Production methods

Penicillin Production

  1. Problem in Penicillin Use:

    • Penicillinase-producing bacteria can inactivate penicillin, limiting its effectiveness.

  2. Requirement for Production:

    • Large amounts of air are essential for penicillin production.

  3. Fermentation Setup:

    • Deep tanks (several thousand gallons in capacity) are used for fermentation.

  4. Preparation of Culture Medium:

    • The medium contains:

      • Corn steep liquor

      • Lactose

      • Glucose

      • Nutrients and salts

      • Phenylacetic acid (or its derivative)

      • Calcium carbonate (acts as a buffer)

  5. Inoculation:

    • The medium is inoculated with a suspension of Penicillium chrysogenum.

  6. Fermentation Process:

    • The mixture is aerated and agitated continuously.

    • The mold grows as pellets throughout the medium.

  7. Duration and Conditions:

    • After about 7 days, growth is complete.

    • The pH rises to 8.0 or above, and penicillin production stops.

  8. Separation of Product:

    • The mould masses are separated from the culture medium using centrifugation and filtration.

Penicillinase-producing bacteria (problem) 
        ↓
Need for high aeration in production
        ↓
Use of deep fermentation tanks (thousands of gallons)
        ↓
Preparation of culture medium:
  → corn steep liquor
  → lactose
  → glucose
  → nutrients and salts
  → phenylacetic acid or derivative
  → calcium carbonate (buffer)
        ↓
Inoculation with Penicillium chrysogenum
        ↓
Continuous aeration and agitation
        ↓
Mould grows as pellets throughout the medium
        ↓
After ~7 days:
  → growth completes
  → pH rises to 8.0 or more
  → penicillin production stops
        ↓
Separation of mould and medium:
  → centrifugation
  → filtration

Cephalosporins Production

  1. Starting Organism:

    • Cephalosporium acremonium is used in fermentation.

  2. Primary Product:

    • Produces Cephalosporin C, but it is not potent for clinical use.

  3. Chemical Modification:

    • Aminoadipic acid side chain is removed from Cephalosporin C.

  4. Formation of Core Compound:

    • This forms 7-α-aminocephalosporanic acid (7-ACA).

  5. Semi-Synthetic Modifications:

    • Side chains are added to 7-ACA to produce clinically useful broad-spectrum antibiotics.

  6. Customization:

    • Side chains can also be modified on both:

      • 6-APA (used in penicillins)

      • 7-ACA (used in cephalosporins)

    • This results in drugs with:

      • Different spectra of activity

      • Different enzyme resistance levels

  7. Outcome:

    • Development of third, fourth, and fifth generation cephalosporins to fight enzyme-producing resistant bacteria.

Cephalosporium acremonium (fermentation)
        ↓
Produces Cephalosporin C (not clinically useful)
        ↓
Remove aminoadipic acid side chain
        ↓
Forms 7-α-aminocephalosporanic acid (7-ACA)
        ↓
Add various side chains to 7-ACA
        ↓
Create broad-spectrum antimicrobials
        ↓
Modify side chains of both:
     → 6-APA (penicillin base)
     → 7-ACA (cephalosporin base)
        ↓
Produce antibiotics with:
     → varied antimicrobial activity
     → improved resistance to microbial enzymes
        ↓
Third, fourth, and fifth generation cephalosporins developed
        ↓
Effective against enzyme-producing, drug-resistant bacteria

Streptomycin Production

  1. Microorganism Used:

    • Streptomyces griseus (an actinomycete) is used.

  2. Inoculum Preparation:

    • Spores are inoculated into a nutrient medium to develop high mycelial biomass.

  3. Transfer to Production Tank:

    • The mycelial inoculum is transferred to the production tank for fermentation.

  4. Fermentation Medium Components:

    • Soybean meal (nitrogen source)

    • Glucose (carbon source)

    • NaCl

    • Conditions:

      • Temperature: 28°C

      • pH: 7.6–8.0

      • High agitation and aeration

  5. Fermentation Duration:

    • The process lasts for about 10 days and has three phases:

Phase 1: Inoculation and growth

  • Rapid microbial growth → high mycelial biomass

  • Proteolysis of soybean meal releases ammonia (NH₃) → pH rises

  • Little or no streptomycin production

Phase 2: Streoptomycin Production

  • Minimal mycelial growth

  • Streptomycin (secondary metabolite) is produced and accumulates

  • Glucose and NH₃ are consumed

  • pH remains stable (7.6–8.0; for optimal antibiotic production)

Phase 3: Decline/ Death Phase

  • Carbohydrates depleted

  • Streptomycin production stops

  • Cell lysis begins

  • pH rises further

  • Fermentation ends

  1. Product Recovery:

    • Filtration separates mycelium from the broth.

    • Streptomycin recovery:

      • Adsorbed on activated charcoal

      • Eluted using acid alcohol

      • Precipitated with acetone

      • Further purified using column chromatography

Streptomyces griseus spores inoculated into medium
        ↓
High mycelial biomass developed in inoculum tank
        ↓
Mycelial inoculum transferred to production tank
        ↓
Fermentation medium: soybean meal + glucose + NaCl
        ↓
Conditions: 28°C, pH 7.6–8.0, high aeration and agitation
        ↓
Fermentation process (~10 days) → 3 phases:

    Phase 1:
        ↓
    Rapid mycelial growth
        ↓
    NH₃ released from proteolysis → pH rises
        ↓
    Little or no streptomycin produced

    Phase 2:
        ↓
    Mycelial growth slows
        ↓
    Streptomycin accumulates in medium
        ↓
    Glucose and NH₃ consumed
        ↓
    pH stable (7.6–8.0)

    Phase 3:
        ↓
    Carbohydrates depleted
        ↓
    Streptomycin production stops
        ↓
    Cell lysis begins → pH rises
        ↓
    Process ends

        ↓
Mycelium separated by filtration
        ↓
Streptomycin adsorbed onto activated charcoal
        ↓
Eluted with acid alcohol
        ↓
Precipitated with acetone
        ↓
Purified using column chromatography

Citric Acid Production by Fermentation

  1. Microorganism Used:

    • Mainly Aspergillus niger (a mold)

    • Other fungi, yeasts, and some bacteria can also produce citric acid.

  2. Fermentation Type:

    • Mold produces citric acid as an overflow product due to disrupted TCA (tricarboxylic acid) cycle.

  3. Raw Materials (Carbon Sources):

    • Beet molasses, cane molasses, sucrose, commercial glucose, starch hydrolysates

    • Sucrose, cane, and beet molasses are the most effective.

  4. Medium Preparation:

    • Raw material is diluted to 20–25% sugar concentration

    • Nitrogen source and salts are added.

    • pH is adjusted:

      • pH ~5 for molasses

      • pH ~3.0 for sucrose

  5. Fermentation Methods:

    • Surface fermentation: medium placed in shallow trays and inoculated with spores.

    • Submerged fermentation: spores cultured in stirred fermenters.

    • Solid-state fermentation: mold grown on solid carrier like bagasse soaked in medium.

  6. End of Fermentation:

    • Calcium hydroxide is added to precipitate calcium citrate.

  7. Recovery & Purification:

    • Calcium citrate is filtered and washed.

    • Treated with sulphuric acid → forms calcium sulfate (precipitate) + citric acid in solution.

    • Citric acid solution is purified with:

      • Ion exchange resins

      • Activated charcoal

    • Citric acid is crystallized.

  8. Uses of Citric Acid:

    • Widely used in:

      • Food and beverage industry

      • Textiles

      • Pharmaceuticals

      • Detergents

      • Air purification (removal of toxic gases)

Aspergillus niger selected as production organism
        ↓
Carbon source selected:
  → sucrose, cane/beet molasses, glucose, starch hydrolysates
        ↓
Sugar diluted to 20–25% concentration
        ↓
Nitrogen source + salts added
        ↓
pH adjusted:
  → pH 5.0 (molasses) or pH 3.0 (sucrose)
        ↓
Fermentation method selected:
  → Surface fermentation (shallow trays)
  → Submerged fermentation (stirred fermenters)
  → Solid-state fermentation (bagasse as carrier)
        ↓
Fermentation completed → citric acid accumulates
        ↓
Add calcium hydroxide → forms calcium citrate
        ↓
Filter and wash calcium citrate
        ↓
Treat with sulphuric acid → forms calcium sulfate + citric acid in solution
        ↓
Purify solution with:
  → Ion exchange resins
  → Activated charcoal
        ↓
Crystallize citric acid
        ↓
Citric acid used in:
  → Food, beverages, pharma, textiles, detergents, air purification

Amino Acid Production by Fermentation

  1. Introduction:

    • Microorganisms can synthesize amino acids from inorganic nitrogen sources.

  2. Overproduction Mechanism:

    • In some microbes, the rate of amino acid synthesis can exceed the cell’s need for protein.

    • Excess amino acids are excreted into the medium, allowing for recovery.

  3. Commercial Viability:

    • Certain microorganisms can produce amino acids in large amounts, making the process suitable for industrial-scale production.

  4. Comparison with Other Methods:

    • Amino acids can also be made via:

      • Protein hydrolysis

      • Chemical synthesis

    • However, microbial fermentation is more economical in many cases.

  5. Key Advantage:

    • Microbial fermentation specifically produces L-amino acids, which are biologically active and naturally occurring (unlike chemical synthesis, which often gives a racemic mixture).

Microorganisms selected for amino acid production
        ↓
Inorganic nitrogen compounds provided as nutrient source
        ↓
Microbes synthesize amino acids during growth
        ↓
Overproduction occurs → excess amino acids excreted into medium
        ↓
Certain microbes produce amino acids in large quantities
        ↓
Amino acids recovered from fermentation broth
        ↓
Compared to:
  → Protein hydrolysis
  → Chemical synthesis
Microbial fermentation is:
  → More economical
  → Produces naturally occurring L-amino acids
        ↓
Used in:
  → Food industry
  → Pharmaceuticals
  → Animal feed supplements

Microbial Synthesis of Vitamin B₁₂ (Cyanocobalamin)

  1. Source of Vitamin B₁₂:

    • Only microorganisms (not fungi or yeasts) can synthesize vitamin B₁₂ naturally.

    • Mainly produced by bacteria and Streptomycetes.

  2. Microorganisms Used:

    • Propionibacterium freudenreichii, P. shermanii,

    • Bacillus megatherium, Streptomyces olivaceus, etc.

    • Propionibacterium species are preferred for commercial production.

  3. Types of Processes:

    • Batch and continuous fermentation methods are both used.

  4. Important Selection Criteria:

    • Microbes must exclusively produce the desired 5,6-dimethylbenzimidazolylcobamide (true vitamin B₁₂).

    • Some microbes produce pseudo-vitamin B₁₂, which is ineffective.

  5. By-product Recovery:

    • Vitamin B₁₂ can also be recovered as a by-product of streptomycin and aureomycin fermentations.

  6. Fermentation Product:

    • Most cobamides are retained inside the cells.

  7. Cell Separation:

    • Centrifugation concentrates bacterial cells to a cream.

    • Filtration removes Streptomycetes.

  8. Vitamin Release:

    • Vitamin B₁₂ is released from cells using:

      • Heat

      • Acid

      • Cyanide (converts coenzyme B₁₂ to cyanocobalamin)

  9. Purification Steps:

    • Adsorption on ion-exchange resin (IRC-50) or charcoal

    • Elution of adsorbed vitamin

    • Partitioning between phenolic solvents and water

    • Crystallization from aqueous-acetone solution

    • Crystals may contain water of crystallization

Microorganisms selected:
  → Propionibacterium freudenreichii, P. shermanii
  → Bacillus megatherium, Streptomyces olivaceus
        ↓
Fermentation process (batch or continuous)
        ↓
Ensure exclusive production of:
  → 5,6-dimethylbenzimidazolylcobamide (true B₁₂)
        ↓
Vitamin B₁₂ produced intracellularly
        ↓
Cells separated from broth:
  → High-speed centrifugation (bacteria)
  → Filtration (Streptomycetes)
        ↓
Release of vitamin from cells by:
  → Acid
  → Heat
  → Cyanide (forms cyanocobalamin)
        ↓
Adsorption of vitamin on:
  → Ion exchange resin (IRC-50) or charcoal
        ↓
Elution of vitamin B₁₂
        ↓
Partition between:
  → Phenolic solvent and water
        ↓
Crystallization from aqueous-acetone
        ↓
Final product:
  → Crystalline cyanocobalamin (Vitamin B₁₂) containing water of crystallization

Step-by-Step: Dextran Production by Fermentation

  1. Introduction:

    • Dextran is a polyglucose used as a blood volume expander and in the treatment of anaemia.

  2. Enzyme Involved:

    • Produced using the enzyme dextransucrase.

    • Substrate: Sucrose

    • Products: Dextran (main) + Fructose (by-product)

  3. Biochemical Reaction:

    Sucrose —(dextransucrase)→ Dextran + Fructose

  4. Microorganism Growth:

    • A dextransucrase-producing microorganism is grown in a fermentation medium.

  5. Fermentation Conditions:

    • Sucrose-rich medium (excess sucrose)

    • Temperature: 25–30°C

    • Initial pH: 7.0–7.2

  6. Enzyme Secretion:

    • As the microorganism grows, it secretes dextransucrase into the medium.

  7. pH Changes:

    • No external pH control.

    • pH drops naturally during fermentation.

    • Enzyme activity peaks at ~pH 5.2, converting excess sucrose into dextran and fructose.

Start with sucrose as the substrate
        ↓
Inoculate with dextransucrase-producing microorganism
        ↓
Fermentation medium prepared with:
  → Excess sucrose
  → Initial pH 7.0–7.2
  → Temperature 25–30°C
        ↓
Microorganism grows and secretes dextransucrase into medium
        ↓
No pH control applied
        ↓
pH drops naturally to ~5.2
        ↓
At pH 5.2, dextransucrase is most active
        ↓
Enzyme catalyzes:
  → Sucrose → Dextran + Fructose
        ↓
Dextran is collected as the main product
        ↓
Fructose is recovered or discarded as by-product