Unit 6: Applied microbiology

Applied microbiology harnesses microorganisms as living factories, converting cheap substrates into high-value products through controlled fermentation. The discipline grew from Pasteur's fermentation studies (1857) into a modern industry producing antibiotics, enzymes, vitamins, and fuels at tonne scale.

• Industrial strain requirements: Genetic stability, high yield on cheap media, rapid growth, non-pathogenicity, and ease of downstream recovery.
• Product classes: Primary metabolites (produced during trophophase/growth, e.g. ethanol, amino acids), secondary metabolites (produced during idiophase/stationary phase, e.g. antibiotics), and enzymes or biomass itself.
• Bioreactor conventions: Batch, fed-batch, and continuous modes; sterile aeration, agitation, pH and temperature control; typical stirred-tank fermenters run 10 L (pilot) to 500,000 L (production).
• Substrates: Molasses, corn steep liquor, whey, and starch hydrolysates supply carbon and nitrogen cheaply.

II. Industrial Microorganisms and Product Formation

The organism–product relationship and kinetics of accumulation.

A. Kinetics of product formation

The timing of product synthesis relative to growth defines process design.

• Growth-associated (Type I): Product forms in parallel with biomass; example — ethanol from Saccharomyces cerevisiae. Rate follows .
• Non-growth-associated (Type II): Product accumulates after growth ceases; example — penicillin from Penicillium chrysogenum in idiophase.
• Mixed (Type III): Partly overlapping; example — lactic acid.

Y(p/x) = mass of product formed / mass of cells formed
µ      = specific growth rate (h⁻¹)
q(p)   = specific product formation rate (g product / g cells / h)

B. Industrial microorganisms

The workhorse genera are selected for proven safety and yield.

• Bacteria: Bacillus subtilis (enzymes), Corynebacterium glutamicum (amino acids), Streptomyces spp. (antibiotics).
• Fungi: Aspergillus niger (citric acid), Penicillium (penicillin), Saccharomyces (ethanol, baking).
• Strain improvement: Random mutagenesis (UV, nitrosoguanidine) followed by screening; recombinant DNA for over-expression and metabolic-pathway engineering.

III. Industrial Microorganisms for the Health Industry

Microbes as sources of therapeutic and prophylactic agents.

A. Purpose and principle

Microorganisms synthesise complex bioactive molecules that are difficult or costly to make chemically.

• Antibiotics: Penicillium chrysogenum → penicillin; Streptomyces griseus → streptomycin; S. venezuelae → chloramphenicol.
• Recombinant therapeutics: Escherichia coli engineered to express human insulin (Humulin, 1982) and growth hormone.
• Vaccines and probiotics: Attenuated cultures and live Lactobacillus strains supporting gut health.

B. Industrial microorganism for health industry

The health sector spans small molecules to protein biologics.

• Secondary-metabolite antibiotics: Produced in idiophase; yields raised from ~60 mg/L (Fleming's original) to over 50 g/L by strain selection and precursor feeding (phenylacetic acid for penicillin G).
• Enzyme therapeutics and diagnostics: Streptokinase (clot lysis) from Streptococcus, asparaginase (leukaemia) from E. coli.
• Steroid intermediates: Microbial transformations supply pharmaceutical steroid precursors (detailed below).

IV. Vitamins

Microbial synthesis of essential micronutrients.

A. Definition and scope

Vitamins are organic micronutrients that microbes overproduce more economically than chemical synthesis for the more complex molecules.

• Vitamin B₁₂ (cobalamin): Produced by Propionibacterium shermanii and Pseudomonas denitrificans; the only vitamin obtained almost exclusively by fermentation because of its cobalt-corrin ring complexity; yields ~150 mg/L.
• Riboflavin (B₂): Overproduced by Ashbya gossypii and Eremothecium ashbyii; the fungus turns cultures bright yellow; industrial titres exceed 20 g/L.
• β-carotene (provitamin A): From Blakeslea trispora, stimulated by mating of (+) and (–) strains.
• Vitamin C route: Sorbitol → L-sorbose by Gluconobacter oxydans, a key oxidation step in the Reichstein process.

V. Steroids

Microbial biotransformation of the steroid nucleus.

A. Purpose and principle

Microbes perform regio- and stereo-specific modifications on the steroid ring that chemical synthesis cannot achieve cheaply.

• Key reaction — 11α-hydroxylation: Rhizopus arrhizus or Rhizopus nigricans hydroxylates progesterone at C-11, a single step replacing ~30 chemical steps and cutting cortisone cost dramatically (1950s breakthrough).
• Δ¹-dehydrogenation: Arthrobacter simplex converts hydrocortisone to prednisolone, raising anti-inflammatory potency.
• Side-chain cleavage: Mycobacterium spp. degrade sterol side chains to yield androstenedione (AD) and androstadienedione (ADD), precursors for sex hormones.
• Significance: Enabled affordable corticosteroids and contraceptives.

VI. Food Industry

Fermentation for preservation, flavour, and nutrition.

A. Scope and principle

Controlled microbial activity preserves food, develops flavour, and adds nutritional value.

• Dairy: Lactobacillus bulgaricus + Streptococcus thermophilus ferment lactose to lactic acid in yoghurt; Penicillium roqueforti ripens blue cheese.
• Bakery and beverages: Saccharomyces cerevisiae leavens bread (CO₂) and ferments beer and wine (ethanol).
• Single-cell protein (SCP): Biomass of Fusarium venenatum (Quorn) or Spirulina as protein source, ~45–55% protein by dry weight.
• Organic acids as additives: Citric acid (Aspergillus niger) and acetic acid (Acetobacter) used as preservatives and acidulants.

VII. Biotransformation

Single-step enzymatic conversion of a substrate to a related product.

A. Definition and features

Biotransformation (bioconversion) uses whole cells or isolated enzymes to modify a compound at a specific site while leaving the rest of the molecule intact.

• Specificity: Regio-, stereo-, and enantio-selective reactions under mild conditions (aqueous, ~30 °C, neutral pH).
• Reaction types: Oxidation, reduction, hydroxylation, dehydrogenation, hydrolysis, and isomerisation.
• Worked example: Sorbitol → L-sorbose by Gluconobacter oxydans — a stereospecific dehydrogenation feeding vitamin-C manufacture; a single microbial step achieves what chemistry cannot selectively.
• Advantages over synthesis: No harsh reagents, fewer by-products, and chirality preserved.

VIII. Enzymes

Microbial enzymes as industrial catalysts.

A. Purpose and principle

Microbial enzymes are preferred industrially for their high yield, ease of genetic manipulation, and independence from seasonal supply.

• Amylases: Bacillus licheniformis α-amylase for starch liquefaction; thermostable to ~90 °C, used in detergents and syrups.
• Proteases: Bacillus subtilisins dominate the detergent market (largest enzyme sector by value).
• Glucose isomerase: Streptomyces enzyme converts glucose to fructose for high-fructose corn syrup; usually immobilised for reuse.
• Immobilisation: Enzymes bound to carriers (entrapment, cross-linking, adsorption) permit continuous operation and recovery.
• Cellulases and pectinases: Trichoderma reesei cellulase for biofuel and textiles; pectinase clarifies fruit juice.

IX. Amino Acids

Fermentative production of dietary and industrial amino acids.

A. Principle

Metabolic regulation is engineered so that end-product feedback inhibition is relieved, allowing over-accumulation.

• L-Glutamic acid (MSG): Corynebacterium glutamicum excretes glutamate when biotin is limited, weakening the membrane; the founding amino-acid fermentation (Japan, 1957), titres >100 g/L.
• L-Lysine: Made by C. glutamicum homoserine-auxotroph mutants that cannot convert aspartate semialdehyde away from lysine, forcing accumulation.
• Regulatory strategy: Auxotrophic and regulatory (analogue-resistant) mutants bypass feedback inhibition and repression.
• Uses: Feed supplements (lysine, methionine), flavour enhancers (glutamate), and infusion fluids.

X. Microbial Fermentations

The unit operations that convert substrate to product at scale.

A. Definition and modes

Industrial fermentation is the large-scale cultivation of microbes under controlled conditions to accumulate a desired product; the term covers both anaerobic and aerobic processes.

• Batch: All nutrients added at start; simple but productivity falls as substrate depletes.
• Fed-batch: Substrate fed incrementally to avoid catabolite repression; standard for penicillin.
• Continuous (chemostat): Fresh medium in, culture out at steady state; dilution rate set below to prevent washout.

D    = dilution rate (h⁻¹)
F    = flow rate of medium (L/h)
V    = working volume (L)
At steady state: µ = D

B. Downstream processing

Recovery often costs more than the fermentation itself.

• Steps: Cell separation (centrifugation/filtration) → cell disruption if intracellular → extraction/precipitation → chromatography → drying.
• Product location: Extracellular products (enzymes, organic acids) are simpler to recover than intracellular ones.

XI. Biohydrogen

Microbial production of hydrogen as a clean fuel.

A. Principle and routes

Biohydrogen is H₂ gas generated by microbial metabolism, valued as a carbon-free energy carrier that yields only water on combustion.

1. Dark fermentation: Anaerobic bacteria (Clostridium, Enterobacter) ferment carbohydrates to H₂ and organic acids without light; fast rates but limited yield (~2–4 mol H₂ per mol glucose).

C6H12O6 + 2H2O → 2CH3COOH + 2CO2 + 4H2   (acetate pathway)

2. Photo-fermentation / biophotolysis: Purple non-sulphur bacteria (Rhodobacter) use light energy to convert organic acids to H₂ via nitrogenase; cyanobacteria and green algae (Chlamydomonas reinhardtii) split water using hydrogenase.

B. Enzymes and limitations

The catalytic machinery dictates the yield ceiling.

• Hydrogenase: Catalyses reversible ; strongly O₂-sensitive.
• Nitrogenase: Drives H₂ evolution in photo-fermentation but is energetically costly (ATP-dependent).
• Limitations: Oxygen sensitivity, low conversion efficiency, and gas-separation cost restrict commercial scale; integrated dark-plus-photo systems are studied to raise total yield.