Production of Streptomycin

introduction image

The production of streptomycin requires the stock culture of Streptomyces griseus, proper nutrient medium and the other growth promoting substances. Streptomycin is an antibiotic which shows broad spectrum against the gram-negative bacteria and the first antibiotic to cure TB.

In 19th October 1943, a scientist Albert Schatz from the Rutgers University was the first to isolate “Streptomycin”. Albert Schatz was the PhD student, working under the guidance of Selman Abraham Waksman. Along with streptomycin, Waksman and his interns have also discovered several other antibiotics like actinomycin, neomycin, clavacin etc.

Streptomycin was the first medicine discovered for the treatment of tuberculosis which was found to be effective. According to the World Health Organisation (WHO), streptomycin is the safest medicine for the treatment of tuberculosis.

Streptomycin has been included in the list of essential medicine that is needed for the health system, by the WHO. In the year 1952, Waksman won Nobel Prize in physiology and medicine for the discovery of streptomycin.

Content: Production of Streptomycin

  1. Definition
  2. Organism Required
  3. Production Process of Streptomycin
  4. Recovery
  5. Uses of Streptomycin

Definition

Production of streptomycin can define as the production of an antibiotic, i.e. “Streptomycin” by the strain of actinomycetes. Streptococcus griseus is used in the large scale production of streptomycin by utilizing carbon, nitrogen, mineral source etc. provided in the fermentation medium.

Streptomycin: Streptomycin can define as the broad spectrum aminoglycoside antibiotic which works actively against the gram-negative bacteria. Streptomycin is commercially available as “Hypochloride” by having chemical formula C21H39N7O12.3HCL.

There are three sugars with carbon, nitrogen, oxygen and hydrogen elements in the structure of streptomycin. Some of the physical and chemical properties of the streptomycin are given below in the table.

Physical and Chemical propertiesStreptomycin
Physical stateSolid
FormHygroscopic powder
OdourFaint amine like odour
TasteSlightly bitter
SolubilitySoluble in water
NatureHygroscopic
Chemical namesAgrimycin, strepcen etc.
Molecular weight581.58 g/mol
pHPolar organic base
On decompositionEmit fumes of nitrogen oxide

Organism Required for Streptomycin Production

Streptococcus griseus is an actinomycete which is used for the preparation of streptomycin. Krainsky was the first scientist who has isolated S.griseus at the time of 1st world war from the Russian soil.

S.griseus is commonly present in the soil and is gram-negative bacteria. It produces grey mycelium during sporulation and also produces grey yellow reverse pigment when growing in the colony.

streptomyces griseus

Streptococcus griseus secretes a toxin commonly refers to as “Valinomycin” which can cause apoptosis in natural killer cells and mitochondrial swelling in peripheral lymphocytes. Thus, due to the presence of Valinomycin, the excessive use of streptomycin can cause neurotoxicity like hearing loss, dizziness etc.

Classification:

  • Domain: Bacteria
  • Phylum: Actinobacteria
  • Order: Actinomycetales
  • Family: Streptomycetaceae
  • Genus: Streptomyces
  • Species: S.griseus

Production Process of Streptomycin

Production of streptomycin is a complex process which undergoes a series of operations under the sterile conditions. There are many steps involved which we will discuss below:

production process of streptomycin

Growth of Streptomyces griseus

Hymns of Streptomyces griseus spores are maintained in the stock soil culture. Then the inoculum of S.griseus is inoculated into the agar media at 28 degrees Celsius.

After that, the spores of S.griseus sporulate and build up the mycelial network in the flask or seed tanks. The sporulation of S.griseus occurs well in a liquid culture medium, and it forms endospores at low nutrient availability. The growth of S.griseus requires a pH range of 5-11.

growth of streptomyces griseus

Preparation of the Medium

The production of streptomycin requires Hockenhull nutrient medium which is having the following media composition:

  • Glucose: 2.5%
  • Soy meal: 4%
  • Sodium chloride: 0.25%
  • Distillers dried soluble: 0.5%
  • pH: 7.6-8.0

For the production of the medium, there must be energy sources like carbon, nitrogen etc.

Carbon source: Monosaccharide like glucose provides the best carbon source that helps in building up of streptomycin. Sugars like fructose, maltose, starch etc. can also be used as the carbon source but oligosaccharides and polysaccharides give low yield.

Nitrogen source: Contents like soy meal, meat extract, ammonium salts etc. can be used for the nitrogen source.

Mineral source: Magnesium, calcium, potassium etc. can be used as a mineral source along with sulphates, phosphates and chlorides.

Growth stimulating source: Sources like L-naphthalene acetic acid and phenylacetic acid can be used as the growth-stimulating factor in the production of streptomycin.

Other than the above sources, compounds like proline, oils, fatty acids, antioxidants are also used to accelerate the production. The precursors are not required for the production of streptomycin.

Fermentation

All the above contents are sterilized and fed into the stirred tank fermentor. The bacterial inoculum of about 4-5% is transferred to the medium.

Inside the fermentor, the temperature is maintained between the range of 25-30 degrees Celsius and pH is maintained between the ranges of 7.6-8. The production of streptomycin requires continuous fermentation, and the growth of S.griseus requires continuous aeration and agitation.

The fermentation process requires 4-10 days. The fermentation process passes through three phases, like:

First phase

During the first phase, the S.griseus grows by utilizing soya meal and releases ammonia by its proteolytic activity. During this phase, the pH rises. The carbon source present in the soy meal and the glucose is slowly utilized by the S.griseus, which produces the streptomycin at a slower rate.

Second phase

Glucose and the ammonia secreted by the utilization of soy meal, the streptomycin is synthesized at a rapid rate. In this phase, the incubation period lasts for 24 hours to 6-7 days.

The pH during this phase remains constant between 7.6 and 8. There is little or no production of mycelial growth.

Third phase

It is the phase of fermentation where the concentration of glucose depletes from the medium due to which the production of streptomycin ceases, and the bacterial cells begin to lyse.

During this phase, the pH of the medium increases by the ammonia forms after the cell lysis. Before lysis, the fermentative medium is harvested for the process of recovery and purification.

Recovery of Streptomycin

The recovery of streptomycin includes the following stages:

Filtration: In the recovery of streptomycin, the bacterial biomass is separated from the fermentative broth by means of Oliver precoated pressure filter. After filtration, the remaining liquid is first acidified, filtered and then neutralized.

Adsorption: Activated carbon is mixed with the clear broth obtained after filtration in a series of three adsorption tanks. Streptomycin and some impurities are separated from the broth.

Then the broth is fed to the pressure filter, which releases out the spent broth. The absorbate is washed with dilute alcohol to remove impurities.

Elution: Streptomycin is eluted from the ion exchange method by the addition of dilute hydrochloric acid. In this process, the impurities are removed like metallic salts from the solution which can contaminate the streptomycin.

Concentration: The remaining solution is concentrated at 60 degrees Celsius under vacumn by the series of three single-phase evaporators.

Crystallization: The streptomycin obtained is first dissolved in methanol and then filtered again. A filtrate is treated with acetone to obtain a precipitate. The precipitate is rewashed with the acetone and vacumn dried.

Final operations

It includes the following steps like:

recovery steps of streptomycin

  1. The crystalline streptomycin is dissolved in the solution containing pyrogen-free water.
  2. Then the solution is passed through the special biological filter like Seitz filter to remove the microorganisms that can contaminate the streptomycin.
  3. The filtrate is then dried in the stainless steel stays from frozen under Vacumn, by which a dried cake is formed.
  4. The dried cake is then crushed into a powdered form by the high Vacumn sublimation using sterile steel balls.
  5. After that, weigh the powdered streptomycin into sterile vials. The whole process is carried out in the air-conditioned room, which provides filtered or stale air by the ultraviolet lamps.
  6. Finally, it is packaged which is carried out in the rooms which have 10% relative humidity.

To avoid or reduce the chances of contamination, special microbial filters, triethylene glycol as a disinfecting agent and the UV lamps are used. All these factors maintain sterile conditions throughout the production process of streptomycin.

Uses of Streptomycin

Clinical science:

  • Streptomycin is very effective against gram-negative bacteria, thus used to treat the infections and diseases caused by gram-negative bacteria.
  • It is widely used in the treatment of severe disease like tuberculosis.
  • Streptomycin is used in conjugation with antimycobacterial drugs, to treat pulmonary infections caused by the Mycobacterium avium complex.
  • It works as an alternative of Penicillin G that is used for the treatment of the rat-bite fever caused by Streptobacillus moniliformis for those patients who are allergic or sensitive to the penicillin.
  • Products like streptomycin-B and dihydroxystreptomycin can be obtained from the streptomycin.
  • Patients suffering from endocarditis, which is caused by Enterococcus, can be treated with streptomycin if they are sensitive to the gentamicin. Tularemia infections, Plague, Brucellosis etc. can also be treated with the streptomycin.

Agriculture science: Streptomycin is used as “Pesticide” that inhibits the growth of bacteria, fungi, algae etc.

Cell culturing: It is used at the time of cell culture, to prevent the growth of undesired microorganisms.

Biochemistry: Streptomycin sulphate is also used in the purification of biomolecules like protein and sometimes nucleic acid.

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