Fermentation and Bioassays Quiz

Fermentation

• Fermentation is a metabolic process that converts sugar to acids, gases, or alcohol.
• It occurs in yeast, bacteria, and oxygen-starved muscle cells.
• It's used to produce specific chemical products, like enzymes, vaccines, antibiotics, and food additives.
• Louis Pasteur made important discoveries about fermentation and its microbial causes.
• Zymology is the science of fermentation.
• Fermentation occurs in the absence of oxygen, when the electron transport chain isn't functional.
• It's the primary method cells use to produce ATP (energy) when oxygen isn't available.
• The process converts NADH and pyruvate into NAD+ and other smaller molecules, depending on the type of fermentation.
• Glycolysis is a common step in all fermentation pathways.
  • C6H12O6 + 2 NAD+ + 2 ADP + 2 P₁ → 2 CH3COCOO⁻ + 2 NADH + 2 ATP + 2 H₂O + 2H⁺
• Pyruvate is the molecule CH3COCOO⁻.
• Pi is inorganic phosphate.
• Two ADP molecules and two Pi are converted to two ATP and two water molecules via substrate-level phosphorylation.
• Two NAD⁺ molecules are reduced to NADH in this process.
• In oxidative phosphorylation, the energy for ATP is created by the electrochemical proton gradient across the inner mitochondrial membrane (or plasma membrane in bacteria) through the electron transport chain.
• Glycolysis also has substrate-level phosphorylation, which generates ATP directly.
• Humans have used fermentation for food and beverage production since Neolithic times (e.g., pickled cucumbers, kimchi, yogurt, and alcoholic beverages).

Definitions of Fermentation

• Preservation methods of food using microorganisms (general use)
• Production of alcoholic beverages or acidic dairy products (general use)
• Any large-scale process using microorganisms (common industrial definition)
• Energy-releasing processes under anaerobic conditions (more scientific)
• Metabolic processes releasing energy from sugars/organic molecules without oxygen, using organic molecules as the final electron acceptor (most scientific)

Examples of Fermentation

• Ethanol fermentation (alcoholic fermentation): Production of ethanol and carbon dioxide
• Lactic acid fermentation: Two types
  • Homolactic fermentation: Produces lactic acid only
  • Heterolactic fermentation: Produces lactic acid, as well as other acids and alcohols

Sugars and Fermentation

• Sugars are the most common substrate for fermentation.
• Typical fermentation products include ethanol, lactic acid, carbon dioxide, and hydrogen gas.
• Other compounds like butyric acid and acetone can also be produced.
• Yeast is responsible for the production of ethanol in beers, wines, and other alcoholic drinks, along with large amounts of carbon dioxide.
• Lactic acid is produced during periods of intense exercise, when oxygen supply is limited.

Ethanol Fermentation

• C6H12O6 → 2 C2H5OH + 2 CO2
• This shows one glucose molecule being converted into two molecules of ethanol and two molecules of carbon dioxide.
• Before fermentation, one glucose molecule is broken down into two pyruvate molecules in a process called glycolysis.

Lactic Acid Fermentation

• Homolactic fermentation is the simplest type.
• Pyruvate undergoes a redox reaction to form lactic acid.
• This process doesn't produce a gas as a by-product.
• Occurs in muscle cells when energy is needed faster than oxygen can be supplied.
• Also occurs in some bacteria (e.g., Lactobacilli) and some fungi.
• In yogurt, lactic acid fermentation leads to the sour taste.

Heterolactic Fermentation

• A slightly more complex type of fermentation, where some lactate is further metabolized to ethanol, carbon dioxide, acetate, etc.
• It's an intermediate between lactic acid fermentation and other types like alcoholic fermentation.

Fermentation in Organisms

• Acidity of lactic acid can hinder other biological processes, but it's beneficial for the fermenting organism.
• High lactic acid concentrations can reduce the rate of fermentation.
• Acetic acid is another product, which is less volatile than ethanol, but produces energy.

Aerobic Respiration

• In aerobic respiration, pyruvate is completely oxidized, creating more ATP and NADH.
• It requires oxygen, and is not used in obligate anaerobes.
• Facultative anaerobic organisms can use either aerobic respiration or fermentation.

Hydrogen Gas Production in Fermentation

• Hydrogen gas can be a product of various fermentations (including mixed/butyric acid/caproate/butanol/glyoxylate fermentations).
• Electrons are transferred to ferredoxin, which then produces hydrogen gas (H₂).
• This pathway can help organisms regenerate NAD+.

Methane Gas Production in Fermentation

• Methane (CH₄) and carbon dioxide (CO₂) are produced through a dismutation process in acetic acid fermentation.
• Methanogens (archaea) catalyze this process in their fermentative metabolism.

History of Fermentation

• Fermentation has been used to produce beverages since the Neolithic period (as early as 7000–6600 BCE).
• Louis Pasteur made important contributions to understanding fermentation by identifying the microbial causes in the mid-1800s.
• Pasteur recognized the important role of microorganisms in food spoilage & fermentation.

Advances and Technologies

• Recent advancements include genetic modification strategies to increase yielding & rate & oxygen tolerance.

Industrial Fermentation

• Intentional use of microorganisms like bacteria/fungi to produce human-useful products.
• Many food and chemical products (acetic acid, citric acid, ethanol) are produced in industrial fermentations.
• Success depends upon concentration of microorganisms, pH, temperature, and oxygen concentration (for aerobic processes).
• Product recovery usually entails concentrating the solution containing the product.
• Four main types of products made in industrial fermentations:
  • Biomass production (viable cellular material)
  • Extracellular metabolites
  • Intracellular components (enzymes/proteins)
  • Substrate transformation (substrate becomes the product through modification)
• Important considerations in industrial fermentations include the organisms used, dissolved oxygen, nutrient levels, and temperature.

Phases of Microbial Growth

• Lag phase: Adaptation period after inoculation.
• Exponential (Log) phase: Rapid growth.
• Deceleration phase: Slowing growth rate.
• Stationary phase: Constant biomass; growth nearly stops.

Fermentation Medium

• Medium has necessary nutrients for the growing organisms, including a carbon source, nitrogen source, water, salts, and micronutrients.
• Wine production uses grape must as a medium.
• Bio-ethanol production often uses inexpensive carbohydrate sources like molasses.

Production of Biomass

• Microbial cells (like single-cell protein) or yeast are sometimes the intended fermentation product.

Production of Extracellular Metabolites

• Some metabolites (like ethanol, citric acid, lysine) are produced during the growth phase (primary metabolites).
• Other compounds (like penicillin, cyclosporin) are made mostly during the stationary phase after the metabolic activity has shifted to different product formation (secondary metabolites).

Transformation of Substrate

• Converting raw materials into final products (e.g., in food and sewage).
• An example is the conversion of organic matter in sewage during treatment to harmless substances.

Food Fermentation

• Many fermented foods (bread, wine, cheese, etc.) were developed long before microorganisms were understood.

Ethanol Fuel

• Fermentation is a part of ethanol production from crops like sugar cane, corn, or potatoes.

Sewage Treatment

• Sewage contains organic matter, broken down by bacterial enzymes.
• Byproducts include gases like methane (used for energy).

Agricultural Feed

• Agroindustrial waste products are fermented to produce animal feed (especially for ruminants).
• Fungi can be used to break down cellulose and improve digestibility.

Bioassays

• Used to determine concentration/purity/biological activity of substances (vitamins, hormones).
• Involve live animals or plant tissues.
• Can be qualitative or quantitative.
• Qualitative bioassays may involve observing physical effects.
• Quantitative assays yield dose-response curves.

Types of Bioassays

• Quantal bioassays: Evaluate "all or none" responses.
• Graded bioassays: Measures the extent of an effect in a scaled manner.

Bioassay Techniques

• Matching bioassay: Measures responses qualitatively, matching responses to standard curves.
• Interpolation bioassay: Determines amount of an unknown to cause a specific effect, based on a standard curve or mathematical models.

Environmental Bioassays

• Environmental bioassays assess toxicity, identify harmful substances, and evaluate biological activity in organisms.
• Radio-immunoassays are one example of alternative techniques.

Quality Control

• Quality control procedures aim to ensure that products meet required standards in commercial or industrial production, including appropriate checks & approvals.
• Three key aspects:
  • Controls and processes (defined, well-managed).
  • Competence of personnel (knowledge, skills, experience).
  • Soft elements (e.g., culture, motivation, relationships)

Shelf Life of Products

• Length of time a product stays suitable for sale or consumption.
• Factors affecting shelf life include storage conditions, temperature, moisture, contamination, microbial growth, chemical reactions
• Expiration dates are guidelines for optimal freshness.
• Some products, like frozen foods, have potentially indefinite shelf life under specific conditions.

Food Spoilage

• Food spoilage is the process where food degrades from its original state to non-edible or less desirable qualities.
• Causes of food spoilage include:
  • Bacteria
  • Yeasts
  • Chemical reactions
  • Oxidation
  • Moisture
  • Temperature
• Prevention methods include:
  • Food rotation (FIFO)
  • Preservatives
  • Refrigeration
  • Freezing
  • Canning
  • Fermentations

Signs of Spoilage

• Appearance changes (color, texture)
• Odor changes
• Taste changes
• Growth of mold/bacteria

Consequences of Spoilage

• Food poisoning (foodborne illness)

Test your knowledge on fermentation processes, significant contributions by Louis Pasteur, and the fundamentals of bioassays. This quiz covers important concepts related to both historical and scientific aspects of fermentation and its applications in biochemistry.