Unit 6: Applied microbiology - Subjective Questions

Industrial microbiology is the branch of applied microbiology that deals with the use of microorganisms for the large-scale production of commercially valuable products such as antibiotics, enzymes, organic acids, vitamins, and beverages.

Characteristics of an ideal industrial microorganism:

• Genetic stability – should not mutate readily and lose productivity.
• High yield – must produce large amounts of the desired product.
• Rapid growth – should grow quickly on inexpensive, readily available media.
• Non-pathogenic – must be safe for workers and the environment.
• Pure culture – should be easily maintained as a pure strain.
• Ease of product recovery – the product should be easy to extract and purify.
• Amenable to genetic manipulation – to improve strains via mutation or recombination.
• Tolerance – able to withstand shifts in pH, temperature, and osmotic pressure.

Common examples include Aspergillus niger (citric acid), Penicillium chrysogenum (penicillin), and Saccharomyces cerevisiae (ethanol, baking).

Microbial products are broadly classified based on the growth phase during which they are produced.

Primary Metabolites:

• Produced during the exponential (trophophase) growth phase.
• Essential for the growth and survival of the organism.
• Product formation is growth-associated.
• Examples: ethanol, lactic acid, acetic acid, amino acids, nucleotides, vitamins.

Secondary Metabolites:

• Produced during the stationary (idiophase) phase, after active growth ceases.
• Not essential for growth but often provide survival advantages (defense, competition).
• Product formation is non-growth-associated.
• Examples: antibiotics (penicillin, streptomycin), alkaloids, toxins, pigments.

Comparison Table:

Microorganisms play a central role in the health/pharmaceutical industry by producing a wide range of therapeutic and diagnostic products.

Major contributions:

• Antibiotics – Penicillium chrysogenum (penicillin), Streptomyces griseus (streptomycin), Streptomyces spp. (tetracycline, erythromycin).
• Vaccines – attenuated or killed microbes, and recombinant subunit vaccines (e.g., Hepatitis B vaccine from yeast).
• Vitamins – Ashbya gossypii (riboflavin, B2), Pseudomonas / Propionibacterium (B12).
• Steroids and steroid hormones – produced via microbial biotransformation (e.g., Rhizopus hydroxylation of progesterone).
• Therapeutic proteins – recombinant insulin, human growth hormone, interferons produced in E. coli and yeast.
• Enzymes – streptokinase (clot dissolution), L-asparaginase (leukemia treatment).
• Diagnostic reagents – enzymes and antibodies for clinical assays.

These products have revolutionized the treatment of infectious diseases, metabolic disorders, and deficiencies.

Vitamin B12 (cyanocobalamin) is a complex cobalt-containing vitamin essential for red blood cell formation and neurological function. It is produced almost exclusively by microbial fermentation, as plants and animals cannot synthesize it.

Microorganisms used:

• Pseudomonas denitrificans
• Propionibacterium shermanii / freudenreichii
• Streptomyces olivaceus

Process details:

• Fermentation is carried out under anaerobic or microaerophilic conditions during the initial phase.
• The medium is supplemented with cobalt salts (essential cofactor) and 5,6-dimethylbenzimidazole (a precursor of the ligand).
• With Propionibacterium, an initial anaerobic phase (72 h) is followed by a mild aeration phase to complete B12 synthesis.
• Yields can reach ~150 mg/L with optimized Pseudomonas denitrificans strains.

Recovery:

• Cells are harvested and treated with heat/cyanide to convert cobalamins into the stable cyanocobalamin form.
• Purification is done by solvent extraction and chromatography.

Microbial fermentation remains the only commercially viable route for B12 production.

Biotransformation (microbial transformation) refers to the use of microorganisms or their enzymes to carry out specific chemical modifications on organic compounds. Unlike fermentation, the substrate is converted to a structurally similar product through one or a few enzymatic reactions.

Key features:

• Region-specific and stereo-specific reactions difficult to achieve by chemical means.
• Reactions include hydroxylation, oxidation, reduction, dehydrogenation, isomerization.
• Carried out under mild conditions (ambient temperature, aqueous medium).

Significance in steroid production:

Steroids like cortisone and hydrocortisone require selective hydroxylation which is extremely difficult chemically.

Example – 11α-hydroxylation of progesterone:

• The fungus Rhizopus nigricans (or Rhizopus arrhizus) introduces a hydroxyl group specifically at the C-11 position of progesterone to yield 11α-hydroxyprogesterone.
• This single microbial step replaced a multi-step (≈30 step) chemical synthesis, drastically reducing cost.

Advantages:

• High specificity, fewer by-products.
• Environmentally friendly, mild reaction conditions.
• Enables production of otherwise inaccessible compounds.

Both processes use microorganisms, but they differ significantly in mechanism and purpose.

Summary: Fermentation is a complete metabolic conversion, whereas biotransformation is a targeted chemical alteration of a specific substrate.

Citric acid is a major organic acid used in food, beverages, and pharmaceuticals. It is produced primarily by Aspergillus niger through the submerged fermentation process.

Biochemistry:

• Glucose is metabolized via glycolysis to pyruvate.
• Pyruvate is converted to acetyl-CoA and oxaloacetate.
• These condense in the TCA cycle to form citric acid.
• Under conditions where aconitase and isocitrate dehydrogenase are inhibited, citric acid accumulates rather than being further oxidized.

Fermentation conditions (critical factors):

• Low pH (~1.6–2.2) to favor citric acid and prevent contamination.
• Trace metal limitation – especially low manganese, iron, and zinc, which is essential for high yield.
• High sugar concentration (14–22% w/v).
• Good aeration (aerobic process).
• Temperature ~ 25–30 °C.

Recovery:

• The broth is filtered to remove mycelium.
• Citric acid is precipitated as calcium citrate by adding lime (Ca(OH)₂).
• Calcium citrate is treated with sulfuric acid to liberate citric acid, followed by crystallization.

Reaction summary:

Microorganisms are extensively used in the food industry for producing, preserving, and enhancing foods.

1. Fermented Dairy Products:

• Yogurt – Lactobacillus bulgaricus and Streptococcus thermophilus.
• Cheese – Lactococcus lactis, Penicillium roqueforti (blue cheese).

2. Bakery Products:

• Bread – Saccharomyces cerevisiae produces CO₂ for leavening.

3. Alcoholic Beverages:

• Beer, wine – Saccharomyces cerevisiae / S. carlsbergensis.

4. Fermented Vegetables:

• Sauerkraut, pickles – Leuconostoc mesenteroides, Lactobacillus spp.

5. Single Cell Protein (SCP):

• Protein-rich biomass from Spirulina, Candida yeasts as food/feed supplements.

6. Food Additives & Flavors:

• Glutamic acid/MSG – Corynebacterium glutamicum.
• Citric acid – Aspergillus niger.
• Vinegar – Acetobacter spp.

7. Probiotics:

• Beneficial live bacteria (Lactobacillus, Bifidobacterium) that improve gut health.

Thus microbes contribute to flavor, texture, preservation, and nutritional enrichment of foods.

Microbial enzymes are commercially important biocatalysts produced by fermentation because microbes offer high yields, ease of manipulation, and cheap production.

Advantages of microbial enzymes:

• High productivity and rapid growth.
• Easy genetic modification for enhanced yield.
• Independent of seasonal/geographic constraints.

Important microbial enzymes and applications:

Production process:

• Enzymes are produced by submerged or solid-state fermentation.
• Extracellular enzymes are recovered from the broth by filtration and precipitation.
• Intracellular enzymes require cell disruption.
• Purification via chromatography and ultrafiltration; enzymes may be immobilized for reuse.

Glutamic acid (as its sodium salt, monosodium glutamate/MSG) is a widely used flavor enhancer, produced industrially by Corynebacterium glutamicum (and Brevibacterium spp.).

Biochemical basis:

• Glucose is metabolized through glycolysis and the TCA cycle.
• α-Ketoglutarate is aminated by glutamate dehydrogenase using NH₄⁺ to form glutamic acid.

Key factor – Biotin limitation:

• C. glutamicum requires biotin for growth but glutamate over-production occurs only under suboptimal (limiting) biotin concentration.
• Biotin limitation alters the cell membrane permeability, allowing glutamate to leak out and accumulate in the medium.
• Alternatively, penicillin or surfactants (Tween 60) may be added to increase membrane permeability.

Fermentation conditions:

• Aerobic, temperature ~30–37 °C, pH ~7.0–8.0.
• Ammonium supplied as nitrogen source.

Recovery:

• Cells are removed; glutamic acid is concentrated and crystallized at its isoelectric point (~pH 3.2), then neutralized with NaOH to yield MSG.

Biohydrogen is hydrogen gas () produced by microorganisms through biological processes. It is regarded as a clean, renewable energy source because its combustion yields only water.

Biological processes for biohydrogen production:

1. Biophotolysis (Photosynthetic):

• Direct biophotolysis – Green algae (Chlamydomonas reinhardtii) split water using light energy; hydrogenase enzyme produces H₂.
  
• Indirect biophotolysis – Cyanobacteria (Anabaena) produce H₂ via nitrogenase, separating O₂ and H₂ evolution temporally.

2. Photo-fermentation:

• Purple non-sulfur bacteria (Rhodobacter sphaeroides) convert organic acids into H₂ using light energy via nitrogenase.
  

3. Dark fermentation:

• Anaerobic bacteria (Clostridium, Enterobacter) ferment carbohydrates to H₂ without light.
  

4. Microbial Electrolysis Cells (MEC):

• Electrochemically active bacteria oxidize organic matter, with a small applied voltage producing H₂ at the cathode.

Advantages: Renewable, uses waste substrates, operates at ambient conditions.

Penicillin is a β-lactam antibiotic and a classic secondary metabolite produced industrially by Penicillium chrysogenum (improved strain of P. notatum).

Fermentation process:

• Carried out by submerged aerobic fermentation in large stirred-tank bioreactors.
• Medium contains corn steep liquor (nitrogen source), lactose (slow-utilized carbon source), and mineral salts.
• Phenylacetic acid is added as a precursor for the side chain of benzylpenicillin (Penicillin G).
• Conditions: pH 6.5–7.5, temperature ~24–26 °C, good aeration and agitation.
• Two phases:
  • Trophophase – rapid growth, little penicillin.
  • Idiophase – slow growth, maximum penicillin synthesis.

Recovery and purification:

• Broth is filtered to remove mycelium.
• Penicillin is extracted with organic solvents (e.g., amyl acetate) at acidic pH.
• Back-extracted into aqueous buffer at neutral pH.
• Crystallized as sodium or potassium salt.

Strain improvement: Mutation and selection increased yields from a few units/mL to over 50,000 units/mL.

These are two major techniques used to cultivate microorganisms industrially.

Summary: SmF is preferred for bacterial products requiring precise control, whereas SSF is economical, especially for fungal enzyme and traditional food fermentations.

Riboflavin (Vitamin B2) is essential for energy metabolism (as a precursor of FAD and FMN). It is produced industrially mainly by microbial fermentation.

Producing microorganisms:

• Ashbya gossypii (filamentous fungus) – the major industrial overproducer.
• Eremothecium ashbyi – another fungal producer.
• Bacillus subtilis – genetically engineered strains.
• Candida famata (yeast).

Fermentation process:

• Aerobic submerged fermentation using cheap carbon sources like plant oils, glucose, or molasses.
• Ashbya gossypii can accumulate very high concentrations (up to several g/L).
• Optimum temperature ~26–28 °C, pH near neutral.

Recovery:

• Riboflavin is recovered by heating the broth to release intracellular vitamin, followed by filtration, precipitation, and crystallization.

Industrial significance:

• Used in food fortification, animal feed, and pharmaceuticals.
• Microbial fermentation has largely replaced chemical synthesis due to being cheaper, greener, and one-step.
• Recombinant Bacillus subtilis strains have made production highly economical.

Immobilized enzymes are enzymes that are physically confined or localized to a defined region of space, retaining their catalytic activity and allowing repeated reuse.

Methods of immobilization:

1. Adsorption:

• Enzyme physically binds to a carrier (activated charcoal, silica, ion-exchange resins) via weak forces.
• Simple and cheap but weak binding; enzyme can leach out.

2. Covalent Bonding:

• Enzyme is covalently attached to a support (agarose, cellulose) through functional groups.
• Strong and stable but may reduce activity.

3. Entrapment:

• Enzyme trapped within a polymer gel matrix (alginate, polyacrylamide).
• Enzyme not chemically altered; but substrate diffusion may be limited.

4. Encapsulation:

• Enzyme enclosed within semi-permeable membrane capsules.

5. Cross-linking:

• Enzyme molecules cross-linked to each other using reagents like glutaraldehyde (carrier-free).

Advantages:

• Reusability of the expensive enzyme.
• Continuous operation possible.
• Increased stability to pH and temperature.
• Easy separation of enzyme from product (no contamination).
• Reduced cost of processing.

Example: Immobilized glucose isomerase in the production of high-fructose corn syrup.

L-Lysine is an essential amino acid widely used as a feed and food supplement. It is produced industrially by fermentation using mutant strains of Corynebacterium glutamicum and Brevibacterium flavum.

Biochemical basis:

• Lysine is synthesized from aspartate via the aspartate pathway.
• Key branch-point enzyme: aspartokinase, which is normally subject to concerted feedback inhibition by lysine + threonine.

Strain improvement strategy:

• Homoserine dehydrogenase-deficient (auxotrophic) mutants are used.
• These mutants cannot synthesize threonine and methionine, so the concerted feedback inhibition of aspartokinase is relieved.
• Threonine is supplied in limiting amounts in the medium, so growth continues but lysine over-accumulates.

Fermentation conditions:

• Aerobic submerged fermentation.
• Carbon source: molasses/glucose; nitrogen: ammonium salts.
• Temperature ~30 °C, pH ~7.0.

Recovery:

• Lysine is recovered by ion-exchange chromatography and crystallized as lysine monohydrochloride.

This regulatory bypass strategy is a classic example of applying metabolic control principles to industrial amino acid production.

A fermenter (bioreactor) is a vessel designed to provide optimal controlled conditions for the growth of microorganisms and the formation of desired products.

Main components:

• Vessel (body): Usually stainless steel, capable of withstanding sterilization; can range from a few liters to thousands of liters.
• Agitator/Impeller: Mixes contents to ensure uniform distribution of nutrients, cells, and oxygen.
• Baffles: Prevent vortex formation and improve mixing.
• Sparger: Introduces sterile air/oxygen from the bottom.
• Temperature control (jacket/coils): Maintains optimum temperature via circulating water.
• pH control system: Sensors with automatic acid/alkali addition.
• Foam control: Antifoam agents or mechanical foam breakers.
• Sampling and inlet ports: For inoculation, feed addition, and sampling.
• Sensors/Probes: Monitor pH, temperature, dissolved oxygen, and pressure.

Operation:

1. The vessel and medium are sterilized (usually in-situ with steam).
2. Sterile medium is inoculated with the culture.
3. Conditions (temperature, pH, aeration, agitation) are monitored and controlled.
4. The process may be batch, fed-batch, or continuous.
5. After fermentation, the product is harvested and subjected to downstream processing.

Maintaining asepsis and precise control are the most critical requirements for successful operation.

These are the three major modes of operating a fermentation process.

1. Batch Fermentation:

• All nutrients added at the start; nothing added or removed (except gases) during the run.
• It is a closed system.
• Microbial growth follows the classic curve (lag, log, stationary, death).
• Advantages: Simple, low contamination risk, easy management.
• Disadvantages: Downtime between batches, lower productivity.

2. Fed-Batch Fermentation:

• Nutrients are added incrementally during the run, but the broth is not removed until the end.
• Volume increases with time.
• Advantages: Avoids substrate inhibition, prolongs product formation phase, higher yields (e.g., penicillin, baker's yeast).

3. Continuous Fermentation:

• Fresh medium is continuously added and equal volume of broth (with product and cells) is continuously removed.
• It is an open system maintained at steady state (e.g., chemostat).
• Advantages: High productivity, constant product quality, ideal for primary metabolites.
• Disadvantages: High contamination and mutation risk over long runs.

Comparison Table:

Microbial steroid transformation involves the use of microorganisms to carry out specific, selective modifications of steroid molecules that are chemically difficult or expensive to achieve. It revolutionized the production of steroid drugs like cortisone, hydrocortisone, and sex hormones.

Significance:

• Enables region- and stereo-specific modifications (e.g., hydroxylation at a specific carbon).
• Replaced long, costly chemical syntheses — e.g., microbial 11α-hydroxylation reduced cortisone synthesis from ~30 chemical steps to a few, drastically lowering cost.
• Operates under mild conditions (aqueous, ambient temperature), reducing hazardous chemical use.

Important types of steroid transformation reactions:

• Hydroxylation – introduction of –OH group (e.g., 11α-hydroxylation of progesterone by Rhizopus nigricans).
• Dehydrogenation – introduction of double bonds (e.g., 1,2-dehydrogenation of cortisone to prednisone by Arthrobacter/Corynebacterium).
• Oxidation – conversion of hydroxyl to keto groups.
• Reduction – conversion of keto to hydroxyl groups.
• Side-chain cleavage – degradation of sterol side chains (e.g., converting cholesterol/phytosterol to steroid intermediates using Mycobacterium).
• Isomerization and epoxidation.

Key organisms: Rhizopus, Aspergillus, Curvularia, Arthrobacter, Mycobacterium.

These transformations are the backbone of the modern steroid drug industry.

Downstream processing (DSP) refers to the sequence of operations required for the recovery, isolation, and purification of a product from the fermentation broth after the fermentation is complete. It often accounts for a large fraction of total production cost.

Major steps of downstream processing:

1. Removal of Insolubles (Solid–Liquid Separation):

• Removal of cells and debris by filtration or centrifugation.

2. Cell Disruption (if product is intracellular):

• Physical (sonication, high-pressure homogenization, bead mill) or chemical/enzymatic methods to release the product.

3. Isolation / Primary Recovery:

• Concentration and initial separation using solvent extraction, precipitation, adsorption, or ultrafiltration.

4. Purification:

• High-resolution separation using chromatography (ion-exchange, affinity, gel filtration) and crystallization to achieve required purity.

5. Polishing / Final Product Formulation:

• Drying (spray/freeze drying), formulation, stabilization, and packaging into the final marketable form.

Factors influencing DSP choice:

• Intracellular vs. extracellular product.
• Required purity (pharmaceutical grade needs higher purity).
• Product stability, concentration, and value.

Importance: Efficient DSP ensures high yield, purity, and economic viability, and is especially critical for high-value products like therapeutic proteins and antibiotics.