Bioprocessing and Biomanufacturing Chapter Notes | Biotechnology for Class 12 - NEET PDF Download

Historical Perspective

• Living organisms, particularly microbes, utilize metabolic processes to produce household products (e.g., curd, yoghurt, idli, kinema) and industrial products (e.g., ethanol).
• Metabolites are chemical compounds produced by living organisms, classified as primary or secondary metabolites.
• Primary metabolites are produced via primary metabolic pathways, essential for cellular functions like growth and development.
• Secondary metabolites are intermediates or indirect products of secondary metabolic pathways, involved in functions such as defense against pathogens, phytoplankton, and herbivores, improving tolerance to abiotic stresses, attracting insects or animals for fertilization and seed dispersal, or deterring unwanted feeders.
• Secondary metabolites have diverse applications in pharmaceuticals, dyes, food additives, enzymes, and vitamins, requiring large-scale production due to insufficient natural quantities.
• Commercial production of secondary metabolites involves bioprocessing, a series of steps to produce purified compounds in bulk (100–10,000 liters), necessitating bioreactors.
• The discovery of penicillin by Alexander Fleming in 1928, a secondary metabolite from Penicillium notatum with antibacterial properties, marked a significant milestone in recognizing the value of biological products.
• Fleming observed that bacteria did not grow near a contaminating mold on a plate, identifying the mold as Penicillium notatum and its metabolite as penicillin.
• Ernest Chain and Howard Florey established penicillin’s potential as an effective antibiotic, used extensively to treat wounded American soldiers in World War II.
• Fleming, Chain, and Florey received the Nobel Prize in 1945 for their contributions to penicillin’s discovery and application.
• The challenge of scaling up penicillin production required systematic processes involving Penicillium species, microbial physiologists, life scientists, and technologists.
• Collaboration among companies, government laboratories, universities, and institutions drove efforts to enhance penicillin production, leading to the emergence of bioprocessing as a field.
• Bioprocessing integrates biological systems or their components (e.g., enzymes, chloroplasts) with chemical engineering processes to produce desired products at a commercial scale.
• The advent of recombinant DNA (rDNA) technology expanded the use of microbes for producing biological materials for human welfare.

Instrumentation in Bioprocessing: Bioreactor and Fermenter Design

• A bioreactor is an engineered vessel, typically made of glass or steel, that supports a biologically active environment for cultivating microbial, plant, or animal cells under aseptic conditions with appropriate nutritional and environmental requirements.
• Bioreactors facilitate biochemical processes involving cell cultures or biochemically active substances derived from organisms, are usually cylindrical, and vary in size.
• Bioreactor requirements include:
  • Maintaining a sterile environment for pure culture growth without contamination.
  • Ensuring an adequate air supply for cellular respiration.
  • Providing uniform mixing of nutrients, cells, and air without causing shear stress to cultured cells.
  • Maintaining optimum temperature for growth and product formation.
  • Monitoring environmental parameters like pH and dissolved oxygen.
• Components of a typical bioreactor include:
  • Agitator shaft: Equipped with an impeller to mix contents, ensuring homogeneous conditions for nutrient and oxygen transport.
  • Sparger: Supplies continuous, sterilized air (oxygen) through microfilters for submerged cell growth in liquid media.
  • Baffle: Prevents vortex formation, which could alter the system’s center of gravity and increase power consumption.
  • Jacket: Circulates water at a controlled temperature to maintain optimal conditions for cell growth and product formation.
  • Sensitivity probes: Monitor temperature and pH of the bioprocess.
  • Digital controller: Connected to probes, regulates temperature via a water bath pumping water through the jacket, and adjusts pH by adding 1 M NaOH or 1 N HCl from connected bottles; displays parameters like temperature, pH, and stirring speed (rpm).
• Types of bioreactors based on design or configuration:
  • Stirred tank reactors: Conventional bioreactors with an agitator shaft and impeller for mixing nutrients, oxygen, and cells; impeller design, shape, and size vary by bioprocess.
  • Air-lift reactors: Use a draft tube to create air currents, lifting fluid broth and cells up and down or inside-out for mixing nutrients and oxygen.
  • Bubble column reactors: Employ air bubbles from a sparger jet for mixing, providing a low-shear environment critical for sensitive cells and high oxygen transfer per unit power input.

Operational Stages of Bioprocess

• Optimizing nutritional conditions and formulating artificial media for culturing organisms, cells, or their components.
• Sterilizing media, bioreactors, and additional tools and equipment.
• Producing pure, active, and healthy inoculum in sufficient quantities.
• Optimizing environmental conditions for growth and product formation.

Downstream processing includes:

• Extracting, recovering, and purifying the product.
• Disposing of effluents produced during the process.

Upstream processing details:

• Raw materials (e.g., microbial, plant, or animal cell biomass) are treated and mixed with ingredients necessary for cell growth.
• Raw materials are converted into a fermentable form suitable for bioprocessing.
• Nutrient media are formulated with chemicals and nutrients for maximum growth and product formation, tailored to specific organisms (e.g., microbial, plant, or animal cell cultures).
• Media formulation considers stoichiometry for growth and product formation: Carbon and energy source + Nitrogen source + other requirements → Cell biomass + products + CO2 + Water + heat.
• Media design minimizes wastage, using the cellular yield coefficient (Y = Quantity of cell dry weight produced / Quantity of carbon substrate utilized) to estimate carbon requirements under aerobic conditions.
• Inoculum must be viable, healthy, fast-growing, and high-producing, developed in solid or liquid (suspension) cultures, typically in shake flasks.
• Inoculum criteria include being in the log/exponential growth phase, exhibiting a short lag phase, being sufficient in quantity, having suitable morphology, being contamination-free, and retaining product-forming capability.
• Aeration supplies oxygen for submerged suspension cultures, with a maximum dissolved oxygen level of approximately 8 g/L in pure water, maintained in bioreactors via spargers.
• Mild agitation ensures uniform distribution of oxygen and nutrients; shear sensitivity varies (animal cells > plant cells > microbial cells) and is controlled by adjusting shaker speed (rpm) or agitator speed in bioreactors.
• Temperature is optimized for growth and product formation, which may differ for each process.
• pH is maintained at an optimal level to support growth and product formation.
• All parameters (media formulation, temperature, pH) are optimized separately before running a bioprocess.
• Sterilization of media, bioreactor vessels, additional materials, and maintenance of aseptic conditions prevent contamination during fermentation.

Modes of bioprocess operation:

• Batch mode: A closed system with a fixed initial nutrient amount; cells pass through growth phases, utilizing nutrients for growth, energy, and product formation, with cell biomass (X), substrate concentration (S), and substrate utilization rate (Qs) varying over time.
• Fed-batch mode: Nutrients are added intermittently or continuously to maintain low residual substrate concentrations, avoiding toxicity; culture volume increases without removal, keeping substrate concentration (S) and utilization rate (Qs) constant while biomass/product increases.
• Continuous mode: Fresh nutrient media and inoculum are added, and used media is removed to maintain a steady state, balancing new biomass formation with cell loss, keeping growth rate, product formation, substrate concentration, and utilization rate constant.

Downstream processing details:

• Involves efficient separation and purification to recover the desired product (cell biomass, extracellular broth components, or intracellular products).
• Processes must be quick, cost-effective, and require minimal investment.
• Physical separation and purification techniques include particulate separation, dialysis, reverse osmosis, solid-liquid separation, adsorption, liquid-liquid extraction, distillation, and drying.
• Solid-liquid separation: Separates solids (biomass, insoluble particles, macromolecules) from culture fluid using filtration or centrifugation.
• Filtration: A cost-effective method for separating large particles (>10 µm) using canvas, synthetic fabrics, or glass fiber; continuous rotary filters are widely used, with ultrafiltration or microporous filtration for smaller particles.
• Centrifugation: Separates particles (100 µm to 0.1 µm) using centrifuges or ultracentrifuges; culture broth may require pre-treatment (heating, pH adjustment, coagulating/flocculating agents).
• Cell disruption: For intracellular products, cells are ruptured using physical (milling, high-pressure homogenization, ultrasonication), chemical (surfactants, alkalis, organic solvents, osmotic shock), or biological (enzymatic cell wall degradation) methods, ensuring minimal damage to the product.
• Recovery: Involves extracting and adsorbing products from dilute aqueous solutions based on intracellular/extracellular location, product concentration, physical/chemical properties, required purity, and impurities.
• Liquid-liquid extraction: Separates components using a solvent where the desired component is preferentially soluble, producing a solute-rich extract and a residual raffinate; requires optimized conditions (temperature, pH, light).
• Purification techniques: Include precipitation (for proteins/antibiotics using salts, organic solvents, or ultrafiltration), chromatography, electrophoresis, membrane separation, dialysis, reverse osmosis, and ultrafiltration.
• Membrane separation processes:
  • Microfiltration: Pore size 0.1–10 µm, molecular weight cutoff <1,000,000 Da, pressure drop 10 psi, retains suspended materials (e.g., bacteria).
  • Ultrafiltation: Pore size 0.01–0.1 µm, molecular weight cutoff 300–300,000 Da, pressure drop 10–100 psi, retains biologicals, colloids, and macromolecules.
  • Reverse osmosis: Pore size <0.001 µm, molecular weight cutoff <300 Da, pressure drop 100–800 psi, retains all suspended and dissolved materials.
• Reverse osmosis: Applies pressure to a salt-containing phase to drive water molecules against the concentration gradient from saline to pure water, requiring pressure slightly above osmotic pressure.
• Dialysis: Removes low-molecular-weight solutes (e.g., organic acids, 100–500 Da; inorganic ions, 10–100 Da) through a selectively permeable membrane, allowing only small molecules to move from high to low concentration regions, as in artificial kidney devices removing urea (MW = 60 Da).

Bioprocessing and Biomanufacturing of Desired Product

• Bioprocessing industries produce valuable products from primary metabolites (e.g., amino acids, organic acids) and secondary metabolites (e.g., antibiotics) using microorganisms, animal cells, plant cells, or their constituents.
• Products include alcohols, antibiotics, amino acids, organic acids, enzymes, vitamins, vaccines, recombinant proteins, pigments, and plant alkaloids, integral to daily life.
• Examples of bioprocessing products:
  • Cell biomass: Baker’s yeast, single-cell protein.
  • Extracellular: Alcohols, organic acids, amino acids, enzymes, antibiotics.
  • Intracellular: Recombinant DNA proteins.
• Major commercial bioprocess products:
  • Ethanol (alcohol): Produced by Saccharomyces cerevisiae (brewer’s/baker’s yeast) via fermentation of malted cereals or fruit juices; wine and beer are non-distilled, while whisky, brandy, and rum are distilled.
  • L-glutamic acid (amino acid): Produced by Corynebacterium glutamicum, used as a flavor enhancer in the food industry.
  • Lactic acid (organic acid): Produced by Lactobacillus delbrueckii.
  • Proteases (enzymes): Produced by Bacillus spp., used in detergents and leather industries.
  • Pectinase (enzyme): Produced by Aspergillus niger, clarifies bottled fruit juices.
  • Penicillin (antibiotic): Produced by Penicillium chrysogenum, the first antibiotic discovered.
  • Vitamin B12: Produced by Propionibacterium shermanii or Pseudomonas denitrificans.
  • Diphtheria vaccine: Produced using Corynebacterium diphtheriae, processed as diphtheria toxoid.
  • Insulin (recombinant protein): Produced by recombinant Escherichia coli using a two-phase cultivation process (glycerol batch and continuous methanol fed-batch).
  • Shikonin (pigment, quinone derivative/naphthaquinone): Produced by Lithospermum erythrorhizon plant cell culture, used as a dye.
  • Taxol (plant alkaloid): Produced by Taxus brevifolia plant cell culture, an anticarcinogenic compound.
• Antibiotics: Penicillin’s discovery revolutionized bioprocessing, followed by the purification of other antibiotics from microbes.
• Amino acids: Lysine and glutamic acid are nutritional supplements and flavor enhancers in the food industry, produced by Corynebacterium glutamicum mutants with reduced synthesis capabilities for specific amino acids or intermediates.
• Organic acids: Produced by various microorganisms: citric acid (Aspergillus niger), acetic acid (Acetobacter aceti), butyric acid (Clostridium butylicum), lactic acid (Lactobacillus sp.).
• Enzymes:
  • Lipases (used in detergents for stain removal), pectinases, and proteases (for juice clarification and leather processing) are produced by fungi (Aspergillus, Mucor, Trichoderma) and Bacillus spp.
  • Streptokinase: Produced by Streptococcus species, genetically modified, used as a “clot buster” for myocardial infarction patients.
• Bioactive molecules:
  • Cyclosporin A: Produced by Trichoderma polysporum, used as an immunosuppressive agent in organ transplants.
  • Statins: Produced by Monascus purpureus, commercialized as cholesterol-lowering agents by inhibiting cholesterol synthesis enzymes.
• Vitamins:
  • Vitamin B12: Initially a by-product of antibiotic fermentation (streptomycin, chloramphenicol, neomycin) using Streptomyces, now produced by high-yielding strains of Propionibacterium freudenreichii, Pseudomonas denitrificans, Bacillus megaterium, and Streptomyces olivaceus.
  • Riboflavin: Produced via biotransformation (glucose to D-ribose by Bacillus pumilus mutants, then chemically converted to riboflavin) or fermentation (by-product in acetone-butanol fermentation by Clostridium acetobutylicum/butylicum, or direct fermentation using Ashbya gossypii).
• Vaccines:
  • Diphtheria vaccine: Uses Corynebacterium diphtheriae to produce diphtheria toxin, processed into toxoid and vaccine.
  • Cell-based vaccines: Use mammalian cell cultures to grow influenza virus for vaccine production, with different companies employing various cell sources.
• Plant cell and tissue culture:
  • Used for commercial production of biochemicals like pigments, quinone derivatives, and plant alkaloids for dyeing, food additives, and pharmaceuticals.
  • Shikonin: First commercially produced dye via Lithospermum erythrorhizon cell culture.
  • Other examples: Berberine (Coptis japonica), ginseng and saponin (Panax ginseng), taxol (Taxus brevifolia).
• Recombinant proteins:
  • Genes of interest are cloned and expressed in heterologous hosts for large-scale production of biological products.
  • Cells with cloned genes are grown on a small scale, and the desired protein is extracted and purified using separation techniques.
  • Insulin production uses recombinant E. coli in a two-phase cultivation process for high yields.
• Ongoing research worldwide aims to commercialize additional bioactive compounds from living organisms, with many compounds in development.