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PROCESS SELECTION

1. Fermentation process

The term “fermentation” comes from a Latin word fermentum (to ferment). The historical
definition describes fermentation as the process in which chemical changes in an organic substrate
occur as the result of action of microbial enzymes. Alchemy called fermentation putrefaction –
natural rotting or decomposing of substances. Nowadays, it is a metabolic process in which
carbohydrates and related compounds are partially oxidized with the release of energy in the
absence of any external electron acceptors – organic compounds produced by breakdown of
carbohydrates. During fermentation, incomplete oxidation of organic compounds occurs and for
this reason less energy is obtained when compared with aerobic oxidation of the compound
(Showell M.S. 1999).

Figure 1 shows the production of penicillin by using fermentation process. The fungus is
grown in a culture medium containing carbohydrates and amino acid. This looks like watery
porridge and is stirred continuously to keep the fungus in contact with fresh supplies if nutrients
mix oxygen into the culture. Roll the fungus up into little pellets (this facilitates the separating of
the liquid part containing penicillin from the fungus lately).

Figure 1 Fermenter for Producing Penicillin

 For first 15 to 24 hour, the fungus just grows then after that it begins to
secret penicillin. Rate of production depends on how much sugar is available. So small amount of
sugar has to be fed all the time that the fungus is producing penicillin. The culture is kept going
until the rate of production is so slow that is not worth waiting more (often after a week). Then
it is filtered, and the liquid is treated to concentrate the penicillin in it (Ron, P., 2014).

Furthermore, Vuppala and Murthy (2015) have been stated that industrial fermentation
organisms which have different organisms, such as bacteria, fungi and plant and animal cells, are
used in industrial fermentation processes. An industrial fermentation organism must produce the
product of interest in high yield, grow rapidly on inexpensive culture media available in bulk
quantities, be open to genetic manipulation and be non-pathogenic that does not cause any
diseases.

2. Purification process

Antibiotic products are available in several different forms. They can be sold in solutions
for syringes, in pill or gel capsule form, or they may be sold as powders, which are used for topical
ointments. Depending on the final form of the antibiotic, various steps must be taken after the
initial isolation of the antibiotic to further enhance the final product. For gel capsules, the powdered
antibiotic is physically filled into the bottom half of a capsule and the top half is mechanically put
in place. For topical ointments, the antibiotic is physically mixed into the ointment. The final
antibiotic product is now transported to the final packaging stations where it is stacked and put in
boxes. The boxes are transported to various distributers, hospitals, and pharmacies. The entire
manufacturing process of fermentation, recovery, and processing may take anywhere from five to
eight days.
 Figure 2 Isolation, Purification and Refining Process

Dayalan, S. A. J. et. al. (2011) stated that purification of penicillin from the production
media began with filtration of the broth as for this mini project that has been choose which is the
production of antibiotic (penicillin). In the first stage, large solids and microbial cells were
separated by filtration, as filtration is the most versatile method for removing the insoluble from
the broth. Penicillin rich aqueous broth was treated with activated charcoal to remove pigments
and impurities. After filtration and carbon treatment, penicillin recovery was done by liquid-liquid
extraction (solvent extraction). Penicillin was extracted from an aqueous phase into the solvent
butyl acetate. Solute recovery was carried out by evaporation of the extracted sample. The
identification of purified penicillin was done by thin layer chromatography (TLC) with benzene:
ethyl acetate: acetic acid (40:40:20) as solvent and visualized in UV illuminator.
3. Crystallization Process

Crystals are highly organized inert matters. If grown without external interference, they
grow in polyhedral shapes and exhibit many degrees of symmetry. Penicillin G is an odourless,
colourless or white crystal, or crystalline powder. Crystallisation is essentially a polishing step that
yields a highly pure product. It is done through phase separation from a liquid to a solid. To begin
crystallisation, we must first have a supersaturated solution. Supersaturation refers to a state in
which there are more dissolved solids in the solvent than can ordinarily be accommodated at that
temperature at equilibrium. Supersaturation can be achieved usually by cooling, drowning, solvent
evaporation, or by chemical reaction. Since the solubility of penicillin in its aqueous solution
decreases with decreasing temperature, as the solution cools, its saturation increases until it reaches
supersaturation and crystallization begins. Drowning is also common of recovery of penicillin G.
It is the addition of a nonsolvent to the solution to decreases the solubility of the solid. A chemical
reaction can be used to alter the dissolved solid to decrease its solubility in the solvent, thus
working toward supersaturation. From here, crystallisation is a two phases process:

Phase 1: Primary Nucleation

Primary nucleation is quite simply the growth of new crystals. A large supersaturation driving
force is required to start this primary step. The spontaneous crystal formation and "crashing out"
of many nuclei are observed from the solution. This step is not fully understood. After primary
nucleation begins, it will continue until the remaining solution concentration is at equilibrium.

Phase 2: Secondary Nucleation

Crystal production is initiated by “seeding” and occurs at a lower supersaturation. Seeding

involves the addition of small crystals to a solution in a metastable area, which results in
interactions between existing crystals, and crystal contact with the walls of the crystalliser. The
crystals will grow on those crystals until the concentration of the solution reaches solubility
equilibrium.

Batch crystallisation is the most the most used method for polishing antibiotics, including
penicillin G. Batch crystallisers simply consist of tanks with stirrers and are sometimes baffled.
They are slowly cooled to produce supersaturation. Seeding causes nucleation and growth is
encouraged by further cooling until the desired crystals are obtained.

While the crystallisation procedures product of very high purity, improves appearance and
has a low energy input, the process can be time consuming due to the high concentration of the
solutions during crystallisation. It can also be profoundly affected by trace impurities and batch
crystallisation can often give poor quality, non-uniform product (McGlade & Lennon, n.d.).

Syed, T. A. I. (2012) stated that crystallization is performed from the solvent or Na, K and
penicillin concentrations need to be adjusted temperature, etc. Excess amount of Na or K are added
to the penicillin crystallization in an agitated vessel. The crystals are separated by a rotary vacuum
filter and are washed and pre-dried using anhydrous butyl alcohol to remove impurities. Large
horizontal belt filters are used for collection and drying of crystals for which usually warm air or
radiant heat is used.

Final spray drying of the procaine antibiotic precipitate is accomplished in a low

temperature freeze dryer. Biomass may be fully recovered and sold as an animal food supplement.
Crystalline penicillin G or V are sold as intermediate or converted to 6-aminopenicillanic acid,
used in the production of semi-synthetic penicillin
Reference

Dayalan, S. A. J., Darwin, P., & Prakash, S. (2011). Comparative study on production, purification
of penicillin by Penicillium chrysogenum isolated from soil and citrus samples. Asian Pacific
Journal of Tropical Biomedicine, 1(1), 15–19. Retrieved from http://doi.org/10.1016/S2221-
1691(11)60061-0 [10 May 2018].

McGlade, E., & Lennon, R. (n.d.). BE401 Industrial Processing Penicillin Recovery Strategies.
Retrieved from http://www.ruairi.info/Penicillin_Recovery.pdf [16 May 2018].

Ron, P. (2014). #27 Use of Microorganisms to Manufacture Antibiotic Penicillin. Biology IGSE
Revision Guide. Retrieved from http://biology-igcse.weebly.com/ [10 May 2018].

Showell M.S. (1999). Enzymes, Detergent. In: Flickinger M.C. and Drew S.W., Encyclopedia of
Bioprocess Technology: Fermentation, Biocatalysis and Bioseparation. New York: John
Wiley and Sons.

Syed, T. A. I (2012) Biochemical Engineering: Principles and Concepts, 254-272. Retrieved from
https://books.google.com.my/books?id=vytUuRlQ9U0C&pg=PA267&lpg=PA267&dq=pro
duction+of+penicillin+using+crystaliization&source=bl&ots=qmy2Kk6Rga&sig=WLHZU
TGx1_LLndDmS1EQj6VPWIE&hl=ms&sa=X&ved=0ahUKEwisrvXr3YnbAhWFe7wKH
RIpBPA4ChDoAQgtMAE#v=onepage&q=production%20of%20penicillin%20using%20cr
ystaliization&f=false [16 May 2018]

Vuppala, G., & Murthy, R. K. (2015). Industrial Fermentation. Journal of Microbiology and
Biotechnology, 4(1), 1–7. Retrieved from https://doi.org/10.1021/ie50251a008 [3 May 2018]