideration here)
• Good bulk liquid mixing characteristics to ensure
homogeneity within the vessel.
• Fermenters produce heat (through dispersion of
kinetic energy and metabolism), therefore the vessel
must be a good heat exchanger.
• The vessel must be able to run aseptically for
prolonged periods of time. (maintenance of positive
pressure within vessel).

Most production fermenters in antibiotics are of

traditional design. (For AS4s)

• Stainless steel construction

• Vessel Height to Tank Diameter 2-4:1
• One Rushton Impeller per Tank Diameter in Height.
• Air sparged into the vessel below bottom impeller
between 0.3 and 1.5 VVM.
• Power consumption in reactor in the order of 1-4
W/L

Production Media

A whole family of production media are required for an

antibiotic producing process.

Each medium is designed to support a particular

stage of the process

Media designed for the early stage of the process will

be generally focussed on achieving optimal spore
germination and strong vegetative growth.

As antibiotics are secondary metabolites, the final

production medium will be focussed on the over
production of the antibiotic often to the detriment of
growth.
Most media used for production are complex in
nature. Although many organisms will produce their
indigenous compound on chemically defined media,
complex media are cheaper to formulate and have
higher yields and productivities.

As with any general growth media it must supply all

the basic constituents for growth/product formation.

• Carbon source
• Nitrogen
• Phosphates
• Trace Elements

In the case of antibiotic fermentations, the use of

specific precursor molecules in media formulations is
significant.

For example: The production of Penicillin G is greatly

improved through the introduction of Phenyl-acetic
acid as a precursor into the fermentation medium.

Nitrogen source, generally cheap supplements

containing corn steep liquor (source of penicillin
precursors: phenylalanine and phenethylamine), soya
flour and fish meal. These materials may often supply
unknown trace elements which aid production.

Technical grade glucose and or starch can be used as

a carbohydrate source. If the process is batch, one
generally finds a mix of carbohydrates present.
Simple carbohydrates to get the process moving and
complex ones to simulate substrate (catabolite)
limitation.

Catabolite Repression
Many antibiotics are not produced in the presence of
excess carbon sources, especially glucose.

In order to overcome catabolite repression, the

addition of the carbon source to the culture must be
carefully controlled. Fed batch is the most common
approach.

Lactose used historically as carbon source in batch

cultures due to its slow rate of hydrolysis (no residual
glucose in fermentation media post hydrolysis)

The presence of excess nitrogen compounds or

phosphates in the fermentation decreases antibiotic
production severely.

Feedback Regulation
If penicillin is added to a culture of penicillin producing
fungi, synthesis of the antibiotic is limited.

Precursors
Secondary metabolites are synthesised from primary
metabolites. The efficient production of antibiotics
requires a steady flow of their precursors.

For example α-aminoadipic acid is an intermediate in

the pathway of lysine biosynthesis.
High levels of lysine in the media shuts off that
pathway by inhibiting the first enzyme in that pathway.

This results in a shortage of all the intermediates in

the pathway including the key intermediate for
penicillin.

Therefore the presence of lysine strongly inhibits

penicillin production.

Production Process Outline- Penicillin

Spores of P. chrysogenum are used to inoculate

100ml of growth medium in a 500ml shake flask.

4 days incubation, contents are transferred to growth

medium in 500L reactor.

After incubation for three days, this culture is used to

inoculate 180m3 reactor.

Final Fermentation completed in 5-6 days.

pH – about 6.5 and the temperature is in the range

23-28C.

Best current strains result in about 10% of the carbon

in the glucose finds its way into penicillin G whose
final concentration may reach almost 30g/L
Penicillin Variants

Most fungi that produce penicillin can easily

incorporate a variety of compounds into the acyl
portion of the penicillin molecule.

Over 100 variants have been produced and each has

been evaluated for a variety of desirable attributes.

All compounds must have the initial beneficial traits of

Penicillin G along with additional advantages.

• Acid Stability (allowing for oral ingestion)

• Lowered allergenicity – 8% of population allergic to
Penicillin G.
• Greater resistance to penicillinase

Penicillin V – Increased acid stability. Allowing for

oral ingestion.

Penicillin O – lower allergic sensitivity.

Semi-Synthetic Variants

The compound 6-aminopenicillanic acid can be

readily acylated by chemical means to produce
semisynthetic variants.

Tens of thousands of compounds have been

synthesised, resulting in new antibiotics with desirable
attributes.
Down Stream Processing of Penicillin

Broth contains 20-35g/L of penicillin

Mycelia are separated from the liquid by filtration

occasionally using filteraids or precoats

Penicillin rich filtrate is cooled to 4C to minimise

chemical and enzyme degradation during solvent
extraction

Sometimes the filtrate is further clarified by a second

filtration with 1-1.5% Hyflo (a filter aid) sometimes with
the precipitation of proteinaceous material by addition
of aluminum sulphate or tannic acid.

In the next stage of the process, penicillin is extracted

into amyl acetate or butyl acetate using a continuous
countercurrent process.

Penicillins are strong acids with pKa values in the

ranve of 2.5-3.1. As the acid forms are soluble in
many organic solvents, they are extracted with high
efficiency into amyl acetate or butly acetate at pH 2.5-
3.0

The extraction is performed in continuous

countercurrent multistage centrifugal extractors.
Efficient extraction uses a solvent to broth ratio of 0.1

One such device is a the Podbielnak extractor. It

consists of a cylindrical drum contining perforate
concentric shells and is rapidly rotated on the
horizontal shaft (2000-5000rpm)

Liquids enter through the shaft; heavy liquids is led to

the centre of the drum and lighter liquid fed to the
periphery of the drum.

The heavy liquid flows radially outwards displacing the

light liquid inwardly and both are led out through the
shaft.

These extractors are ideal for liquids with small

density differences where short residence times are
essential.

The penicillin containing solvent is then treated with

0.25-0.5% carbon to remove pigments and other
impurities.

Penicillin is then back extracted into water by the

addition of alkali (potassium or sodium hydroxide) or
buffer at pH 5.0-7.5

The volume ratio of water to solvent in this stage of

the process is 0.1-0.2 in continuous multistage
extractor.

Penicillin can be precipitated form the resultant

aqueous phase.

Penicillin crystals are washed and predired with

anhydrous l-propanol, n-butanol or other volatile
solvent.

Final Drying is accomplished using vacuum, warm air

or radiant heat on large horizontal belt filters.

Other Antibiotics Produced by Fungi

Cephalosporins

Molecule is relatively similar in structure to penicillin.

Produced from selected strains of Cephalosporium

Chemical structure is less susceptible to hydrolysis of

the beta lactam ring than penicillin, by Staphylococci,
but are readily hydrolysed by enzymes from gram
negative bacteria.

Fermentation processes similar to those employed for

penicillin. Hoeever, even after extensive investigation,
yields are considerably lower that those obtained in
penicillin fermentation.

Several semi-synthetic derivatives of Cephalosporin in

commercial production.
Streptomycetes
Actinomycetes represented by Streptomycetes,
include more that thirty genera of gram positive
bacteria that show branching, filamentous or
irregularly rod shaped morphology.
The term acintomycete first originated in 1887,
coming from the Greek root, “Ray Fungus”

However, their taxonomic position is now well

established within the the kingdom of the Prokaryote.

That said, Streptomycetes are boundary organisms

from a morphological, physiological and metabolic
point of view.

The outstanding property of Streptomycetes is their

ability to synthesize a variety of antibiotics.

To date over 6000 antibiotics of microbial origin have

been isolated. 60% of actinomycete origin, the rest
from fungal and bacterial sources.

Out of these 70 compounds have found practical

application in human and animal medicine.

90% of these compounds come from the

Actinomycetes.

Antibiotics from Streptomycetes include almost all

known structural classess of commercially important
antibiotics.
Impact of Antibiotics Produced by Actinomycetes
on Human Health

Tuberculosis, caused by Mycobacterium tuberculosis,

serious disease until the advent of streptomycin.

Enterotyphus, 50% mortality rate, cured by

chloramphenicol,

Syphilis and bacterial dysentery are now only

encountered rarely.

1946-1980 – Average life expectancy of the Japanese

male increased from 43 to 73. Much of this increase
due to antibiotics.

Infant mortality down to 1% of what it was in 1950.

In addition to the obvious medical benefits, antibiotics

help stabilise food supply by controlling animal, fish
and plant diseases, leading to higher agricultural
productivity.
Genetic Approaches to Antibiotic Process
Optimisation

In the last several years, important advances have

been made in the technical procedures for genetic
recombination and gene cloning in Streptomyces.

However, most antibiotic synthesis processes are

complex multistep processes, often poorly
understood.

Therefore, random chemically induced mutations

continues to be the most widely applied and
successful genetic procedure to improve the antibiotic
productivity.

In spite of the enormous economic importance of

chemical mutation on antibiotic productivity, little is
known about the fundamental mechanism of mutation.

Mutations and Genetic Instability

Spontaneous mutations include a variety of molecular

insults, including deletions, duplications,
transpositions, insertions, base pair substitutions and
reading frame shifts.
Induced Mutations

• UV raditation
• Chemical Mutations
• 4-Nitroquinoline-1-oxide

• Hydroxylamine

• Methyl Methanesulfonate

• Ethyl Methanesulfonate

• N-methyl-N'-nitro-N-nytrosoguanidine

Mutagenesis and Strain Development

Mutation induction in Streptomyces is a complex

process. Not all mutagens are capabile of inducing a
high level of mutation in all streptomycetes.

• Treat spore suspension with chemical or UV

mutagen.
• Isolate improved mutants using screening
techniques.

Shake Flask Screens are the most popular method for

isolation of improved mutants and is still used
frequently today.

• Treat with mutagen

• Plate out on growth medium at low density
• Use agar plugs to isolate single colonies
• Innoculate liquid cultures with test colony.
• Screen for increased productivity.
Plate Based Techniques

• As before, treat spore suspension and plate at low

density.
• Culture the spores into colonies on the plate.
• Overlay the colonies with membrane innoculated
with test organism.
• Zones of exclusion are measured using image
analysis.
• Largest zone of exclusion, highest concentration of
antibiotic diffused into the surrounding agar.