Streptomycin - Biosynthesis and Large scale production

1. INDUSTRIAL PRODUCTION OF
STREPTOMYCIN
By
Mr. K.VIJAY M.Sc., TNSET, NET(LS), (Ph.D.)
Assistant Professor,
Sacred Heart College,
Tirupattur,TN,India.

2. STREPTOMYCIN
Streptomycin is produced by either of the two Streptomyces species, namely :
Streptomyces griseus and S. humidus. The antibiotic was particularly active
against Gram +ve actinobacteria e.g., Mycobacterium tuberculosis.
Besides, it is also found to exert its activity against Gram +ve bacteria, and
used therapeutically for curing infections by such organisms that distinctly show
resistance to penicillin.
The wonderful epoch making discovery of streptomycin was meticulously
carried out by three prominent researchers: Schatz, Bugie and Waksman
(1944) ; and one of their first and foremost soil isolate S. griseus was, in fact, a
mother strain still being employed largely as industrial strains across the globe
even today.
However, constant research in selective mutation and purification of various
strains helped to enhance excellent achievable yields.

3. Chemical Structure
The chemical structures of two basic compounds, viz., streptomycin and
dihydrostreptomycin are as given below:

4. One regulatory gene (strR) and four biosynthesis genes (strB1, strF, strN, and strS)

5. Dihydrostreptomycin may be prepared by the chemical reduction of the carbonyl
moiety present in the L-streptose segment of the streptomycin molecule as shown
above.
The various salts of streptomycin and dihydrostreptomycin are as stated below :
Streptomycin :
(i) Trihydrochloride : C21H39N7O12 . 3HCl
(ii) Trihydrochloride-cadium : (C21H39N7O12)2 . CaCl2
chloride double salt
(iii) Pantothenate : C21H39N7O12. C9H17NO5
(iv) Sesquisulfate : (C21H39N7O12)2 . 3H2SO4
Dihydrostreptomycin :
(i) Trihydrochloride : C21H41N7O12 . 3HCl
(ii) Sesquisulfate : (C21H41N7O12)2 . 3H2SO4
(iii) Pantothenate : C21H41N7O12 . C9H17NO5

6. Choice of Medium
The choicest medium for the fermentative process of streptomycin production
essentially comprise of
Carbon Source : e.g., dextrin, glycerol, glucose, starch, and similar
economically viable substances.
Nitrogen Source : e.g., naturally occurring processed agricultural products :
cottom seed meal, soyabean meal, cornsteep liquor solids, casein-hydrolysate,
yeast and its prepared extracts ; and pure inorganic salts : ammonium sulphate
[(NH4)2 SO4], ammonium nitrate [NH4NO3].
Vegetable/Animal Fat : e.g. soyabean oil, linseed oil, fatty acids having more
than 14C chain lengths plus their corresponding esters, and lard oil.
Typical industrial medium best suited for the fermentation of streptomycin as
given below :

7. Inoculum
High-yielding Streptomyces griseus spores accomplished through
meticulous mutation procedures are stringently maintained either soil
stocks or duly lyophilized in an appropriate carrier, for instance : sterile
skimmed milk.
Consequently, the spores obtained from these stock cultures are carefully
inoculated into a ‘sporulation medium’ strictly under aseptic conditions. It has
been observed that it helps for sufficient ‘sporulated growth’ to initiate the much
desired liquid buildup of mycelial inoculum in flasks or inoculum tanks
respectively.
Streptomycin Production
Streptomycin production outputs in bioreactors invariably respond to relatively
high degree of aeration as well as agitation. It has been duly established
that the ‘optimum production parameters’ for streptomycin are :
Fermentation temperature : Varies between 25-30°C (~ 28°C)
pH : Ranges between 7.0 - 8.0 (Max. between 7.6-8)
Duration : Varies between 5-7 days (yield > 1200 mcg / mL)

8. Importantly, streptomycin is fairly rough and tough, and hence hardly gets
destroyed by the presence of contaminating microorganisms as is the case with
penicillin. Nevertheless, contaminants definitely minimise yields to a
considerably extent.
(1) The actinophage infections may prove to be harmful and serious in nature
for both the inoculum and production vessels, because the streptomycete
rapidly undergoes cleavage thereby reducing yields substantially.
(2) Development and application of ‘tailor-made’ strains of S. griseus,
specifically resistant to certain more common phages, are being used
nowadays globally.
Production : The classical and widely promulgated commercial fermentation
operation for the production of streptomycin essentially passes through three
cardinal phases.

9. Phase-1 : It extends up to only 24 hours wherein the rapid growth commences
producing the large proportion of mycelium required for the fermentation. The
proteolytic characteristic property of S. griseus predominantly sets free NH3
right into the medium from the soyabean meal, and thus the carbon-
enriched nutrients present in the soyabean meal are adequately consumed for
the vigorous progressive growth. Nevertheless, the glucose up-take of the
medium is rather on a very low during this particular phase, and perhaps
that could be the reason for reasonably lower (slight) streptomycin
production. Interestingly, the ensuing pH of the medium rises from 6.7/6.8 to
nearly 7.5.
Phase-2 : It is the most crucial and critical stage since during this phase
streptomycin is eventually generated at a tremendously rapid rate that usually
extends from 1 day to almost 6/7 days of incubation under perfect sterile
environment. Because there is little growth of mycelium, the weight of
mycelium almost remains constant. In fact, three events take place precisely in
this specific phase (a) NH3 is fully consumed ; (b) glucose also being used-
up to the maximum extent (c) pH stands constant between 7.6 to 8.0.
Phase-3 : With the virtually complete depletion of ‘sugar’ from the
fermentation medium the streptomycin production almost comes to a standstill
situation. At this point in time, the ensuing fermentation product is harvested
before the commencement of this phase of senescence.

10. Harvest-Recovery-Purification: Once the fermentation attains completion, the
resulting mycelium is duly separated from the ensuing fermented broth
by filtration; and thus, the streptomycin is finally recovered by one of the
two methods described below based on the specific industrial concern.
Method-I : The streptomycin produced is adequately adsorbed from the
fermented broth onto activated carbon particles, and subsequently
subjected to elution from the carbon particles by means of diluted mineral
acid till streptomycin gets eluted almost completely. The eluted product is
precipitated by suitable solvents, filtered, and dried under vacuum before
further purification.
Method-II : Fermentation broth is first acidified and subsequently filtered
and neutralized. The resulting clear broth is forced via a packed column of
cation-exchange resin to allow the adsorption of streptomycin on it
completely. The column is washed with water (Demineralized) soon after the
completion of adsorption, and finally eluted with diluted HCl, and the liquid
containing streptomycin is concentrated under vacuum almost to dryness.
The crude antibiotic is dissolved in methanol and filtered, and acetone is
now added so as to allow the complete precipitation of streptomycin.
In the final treatment the ensuing precipitate is washed thoroughly with acetone
and dried in vacuum before being solubilized in MeOH for the ultimate
preparation of the desired streptomycin-calcium chloride complex in its
purest form.

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

12. A Typical Antibiotic Fermentation Plant