Fermentation

Definition: Fermentation is a metabolic process that converts sugar to acids, gases,

or alcohol. It occurs in yeast and bacteria, and also in oxygen-starved muscle cells,
as in the case of lactic 1 acid fermentation.

 Historical Significance: Fermentation has been used for millennia in food

preservation and production (e.g., brewing, baking, dairy).
 Modern Applications: Beyond food, fermentation is crucial in the
industrial production of pharmaceuticals (e.g., antibiotics), enzymes,
biofuels, and various other biochemicals.
 Types of Fermentation (based on oxygen requirement):
o Aerobic Fermentation: Requires the presence of oxygen. Typically
used for biomass production or certain biotransformations.
o Anaerobic Fermentation: Occurs in the absence of oxygen.
Substrate is the final electron acceptor. Examples include alcoholic
and lactic acid fermentation.
 Metabolic Pathways: Understanding the underlying biochemical pathways
(e.g., glycolysis, citric acid cycle, electron transport chain, specific
fermentation pathways) is essential.

2. Fermenter Design and Operation (Bioreactors)

 Definition: A fermenter or bioreactor is a vessel in which biological

reactions are carried out under controlled conditions.
 Key Components of a Fermenter:
o Vessel: Typically made of stainless steel, designed for sterilization
and containment. Size varies from laboratory scale to industrial scale
(thousands of liters).
o Agitation System (Impeller): Ensures uniform mixing of the culture
medium, facilitates nutrient and oxygen transfer, and prevents
sedimentation. Different impeller designs exist (e.g., Rushton turbine,
propeller).
o Aeration System (Sparger): Introduces sterile air or oxygen into the
culture medium for aerobic fermentations. Sparger design affects
bubble size and oxygen transfer efficiency.
 o Baffles: Prevent vortex formation and enhance mixing efficiency.
o Temperature Control System: Maintains the optimal temperature
for microbial growth and product formation (e.g., heating jackets,
cooling coils).
o pH Control System: Monitors and adjusts the pH of the culture
medium using acid or base addition.
o Foam Control System: Prevents excessive foam formation, which
can hinder oxygen transfer and cause contamination. Mechanical (e.g.,
impellers) or chemical (antifoam agents) methods are used.
o Sampling Ports: Allow for aseptic sampling to monitor cell growth,
substrate consumption, and product formation.
o Sensors: For real-time monitoring of critical parameters like
temperature, pH, dissolved oxygen, and nutrient levels.
 Types of Fermenters:
o Stirred Tank Bioreactors: Most common type, with mechanical
agitation.
o Airlift Bioreactors: Mixing and aeration are achieved by introducing
air at the bottom of the vessel, creating density gradients.
o Packed Bed Bioreactors: Microorganisms are immobilized on a solid
support matrix.
o Fluidized Bed Bioreactors: Solid particles with immobilized
microorganisms are suspended in an upward flow of liquid or gas.
o Photobioreactors: Designed for photosynthetic microorganisms,
utilizing light as an energy source.
 Modes of Operation:
o Batch Fermentation: All components are added at the beginning, and
the process runs for a fixed time. Product is harvested at the end.
o Fed-Batch Fermentation: Nutrients are added incrementally during
the fermentation process to maintain optimal growth and production.
Volume increases over time.
o Continuous Fermentation: Fresh medium is continuously added,
while spent medium and product are continuously removed,
maintaining a steady state.

3. Aeration and Agitation

 Oxygen Transfer: Oxygen has low solubility in aqueous media, making its
transfer to microorganisms a critical limiting factor in aerobic fermentations.
 Factors Affecting Oxygen Transfer Rate (OTR):
 o Gas-Liquid Interfacial Area: Influenced by sparger design, bubble
size, and agitation.
o Oxygen Concentration Gradient: Difference in partial pressure of
oxygen between the gas and liquid phases.
o Mass Transfer Coefficient (kLa): Represents the efficiency of
oxygen transfer. Affected by agitation speed, aeration rate, and fluid
properties.
 Agitation:
o Purpose: To ensure homogeneity of the culture medium, enhance
nutrient and oxygen transfer, and maintain cell suspension.
o Agitation Rate: Optimal rate depends on the microorganism, culture
volume, and impeller design. Excessive agitation can cause shear
stress and damage cells.
 Scale-Up Considerations: Maintaining adequate oxygen transfer and
mixing becomes increasingly challenging at larger scales.

4. Temperature and pH Control

 Temperature:
o Importance: Enzyme activity and microbial growth rates are highly
temperature-dependent. Optimal temperature varies for different
microorganisms.
o Control Mechanisms: Heating jackets, cooling coils, and sometimes
internal heat exchangers are used to maintain the desired temperature.
 pH:
o Importance: pH affects enzyme activity, nutrient availability, and
cell membrane stability. Optimal pH range is specific to the
microorganism and the process.
o Control Mechanisms: Addition of acidic or basic solutions (e.g.,
NaOH, KOH, H₂SO₄, HCl) using automated control systems based
on pH sensor readings. Buffers may also be included in the medium.

5. Product Extraction (Downstream Processing)

 General Steps: After fermentation, the desired product needs to be

separated and purified from the complex broth containing cells, media
components, and other byproducts.
 Solid-Liquid Separation:
o Centrifugation: Used to separate cells or other particulate matter
from the liquid.
 o Filtration: Passing the broth through a porous membrane to remove
solids. Different types of filters (e.g., depth filters, membrane filters)
are used.
 Cell Disruption (if product is intracellular):
o Mechanical Methods: Bead milling, high-pressure homogenization,
sonication.
o Non-Mechanical Methods: Enzymatic lysis, chemical lysis.
 Product Isolation and Purification:
o Solvent Extraction: Separating the product based on its solubility in
a particular solvent.
o Precipitation: Adding salts or organic solvents to reduce product
solubility and cause it to precipitate.
o Chromatography: Separating molecules based on their physical and
chemical properties (e.g., size exclusion, ion exchange, affinity
chromatography).
o Membrane Separation: Using semi-permeable membranes to
separate molecules based on size and charge (e.g., ultrafiltration,
nanofiltration, reverse osmosis).
 Product Formulation and Finishing: Drying, crystallization, sterilization,
and packaging to obtain the final product.

6. Typical Industrial Fermentations

 Microbial Production of Alcohol (Ethanol):

o Microorganisms: Saccharomyces cerevisiae (yeast).
o Substrates: Sugars (e.g., glucose, sucrose, starch hydrolysates).
o Process: Anaerobic fermentation yielding ethanol and carbon dioxide.
o Applications: Alcoholic beverages, biofuels, industrial solvents.
 Microbial Production of Acids:
o Lactic Acid: Lactobacillus species. Used in food preservation,
bioplastics.
o Citric Acid: Aspergillus niger. Used as a food additive, in
pharmaceuticals.
o Acetic Acid: Acetobacter species. Production of vinegar.
 Microbial Production of Enzymes:
o Amylases, Proteases, Lipases: Produced by various bacteria and
fungi (e.g., Bacillus, Aspergillus). Used in detergents, food
processing, textiles.
 Microbial Production of Antibiotics:
o Penicillin: Produced by Penicillium chrysogenum.
 o Streptomycin: Produced by Streptomyces griseus.
o Process: Complex secondary metabolite production, often requiring
specific growth conditions and strain optimization.

7. Preparation of Fermented Foods

 Bread and Baked Goods:

o Microorganism: Saccharomyces cerevisiae (baker's yeast).
o Process: Yeast ferments sugars in the dough, producing carbon
dioxide that causes leavening.
 Buttermilk and Sour Cream:
o Microorganisms: Lactococcus lactis, Streptococcus cremoris.
o Process: Lactic acid fermentation of milk, leading to acidification and
characteristic flavor and texture.
 Cheeses:
o Microorganisms: Various bacteria and fungi, depending on the
cheese type (e.g., Lactobacillus, Streptococcus, Penicillium).
o Process: Complex fermentation and enzymatic processes involving
milk coagulation, curd formation, and ripening.
 Vinegar:
o Microorganisms: Acetobacter species.
o Process: Two-stage fermentation: first, yeast ferments sugars to
ethanol (as in alcoholic fermentation), then Acetobacter oxidizes
ethanol to acetic acid.
 Wine:
o Microorganism: Saccharomyces cerevisiae (wine yeast).
o Process: Fermentation of grape juice sugars to ethanol and carbon
dioxide.
 Yogurt:
o Microorganisms: Streptococcus thermophilus and Lactobacillus
bulgaricus.
o Process: Symbiotic lactic acid fermentation of milk, resulting in a
thickened texture and characteristic flavor.
 Soy Sauce:
o Microorganisms: Aspergillus oryzae or Aspergillus sojae, and
Zygosaccharomyces rouxii.
o Process: Complex fermentation of soybeans and wheat with salt,
involving enzymatic breakdown of proteins and carbohydrates,
followed by lactic acid and alcoholic fermentation.
8. Microbial Toxins and Insecticides

 Microbial Toxins (Mycotoxins):

o Production: Produced by certain molds (fungi) growing on food
crops (e.g., aflatoxins by Aspergillus flavus, ochratoxin A by
Penicillium and Aspergillus).
o Significance: Can contaminate food and feed, posing health risks to
humans and animals.
o Control Measures: Proper storage conditions, good agricultural
practices, and detoxification methods.
 Microbial Insecticides:
o Examples: Bacillus thuringiensis (Bt) produces crystal proteins (Cry
toxins) that are toxic to specific insect pests.
o Advantages: Often more specific and environmentally friendly than
synthetic chemical insecticides.
o Applications: Used in agriculture and forestry for pest control.
o Production: Grown in large-scale fermenters, and the spores and/or
toxins are formulated into insecticide products.