Production of Benzylpenicillins [Penicillin G]

7.1.3. Production of Benzylpenicillins [Penicillin G]

Alexander Fleming’s originally isolated strain of Penicillium notatum (Straub) afforded actually very low yield of penicillin. Vigorous search for improvement of strain revealed the isolation of P. chrysogenum which distinctly gave much higher yields of penicillin. Importantly, the newer strains of Penicillium could even produce upto 180 folds higher yields in comparison to the original isolate that are solely based upon the novel phenomenon of ‘mutation’ or the so-called ‘genetic engineering’ methodologies.

In actual practice, penicillin is commercially produced in submerged vat cultures employing a highly purified and selected strain of P. chrysogenum, whereby the ultimate yield of the targetted product (penicillin) has been enhanced almost three folds i.e., from 10 mcg. mL –1 to 30 mcg. mL –1 . Interest- ingly, these modified, researched, purified strains of Penicillium do exhibit a number of marked and pronounced characteristic features, such as : high-titre values, improved growth, immense tolerance to the side-chain precursors, acetyltransferase activity, ability to store intracellular requirement(s).

The various steps that are associated intimately with the production of Benzylpenicillins [Peni- cillin G] are stated as under :

7.1.3.1. Inoculum

Penicillium notatum (i.e., Fleming’s initial/original strain) together with other ‘early isolates’ afforded exclusively low yields of penicillin ; besides, they responded very sluggishly to the submerged culture techniques particularly. Contrary to this, an early strain of P. chrysogenum (NRRL, 1951), duly isolated from the moldy fruits, was observed to yield much higher yields of penicillin. Consequently, the high-yield strain was duly subjected to careful treatment with a broad-spectrum of time-tested mutagenic agents, for instance : UV-radiations, X-rays, and mechlorethamine (MBA)—a nitrogen mustard. Obviously, these mutagenic agents helped a long way in the appropriate selection of several higher yielding mutants in particular ; and, in general, the judicious application of these ensuing mutagenic agents in sequence, along with certain repetitive treatments, ultimately grave rise to the newer strain Q-176, that essentially had the ability of producing maximum yields of penicillin.

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Q-176 strain produced > 1000 Units . mL –1 NRRL-1951 strain produced ~ 200 Units . mL –1

Drawback : Both Q-176 and NRRL-1951 strains gave rise to the formation of a yellow water- soluble pigment known as chrysogenin that prominently introduced a distinct yellow tint to the final product of penicillin. Therefore, it was almost necessary to intensify the studies in the direction of mutation and selection to lay hand on such modified strains that failed to produce the undesired yellow pigment.

Developments in Better Penicillin-Producing Strains : It is worthwhile to observe at this point in time that a major segment of strain-development programmes ultimately culminated with the latest high-yielding industrial strains for the penicillin production. However, one may serenely take notice of the fact that all of these modified strains are truly the descendant variants of the mother strain Q-176.

Asexual Reproduction : Nevertheless, the penicillin-producing strains of Penicillium are found to be due to asexual reproduction ; and, therefore, the scope of the ‘conventional methods of genetic analysis’ may not be applicable to them at all.

Parasexual Recombination : The incidence of a specific type of combination usually termed as ‘parasexual recombination’ may take place by the help of prevalent resultant-segregation as well as

recombination of genes.

Meticulous and intensive studies carried out by several researchers, namely : Roper (1952)*, Sermonti (1956)**, and Pontecorvo (1956)*** have evidently demonstrated that in the event when two genetically altogether different strains of Penicillium are allowed to grow simultaneously, the hyphae**** of the two strains in question will exhibit a tendency to fuse at a number of points. The formation of diploid nucleus***** shall come into being when the corresponding cells duly generated from the aforesaid ‘union’ essentially comprise of nuclei from each of the respective fungal strains and invariably two nuclei strategically located in the close proximity within the cell ultimately get fused. In case, the ensuing diploid nucleus just per chance gets into a respective conidium******, that happens to be uninucleate in nature, the eventual formation of an altogether new strain would be perpetuated.

Formation of Haploid Nuclei******* : Further effective division by diploid nuclei meiotically may give rise to the formation of haploid nuclei essentially having distinct genetic combinations. Interestingly, this very technique has a lot of potential and scope for future development of remarkably newer and useful industrial strains of penicillin-yielding fungi.

* Roper JA, Experientia, 8 : 14-15, 1952. ** Sermonti G, J. Gen. Microbiol, 15 : 599-608, 1956. *** Pontecorvo G, Ann. Rev. Microbiol. 10 : 393-400, 1956. **** Filaments of mold or parts of a mold mycelium. ***** A nucleus having two sets of chromosomes ; said of somatic cells, that contain twice the number of

chromosomes present in the egg or sperm. ****** Asexual source of fungi. ******* Nuclei possessing half the diploid or normal number of chromosomes found in somatic or body cells.

Such is the case of the germ cells-ova or sperm-following the reduction divisions in gametogenesis.

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7.1.3.2. Production Media

Though the precise and exact compositions of the penicillin-production media really employed in any industry are more or less impossible to quote and determine, by virtue of the fact that such information(s) are regarded to be the ‘trade secrets’ or patented by the actual users. Nevertheless, a large segment of these commonly used media invariably comprises of such ingredients as : cornsteep

liquor solids, lactose, glucose, calcium carbonate, potassium dihydrogen phosphate [KH 2 PO 4 ], edible oil, and a penicillin precursor. Jackson (1958)* promulgated a very useful and typical medium having essentially the following composition :

S.No. Ingredients

Quantity (%)

Remarks

1. Fermentable carbohydrates — Corn steep liquor solids

2. Organic nitrogen source

q.s.

3. Phenyl acetic acid

q.s.

Penicillin precursor

4. Potassium dihydrogen

phosphate [KH 2 PO 4 ]

5. Calcium carbonate

1 Acts as buffer

6. Edible oil

7. Organic salts

q.s.

Maintain salt-balance in medium

Note : (1) The pH after sterilization is carefully maintained between 5.5 to 6.0. (2) Higher lactose content ranging between 4 to 5% is desired with vigorously increased

aeration and agitation environments maintained within the fermentor (i.e., bioreactor). (3) The ‘production media’ contains both ‘lactose’ and ‘precursor’ which are not included

in the inoculum media.

7.1.3.3. Biomass** Production

It has been amply demonstrated that the ensuing production of penicillin exclusively depends upon the prevailing biomass production ; and, therefore, it is absolutely desirable to achieve a relatively high biomass concentration in the fermentor (bioreactor). The very presence of carbon compounds (car- bohydrates) besides other nutrients and additives is grossly responsible for the initial growth of the organism(s) almost achievable near the maximum specific growth rate. Importantly, the rapid growth

rate prominently gives rise to an appreciable enhancement in the initial O 2 -uptake rate as well as the subsequent CO 2 -evolution rate accordingly. It is, however, pertinent to mention at this juncture that

* Jackson T, Development of Aerobic Fermentation Processes. In : Biochemical Engineering [R. Steel ed], Heywood and Co., LTD, London, pp : 183-221, 1958.

** All of the living organisms present in a specified area.

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the ultimate penicillin production may be enhanced by suitably augmenting and greatly improving mycelial* biomass, which could be accomplished appropriately by boosting up both the speed and rate of agitation.

7.1.3.4. Course of Typical Penicillin Fermentation

The actual course involved in a typical penicillin fermentation on account of several undergoing chemical changes is represented explicity in Fig. 3.20.

Mycelial N

Time (Hours)

Fig. 3.20. Various Chemical Changes Involved in a Typical Penicillin Fermentation with Added Phenylacetic Acid Precursor.

[Adopted from : Brown WE and Peterson WH, Ind. Engg. Chem. 42, 1773, 1950] Salient Features : The various salient features intimately associated with the chemical changes

incurred in Fig. 3.15 are enumerated below : (1) At the initial stage of fermentation pH remains rather constant, whereas the cornsteep liquor-

cabon entities, glucose, and ammonia are being utilized simultaneously. * The mass of filaments (hyphae) which constitutes the vegetative body of fungi, such as : molds.

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(2) Optimum pH range for penicillin production rises maximum between 7 to 7.5 by a sequence of events, namely : carbon compounds (i.e., carbohydrates) utilized and depleted — portion of lactic acid (from cornsteep liquor) being consumed — ammonia (NH 3 ) released by deamination of aminoacids from cornsteep liquor.

(3) At this criticial point in time pH remains virtually steady and constant as the method makes use of the lactose to form penicillin, further rise in pH arrested due to the fact that prevailing mold gets absolutely starved (of nutrients).

(4) Completion of fermentation is indicated by pH rise to 8 or even higher by virtue of consider-

able depletion of ‘lactose’ which smartly brings about autolysis of the mycelium.

(Note : In usual practice, the penicillin fermentation is arrested and harvested before this specific and critical stage is achieved.)

(5) First 20 to 30 hours : i.e., during cornsteep liquor solid and carbohydrate consumption the fungal growth turns out to be distinctly thick and heavy due to three possible reasons, namely : (i) disperse strands (of DNA) ; (ii) clusters of mycelium ; and (iii) availability of definitive pellets of mycelium (ranging between 0.5 to 2 mm diameter).

(6) Fig. 3.15 reveals vividly that the yields of penicillin are found to be ‘linear even at 22 hours, but in actual practice they range between 48 to 96 hours.

(7) Ultimate yield of penicillin varies between 3 to 5% solely based upon carbohydrate actually

consumed , and almost attains a level in excess of 1500 Units . mL –1 .

(8) Sylvester and Coghill* (1954) have arrived at the following statistically averaged estimation with regard to the yield of penicillin :

Aim : To produce 1000 Gallons of fermented culture (approx. equivalent to 5-6 lbs of penicillin) by submerged-culture process.

S.No.

1. Various nutrients

lbs

(e.g., cornsteep-liquor solids, lactose, glucose etc.)

2. Live LP-Steam

5. Air (Compressed and Sterile)

LP = Low Pressure ; DM = Demineralized Water ; Gallon = 4.5 L or Imperial Gallon =

3.75 L ; kg = 2.45 lbs ;

(9) pH plays an extremely vital and critical role particularly in the course of penicillin fermenta- tion since penicillin is quite sensitive to relatively low pH values. Besides, penicillin is equally sensitive to pH values above 7.5, specifically in the presence of NH + 4 ion. Therefore,

* Sylvester JC and RD Coghill : The Penicillin Fermentation., Jn : Industrial Fermentation, Vol. II (Underkofler LA and RJ Hickey eds)., Chemical Publishing Co., Inc., New York, pp. 219-263, 1954.

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it is quite necessary and mandatory to maintain pH ~ 7 (i.e., near neutrality) by incorporating calculated quantum of CaCO 3 and MgCO 3 into the medium, and also using phosphate buffer.

[Note : (i) A little rise in pH is not so alarming since during this stage very little NH 3 (gas) gets released to increase the prevailing pH values. (ii) Fluctuation in pH may be adequately controlled by the addition of calculated amount

of either NaOH or H 2 SO 4 .]

(10) Overall Performance : The various constituents present in the medium exert a remarkable effect on the overall penicillin yields as stated briefly below : Cornsteep-Liquor Solids — Gives rise to NH 3 needed in the early stages of fermentation

along with certain carbon-nutrients.

Glucose — Gets readily used-up to afford requisite mycelial growth but permits and restricts very little penicillin production. Lactose

— Gets only gradually degraded to glucose and glactose : and perhaps this rather not-so-rapid availability of glucose from lactose affords the much desired starvation environments ur- gently needed for penicillin production.

Liquid nutrients — Liquid nutrients (i.e., fatty oils*) are fully consumed by the respective fungus during penicillin production. However, some of the ‘oil’ is incorporated into the fermentation medium to serve as ‘antifoaming agent’. Most probably these oils (liquid nutrients) are duly subjected to degradation by the correspond- ing ‘fungus’ either to the 2C-acetate or similar compound level before being utilized in the actual formation of mycelium and

penicillin.

7.1.3.5. Penicillin Nucleus : Two Amino Acids

One may observe the presence of two specific amino acids embedded into the penicillin nucleus, namely : L-cysteine and D-valine as depicted below :

NH 2 L-Cysteine

PENICILLIN NUCLEUS

* Fatty Oils : include lard oil (animal fat), soyabean oil, linseed oil, and fatty acids of more than 14 C-chain lengths and their corresponding esters.

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It has been proved beyond any reasonable doubt that the adequate supplementation of L-cysteine and L-cystine i.e., the two S-containing amino acids, predominantly enhanced the overall yields of penicillin to a much greater level and extent that occurred by the addition of all types of pure ‘inorganic S-containing ’ compounds.

Arnstein* (1954) further substantiated and expanded the aforesaid findings by employing isotopically labeled L-cysteine (viz.,

β 35 – 14 C, S, and 15 N) to demonstrate precisely that the prevailing mold could induct this ‘specific amino acid’ directly right into the ‘penicillin nucleus’. Likewise, fur-

ther researches carried out in this direction by Arnstein et al. using isotopically labeled D- and L-valine together with other ‘inhibitor studies’, invariably established the fact that the C-skeleton of L-valine gets duly incorporated very much into the ‘penicillin nucleus’.

7.1.3.6. Role of Enzyme Penicillinase

Penicillinase is an extracellular hydrolyzable enzyme adaptively generated by the specific mem- bers belonging to the ‘coliform group of organism’,in general, by most Bacillus species, and also certain strains of staphylococcus. Penicillinase actually hydrolyzes penicillin into penicilloic acid (a dicarboxylic acid) as given under :

P en ic illin as e

CH 3 N

CH 3 [H O H ; H y d ro ly sis]

R C NH CH

COOH HO

H 1 2 P en ic illin

P e n ic illo ic A c id Characteristic Features : Penicillinase is vehemently present in a plethora of penicillin-resist-

ant pathogenic strains of Staphylococcus aureus ; and, therefore, is largely responsible for causing over- whelmingly penicillin-resistance in the course of an infection. In addition to this the ‘enzyme’ aids in the rapid degradation of penicillin in the penicillin-fermentation medium,in the event of a possible

contaminant which particularly produces the enzyme that not only has an easy access to, but also capa- ble of growing in the fermentation broth.

7.1.3.7. Penicillin Production and Recovery

Principle : Penicillin in the anionic (acid) form is prove to extraction by solvent(s) as shown below :

P e n icillin [A n io n ic (ac id ic ) fo rm ]

* Arnstein HRV., Biochem. J. 57 : 360-368, 1954.

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The corresponding solution in an organic solvent may be back-extracted conveniently as its corresponding salt into an aqueous solution. This, in fact, constitutes the fundamental basis for the ‘recovery’ as well as subsequent means of ‘purification’ of penicillin from the respective duly harvested culture-broths.

Production and Recovery : A general and basic flowsheet diagram for the large-scale recovery and purification of ‘antibiotics’ is illustrated in Fig. 3.21. The various steps that are usually followed in

a sequential manner are described as under : (1) Once the entire fermentative procedure is accomplished i.e., at harvest, the completed peni-

cillin fermentation culture is subjected to filteration by the help of heavy duty rotary vacuum filter to get rid to the mycelium plus other unwanted solid residues.

(2) The pH of the clear filtered fermented broth is carefully brought down between 2 to 2.5 by the addition of a calculated amount of either phosphoric acid [H 3 PO 4 ] or sulphuric acid [H 2 SO 4 ] so as to convert the resulting penicillin to its anionic form, as shown above.

(3) The resulting fermented broth (pH 2 – 2.5) is extracted immediately by using a Pod bielniak countercurrent solvent extractor,* with an appropriate organic solvent e.g., amyl acetate, butyl acetate, or methyl isobutyl ketone.

(4) Penicillin, thus obtained, is back extracted into aqueous medium from the corresponding organic solvent by the careful addition of requiste quantum of KOH or NaOH to give rise to the formation of the corresponding potassium or sodium salt of the penicillin.

(5) The resulting aqueous solution, containing the respective salt of penicillin, is again acidified and reextracted with the organic solvent methyl isobutyl ketone.

(6) In fact, these shifts taking place between ‘aqueous’ and ‘solvent’ medium help in the ulti- mate process of purification of the penicillin.

(7) The resulting solvent extract is finally subjected to a meticulous back-extraction with aque- ous NaOH preferably, a number of times till extraction of penicillin is completed ; and from this combine of aqueous extractions different established procedures are adopted to afford the penicillin to crystallize out either as sodium or potassium penicillin.

(8) The crystalline penicillin thus obtained is washed, dried under vacuum, and the final product must conform to the requirements/specifications laid down by various Official Compendia.

* Cassida LE Jr. Industrial Microbiology, New Age International Publishers, New Delhi, p-244, 2004.

Fermentation solids

Filter Liquid-Liquid Centrifugal SOLID-LIQUID EXTRACTION

Filtered fermentation liquor

extractors Extraction

Filter Eluant

ADSORP TION

PRECIPITATION EXTRACTION

Solvent WASTE LIQUORS

tank Precipitation

Adsorption

tank

Solvent stripping

tank E

Filter

PARTIALLY PURIFIED ANTIBIOTICS

Spray dryer

Continuous Mixed solvents dryer

Sterile or

columns ANIMAL FEED SUPPLEMENTS

refining

area

BULK ANTIBIOTICS

Fig. 3.21. General and Basic Flowsheet Diagram for Large-scale Recovery and Purification of Antibiotics.