Semester 6
BP605T
Fermentation Technology & Blood Products
The concluding unit covers the industrial production side of biotechnology: fermentation methods and general requirements (media composition, equipment, sterilization, aeration, agitation), large-scale fermenter design with process controls, and the detailed production processes for five important fermentation products — Penicillin, Citric acid, Vitamin B₁₂, Glutamic acid, and Griseofulvin. The unit also covers blood product technology — collection, processing, and storage of whole blood, plasma, and plasma substitutes.
Syllabus & Topics
- 1Fermentation – Introduction & Methods: Fermentation = controlled cultivation of microorganisms (or their enzymes) for production of useful products (antibiotics, vitamins, amino acids, enzymes, organic acids). Types: (1) Submerged fermentation (SmF): organisms grow in liquid nutrient broth with agitation and aeration. Most common for antibiotics, enzymes. (2) Surface/solid-state fermentation (SSF): organisms grow on moist solid substrate surface. Used for: citric acid (Koji process), enzymes, vinegar. (3) Based on process: Batch (closed — all nutrients added initially, product harvested at end), Fed-batch (nutrients added during process — extends production phase), Continuous (nutrients added and product removed continuously — steady state).
- 2Fermentation Media: Growth medium provides nutrients for microbial growth and product formation. Components: (1) Carbon source: glucose, molasses, starch, corn steep liquor (CSL), sucrose, lactose. Serves as energy source + carbon skeleton. (2) Nitrogen source: ammonium salts (NH₄)₂SO₄, nitrates, soybean meal, yeast extract, peptone, corn steep liquor. (3) Minerals: Mg²⁺ (enzyme cofactor), Fe²⁺, Zn²⁺, Ca²⁺, K⁺, PO₄³⁻ (DNA/RNA synthesis). (4) Vitamins/growth factors: biotin, thiamine (some organisms require). (5) Precursors: compounds incorporated directly into product (phenylacetic acid → penicillin G side chain). (6) Inducers: trigger metabolite production. (7) Antifoaming agents: silicone oils, vegetable oils → prevent foaming in aerated cultures. Media design: balance growth (trophophase) vs production (idiophase) phases.
- 3Fermentation Equipment & Sterilization: Fermenter (Bioreactor): vessel for conducting fermentation. Materials: stainless steel (316L grade — corrosion resistant), borosilicate glass (lab scale). Accessories: impeller (agitation), sparger (aeration), baffles (prevent vortex), heating/cooling jacket/coils, pH/temperature/DO probes, sampling port, inoculation port. Sterilization — critical for aseptic operation: (1) Heat sterilization: autoclaving at 121°C, 15 psi, 15-20 min (batch sterilization of media). Continuous sterilization: flash heating to 140°C for seconds → superior nutrient preservation. (2) Filter sterilization: for heat-sensitive media, air (0.2 μm membrane filters for air — HEPA filters). (3) Radiation: UV for surfaces. (4) Chemical: formaldehyde/ethanol for fermenter surfaces.
- 4Aeration & Agitation: Aeration: supply of oxygen to aerobic cultures. DO (Dissolved Oxygen) is critical — many fermentation organisms are obligate aerobes. Methods: sparging sterile air through ring sparger at bottom of fermenter. Oxygen transfer rate (OTR) depends on: kLa (volumetric mass transfer coefficient) × (C* − CL). Agitation: mixing by impeller rotation. Functions: (1) ↑oxygen transfer (breaks air bubbles → ↑surface area). (2) Uniform distribution of nutrients and organisms. (3) Heat transfer (prevent thermal gradients). (4) Prevent cell sedimentation. Impeller types: Rushton turbine (most common — radial flow), marine propeller (axial flow), pitched blade turbine. Baffles: metal strips on vessel wall → prevent vortex formation → improve mixing. Agitation rate must be optimized: too high → shear damage to cells (especially mycelial organisms).
- 5Large-Scale Fermenter Design & Controls: Industrial fermenters: 10,000 to 200,000 L capacity. Stirred-tank reactor (STR): most common design — cylindrical vessel with flat/dished bottom, agitator shaft with multiple impellers, baffles, sparger, jacket. Height:Diameter ratio typically 2-3:1. Controls (instrumentation): (1) Temperature: controlled by cooling water jacket/coils (fermentation is exothermic → heat must be removed). (2) pH: maintained by automatic addition of acid (H₂SO₄) or base (NaOH/NH₄OH). (3) Dissolved Oxygen (DO): monitored by polarographic/galvanic probes → controlled by airflow rate and agitation speed. (4) Foam: detected by foam probe → automatic antifoam addition. (5) Agitation speed: variable speed motor. (6) Pressure: maintained slightly positive (0.2-0.5 atm) to prevent contamination. Additional: feed rate control (fed-batch), exit gas analysis (CO₂, O₂), weight/volume.
- 6Penicillin Production: Organism: Penicillium chrysogenum (improved from Fleming’s P. notatum by mutagenesis — current strains produce 50,000× more). Medium: Corn steep liquor (nitrogen, precursor), Lactose (carbon — slow utilization promotes penicillin production), Phenylacetic acid (PAA — precursor for Penicillin G side chain), minerals, CaCO₃ (buffer). Process: submerged fermentation (STR), aerobic, 25-26°C, pH 6.5, 5-7 days. Batch/fed-batch (lactose + PAA fed continuously). Growth phase (trophophase, 24-48 h) → production phase (idiophase — penicillin is a secondary metabolite). Downstream processing: filter mycelium → extract penicillin from broth by solvent extraction (butyl/amyl acetate at pH 2.5) → back-extract into aqueous at pH 7 → crystallize as potassium/sodium salt.
- 7Citric Acid Production: Organism: Aspergillus niger. Largest volume organic acid produced by fermentation (~1.5 million tons/year). Medium: Sucrose or molasses (high sugar, 15-20%), low iron (critical — ↓Fe → ↓aconitase activity → citric acid accumulates in TCA cycle), low pH (2-3, prevents oxalic acid). Process: (1) Surface (Koji) process: shallow trays, 25-30°C, 7-10 days. (2) Submerged fermentation: STR, 30°C, pH 2-3, 6-8 days, highly aerated. (3) Solid-state fermentation: on sugarcane bagasse/beet pulp. Downstream: filter → precipitate as calcium citrate (add Ca(OH)₂) → treat with H₂SO₄ → free citric acid → crystallize. Uses: food (acidulant, preservative), pharmaceuticals (effervescent salts, anticoagulant — citrate), cosmetics.
- 8Vitamin B₁₂ Production: Organism: Pseudomonas denitrificans or Propionibacterium shermanii. Vitamin B₁₂ (Cyanocobalamin): complex cobalt-containing vitamin. Exclusively produced by microorganisms (not by plants or animals). Medium: sucrose/glucose, corn steep liquor, cobalt chloride (essential — incorporated into corrin ring), DMBI (5,6-dimethylbenzimidazole — precursor for nucleotide ligand). Process: aerobic fermentation (Pseudomonas) or anaerobic → aerobic switch (Propionibacterium), 28-30°C, 3-4 days. B₁₂ produced intracellularly → cells harvested → extracted with hot water/cyanide → purified by chromatography. Global production: ~35 tons/year. Entirely by fermentation (chemical synthesis too complex — 70+ steps).
- 9Glutamic Acid & Griseofulvin Production: Glutamic acid: Organism: Corynebacterium glutamicum. Medium: glucose, urea/ammonium salts, biotin (suboptimal concentration — key: biotin limitation → alters membrane permeability → glutamic acid excreted). Process: aerobic, 30-35°C, pH 7, 48-72 h. Excess glucose → α-ketoglutarate → transamination → glutamic acid. Used as: MSG (monosodium glutamate — flavor enhancer). Global production: >3 million tons/year. Griseofulvin: Organism: Penicillium griseofulvum (or P. patulum/janczewskii). Antifungal antibiotic (disrupts fungal mitotic spindle). Medium: glucose/sucrose, corn steep liquor, chloride ions (enhance production). Process: submerged fermentation, aerobic, 25°C, pH 6.5, 10-14 days. Downstream: solvent extraction, crystallization.
- 10Blood Products – Collection & Processing: Whole blood collection: from voluntary donors; 450 mL per donation into anticoagulant (CPD — citrate phosphate dextrose or CPDA-1 with adenine). Storage: 2-6°C in blood bank refrigerator. Shelf life: 21 days (CPD), 35 days (CPDA-1). Component separation (by centrifugation): Whole blood → centrifuge → Packed RBCs (bottom) + Platelet-Rich Plasma (top). PRP → centrifuge → Platelet Concentrate + Plasma. Plasma → freeze → Fresh Frozen Plasma (FFP). FFP → thaw at 4°C → Cryoprecipitate (pellet: Factor VIII, Fibrinogen, vWF, Factor XIII) + Cryo-poor plasma. Each donation → multiple components → multiple patients treated.
- 11Dried Plasma & Plasma Substitutes: Dried human plasma: FFP lyophilized (freeze-dried) → powder. Reconstitute with sterile water before use. Advantages: long shelf life (5 years vs 1 year for FFP), no need for frozen storage, easy transport. Uses: volume expansion, coagulation factors. Plasma substitutes (volume expanders): used when blood/plasma not available. (1) Colloids: Dextran (dextran-40 for microcirculation, dextran-70 for volume expansion — polysaccharide from Leuconostoc mesenteroides), Hydroxyethyl Starch (HES — hetastarch), Modified gelatin (Polygeline/Haemaccel — from bovine gelatin), Human Albumin (5% or 25%). (2) Crystalloids: Normal Saline (0.9% NaCl), Ringer’s Lactate (NS + KCl + CaCl₂ + sodium lactate), Dextrose solutions. Clinical: crystalloids for initial resuscitation → colloids/blood products for ongoing hemorrhage.
Learning Objectives
Fermenter Design: Draw and label a stirred-tank fermenter showing all components and control systems.
Penicillin Process: Describe the complete production process for Penicillin G including organism, medium, conditions, and downstream processing.
Citric Acid: Explain why low iron concentration is critical for citric acid accumulation in Aspergillus niger fermentation.
Blood Components: Describe the separation of whole blood into its components by differential centrifugation.
Plasma Substitutes: Compare colloid vs crystalloid plasma substitutes with examples and indications.
Exam Prep Questions
Q1. Why is Penicillin a ‘secondary metabolite’?
Metabolites are classified as: Primary (produced during growth/log phase — essential for growth: amino acids, nucleotides, vitamins) and Secondary (produced in stationary/idiophase — NOT essential for growth, often have ecological functions like defense). Penicillin is produced by P. chrysogenum primarily in the idiophase (after active growth slows). The organism doesn’t need penicillin for its own growth — it produces it to kill competing bacteria in its environment. This is why fermentation conditions must support both a growth phase AND an extended production phase.
Q2. Why is biotin limitation important in glutamic acid production?
Biotin is a cofactor for fatty acid synthesis (acetyl-CoA carboxylase). When biotin is limited, fatty acid synthesis is impaired → cell membrane becomes more permeable (incomplete lipid bilayer) → glutamic acid, which normally accumulates intracellularly, LEAKS OUT into the medium → easy recovery. At optimal biotin levels, the membrane is intact, glutamic acid stays inside the cell and is further metabolized. This biotin limitation strategy is the key to industrial glutamic acid production by Corynebacterium glutamicum.
Q3. What is the difference between Packed RBCs and Whole Blood transfusion?
Whole blood: contains RBCs + WBCs + platelets + plasma with all proteins. Rarely used now. Packed RBCs: whole blood centrifuged, plasma removed → concentrated RBCs (Hematocrit ~70–80%). Advantages: (1) Reduces volume (safer in heart failure patients). (2) Minimizes transfusion reactions (fewer WBCs, less plasma). (3) One whole blood donation → multiple components for multiple patients. (4) Stores better. Modern blood banking follows “component therapy” — give only what the patient needs.