Fermentaion Technology:

Penicillin production:
Penicillin exhibits the properties of a typical secondary metabolite, being
formed at or near the end of exponential growth. Its formation depends on medium
composition and dramatic overproduction is possible.
However, P. notatum, the organism originally found to produce the antibiotic,
generated little more than 1mg/L from the surface cultures initially used for
penicillin production. A 20–25-fold increase in yield was achieved when corn
steep liquor was incorporated into the fermentation medium. This byproduct of
maize processing contains various nitrogen sources, along with growth factors and
side-chain precursors, and remains as a major ingredient of most penicillin
production media. Even greater penicillin yields were obtained from a closely
related species, Penicillium chrysogenum,
Further increases in yield were achieved when production went over to submerged
fermentation. The wartime requirements for penicillin stimulated the rapid
development of a large-scale submerged culture system using stirred tank reactors
(STRs).
The traditional approach to improving penicillin yields involved random
mutation and selection of higher producing strains. Resulting mutants were grown
in liquid medium and culture filtrates were assayed for penicillin. This was slow
and painstaking as large numbers of strains had to be tested. Penicillin
fermentations now produce yields in excess of 50 g/L, a 50 000-fold increase from
the levels first produced by Fleming’s original isolate. The contribution of classical
methods of strain improvement has so far outweighed all other approaches,
including more recently available genetic manipulation techniques. The latter have
contributed more to our understanding of the complex mechanisms of penicillin
biosynthesis, particularly the genetic arrangement of improved strains, the
identification of problems in penicillin synthesis and the regulation of secondary
metabolism in overproducing strains.
Different biosynthetic penicillins can be formed by the addition of side-chain
precursors to the fermentation medium and that natural penicillins can be modified
chemically to produce compounds with improved characteristics. Most penicillins
are now semi-synthetic, produced by the chemical modification of natural
penicillins, obtained by fermentation using strains of P.chrysogenum.
Modification is achieved by removing their natural acyl group, leaving 6-APA(6-
amino pencillanic acid) , to which other acyl groups can be added to confer new
properties.

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These semi-synthetic penicillins, such as methicillin, carbenicillin and ampicillin
exhibit various improvements, including resistance to stomach acids, a degree of
resistance to penicillinase and an extended range of activity against some Gram-
negative bacteria.
Penicillin production is usually via a fed-batch process carried out aseptically in
stirred tank fermenters of 40000–200000L capacity, although airlift systems are
sometimes used. The fermentation involves an initial vegetative growth phase
followed by the antibiotic production phase. Throughout the process, the oxygen
level is very important and must be maintained at 25–60mmol/L/h. These
processes are maintained at 25–27°C and pH 6.5–7.7, the specific conditions
depending upon the P.chrysogenum strain used.
Various carbon sources have been adopted for penicillin production, including
glucose, lactose, sucrose, ethanol and vegetable oils. About 65% of the carbon
source is metabolized for cellular maintenance, 25% for growth and 10% for
penicillin production. In the past, a mixture of glucose and lactose was used, the
former producing good growth, but poor penicillin yields. The mode of ‘feeding’of
a particular carbon source is vitally important, as it can influence the production of
this secondary metabolite. Corn steep liquor is still used as a source of nitrogen,
additional nutrients and side-chain precursors. Its acidic nature creates a
requirement for calcium carbonate (1%, w/v) and a phosphate buffer to neutralize
the medium, thereby optimizing its pH for penicillin production. Ammonia,
mineral salts and specific side-chain precursors,
e.g. phenyl acetic acid or phenoxyacetic acid, may also be added. However, as
some precursors are toxic, they must be fed continuously at non-inhibitory
concentrations. Inoculum development is usually initiated by adding lyophilized
spores to a small fermenter at a concentration of 5*103 spores/ml. Fungal
mycelium may then be grown up through one or two further stages until there is
sufficient to inoculate the production fermenter. Initially, there is a vegetative
growth phase devoted to the development of biomass, which doubles every 6 h.
This high growth rate is maintained for the first 2 days. To ensure an optimum
yield of penicillin in the following production phase, the mycelium must develop
as loose pellets, rather than compact forms. During the following production phase,
the carbon source is fed at a low rate and penicillin production increases. This
continues for a further 6–8 days, provided that appropriate substrate feeds are
maintained.
Penicillin is excreted into the medium and is recovered at the end of fermentation.
Whole broth extraction may be performed, but can lead to downstream processing
problems, as additional materials leach from the mycelium. Usually, penicillin
recovery follows removal of mycelium using rotary vacuum filters, the efficiency

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of which may be affected by the culture media composition, particularly its
proteinaceous components.
Recovered mycelium is then washed to remove residual penicillin, prior to its use
as animal feed or fertilizer. Antibiotic recovery is often by solvent extraction of
the cell-free medium, which gives yields of up to 90%. This involves reducing the
pH of the filtered medium to 2.0–2.5 by addition of sulphuric or phosphoric acid,
followed by a rapid two-stage continuous countercurrent extraction at 0–3°C using
amyl acetate, butyl acetate or methyl isobutyl ketone. The low temperature is
necessary to reduce damage to penicillin due to the low pH.
Alternatively, ion-pair extraction may be used at pH 5–7, in which range penicillin
is stable. Any pigments and trace impurities are removed by treating with activated
charcoal. The penicillin is then retrieved from the solvent by addition of sodium or
potassium acetate. This reduces the solubility of the penicillin and it precipitates as
a sodium or potassium salt. Resultant penicillin crystals are separated by rotary
vacuum filtration. Solvent is recovered from the separated liquor and any other
materials used, such as the charcoal, which is very important in terms of the overall
economics of the process. Penicillin crystals are mixed with a volatile solvent,
usually anhydrous ethanol, butanol or isopropanol, to remove further impurities.
The crystals are collected by filtration and air dried. At this stage the penicillin is
99.5% pure. This product may be furtherprocessed to form a pharmaceutical grade
product or is used in the production of semisynthetic penicillins.

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Fig. 1. Production of penicillin.

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Development of large – scale fermentation technology:

The most obvious benefits that can be achieved from integrating fermentation and
downstream processing are minimizing waste, raw materials, capital and energy.
In general, large-scale fermentation development comprises of the following steps:
1. Organism selection
2. Metabolic and cellular engineering:
3. Fermentation process development:
• culture and media optimization (from complex to defined minimal media)
• optimization of cultivation parameters that take into account product recovery
and purification (minimize byproduct formation, minimize chemical inputs, and
develop high-cell-density cultivation)
• incorporation of cell retention/recycling
4. Introduction of downstream unit operations within a fermentation process:
• examples are extractive fermentation, electrodialysis and in-line membrane
separation technologies.

Integration:
Integration can be approached from different angles, relate to the steps involved in
large-scale fermentation development.
Simplify the fermentation broth — In principle, any ingredient added to the
broth that does not end up as product will have to be removed. It therefore
behooves the fermentation- process designer to eliminate any unnecessary
ingredients from the broth. For example, many fermentation processes employ
complex media, such as yeast extract or corn steep liquor (initial waste stream of
corn wet milling operation; generally used as a rich source for nitrogen in
fermentation media). These media are inexpensive and ample in nutrients.
Designing defined fermentation media from salts and vitamins requires a
considerable development effort to provide a recipe capable of supporting
microbial production at the desired levels. However, it is sometimes worth
considering the tradeoff between slightly reduced fermentation performance and a
greatly simplified downstream process. In addition, if a complex medium
component must be used, the component may contain elements that do not directly
benefit the fermentation, yet provide a separation challenge downstream. In some
cases, it may be feasible to separate out these non-beneficial nutrients before they
reach the broth.
In some cases, different forms of the product are easier to separate downstream
than others. A common example of this is organic acids, where the free-acid form
of the product may be easily extracted from a fermentation broth, while the salt is

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not easily removed. If fermentation is designed separately from downstream
processes as is usually the case, an acidification step will be required downstream.
However, this requires that a neutralizing base be added to the broth, which must
later be removed by adding an acid, with both base and acid becoming a waste salt
that must then be disposed of. This inefficiency can be avoided if the fermentation
can be carried out at a low enough pH to provide the product predominantly as the
acid. The resulting savings in acid, base and waste disposal costs may offset a
considerable amount of decline in fermentation performance resulting from the
more unfavorable acidic fermentation conditions.

Reuse the fermentation broth components — significant improvements in

fermentation raw-material yield and production rate can be effected by reusing
components of the broth. For example, recycling cells, although a technical
challenge holds promise for improving fermentation efficiency.
Separation of the biomass from the broth requires that the cell fraction be treated
cleanly to prevent contamination of subsequent fermentations, and that the cells
not be subjected to unnecessary stresses to maintain their viability.
However, any live biomass that can be reused reduces the amount of new substrate
that is required for biomass growth, as well as reducing the challenges of biomass
disposal. Also, an increased concentration of cells can increase the production rate
in the fermenter, which will reduce fermentation capital costs. Even if the biomass
cannot be recycled viably, if it can at least be recycled cleanly, it can become a
nutrient source for later biomass production.
Another strategy is reusing some or all of the broth after product separation. Often,
optimum product synthesis and biomass growth take place when medium nutrients
are present in excess. However, this results in nutrients being left over at the end of
fermentation. Reuse of the broth allows a reduction of nutrient addition to the next
batch, as well as avoiding the cost of treating a high biochemical oxygen demand
(BOD) waste stream.

Remove the product during fermentation— in many fermentations, the product

acts as an inhibitor to the production reactions. This can limit the concentration
that can be achieved in the fermenter. However, as said above, the product
concentration in the finished fermentation broth is a clear inverse indicator of cost.
Removing the product during fermentation increases the yield by allowing more to
be produced from a given amount of biomass, plus increases the production rate by
reducing the accumulation of an inhibitory product. Using continuous extraction, a
side-stream can be pumped out of the unit and the extracted broth returned to it.
Further, two-phase fermentations have been developed to extract the product from

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a biomass-containing aqueous phase into an organic phase, which can then be
removed on-line.

Reduce the water content of the broth — typically, as much as 90% or more of
the broth is water, which must be removed. This is not only costly to separate, but
also produces a large aqueous stream that must then be disposed of or recycled.
Integrative approaches to water reduction include increasing the biomass
concentration (i.e., high-cell-density (HCD) fermentation), engineering the
organism to tolerate higher product concentrations, and removing inhibitory
elements from the fermentation recipe.