Microbiology: Enzyme and Bacterial Growth Quiz

### Substrate Inhibition

• Substrate binds to active site of the enzyme
• Inhibition increases with substrate concentration (above a threshold)

Product Inhibition

• The product of the reaction (the molecule that is made) binds to the enzyme's active site
• Blocks the active site and slows the reaction

Inhibition by Toxic Compound

• A toxic compound binds to an enzyme and disrupts its function

### Bacterial Cell Growth

• Bacteria grow in a variety of conditions
• Temperature, oxygen requirements, nitrogen sources, mineral content, and water quality all impact bacterial growth

### Temperature Requirements

• Bacteria can be classified based on their optimal growth temperature
• Psychrophiles prefer cold temperatures (-5°C to 15°C)
• Mesophiles grow best at moderate temperatures (25°C to 45°C)
• Thermophiles thrive in hot temperatures (45°C to 70°C)
• Hyperthermophiles grow in extremely hot conditions (70°C to 110°C)

### Oxygen Requirements

• Bacteria can be classified based on their need for oxygen
• Obligate aerobes require oxygen for growth
• Microaerophils need low oxygen concentrations
• Obligate anaerobes cannot grow in the presence of oxygen
• Aerotolerant anaerobes can tolerate but do not use oxygen
• Facultative anaerobes can grow with or without oxygen

Nitrogen Sources

• Inorganic Nitrogen includes sources:
  • Ammonia gas
  • Ammonium chloride
  • Ammonium sulphate
  • Ammonium nitrates
  • Sodium nitrates
• Using ammonium salts will result in pH drift towards acidity
• Using sodium nitrate results in pH drift towards alkalinity
• Organic Nitrogen
  • Supplied by amino acids, proteins, and urea
  • Results in faster growth
  • Commonly added as complex sources like:
    • Soybean meal
    • Corn steep liquor

### Water Quality

• Assessing Water Suitability:
  • Check pH level
  • Measure dissolved salts
  • Assess effluent contamination
• In Older Practices water mineral content was important
  • High calcium levels were important for making dark beer
  • High carbonate levels were important for making stouts.
• Modern Practices:
  • Deionization of water is common
  • Reuse of water can reduce water cost and effluent treatment expenses

### Elemental Composition of Bacteria

• Carbon (C) (50-53%) Key component of organic molecules
• Hydrogen (H) (7%) Vital for water, organic molecules, and energy production
• Nitrogen (N) (12-15%) Important for proteins, nucleic acids, and vitamins
• Phosphorus (P) (2-3%) Essential for DNA, RNA, ATP, and cell membranes
• Sulphur (S) (0.2-1.0%) Found in amino acids, vitamins, and enzymes
• Potassium (K) (1.0-4.5%) Important for enzyme function, carbohydrate metabolism, and osmotic balance
• Sodium (Na) (0.5-1.0%) Necessary for cell transport and osmotic balance
• Calcium (Ca) (0.01-1.1%) Found in cell walls and involved in various cellular processes
• Magnesium (Mg) (0.1-0.5%) Important for enzyme function and chlorophyll production
• Chloride (Cl) (0.5%) Involved in osmotic balance
• Iron (Fe) (0.02-0.2%) Crucial for electron transport in respiration

### Mineral Sources

• Microorganisms need minerals for growth, metabolism, and product formation
• Essential Minerals:
  • Magnesium
  • Phosphorus
  • Potassium
  • Sulphur
  • Calcium
  • Chlorine
• Trace Minerals:
  • Cobalt
  • Copper
  • Manganese
  • Iron
  • Molybdenum
  • Zinc

### Carbon Sources

• Carbon is used to metabolise ATP and other energy molecules
• Commonly Used:
  • Glucose
  • Sucrose
  • Galactose
  • Sorbitol
  • Maltose

### Organic Supplements

• Amino Acids
  • Provide nitrogen and support metabolism and biosynthesis of proteins, lipids, and nucleotides
  • Typical source is casine (milk protein) hydrolysates
• Vitamins
  • Important for metabolic processes
  • Vitamin B1 (thiamin)
  • Vitamin B6 (nicotinic acid pyridoxine)
  • Myo-inositol (not a vitamin but used for plant culture media)
• Activated Charcoal (AC)
  • Binds hydrophobic compounds that can inhibit growth
  • Used for detoxification
• Gelling Agents (Support Systems)
  • Solidified surface typically made from complex carbohydrates extracted from seaweed (agar)
  • Provides structure for microbial growth
  • Alternatives:
    • Gums from plants
    • Agarose

### Plant Culture Media

• Growth Regulators:
  • Auxin
  • Cytokinin
  • Gibberellins
  • Abscisic acid
  • Ethylene

### Auxin

• Promotes cell division and growth
• Most auxins are synthetic
• Naturally produced 1H-indol-3-acetic acid (unstable to heat and light)
• Produced in apical and root meristems, seeds, and developing fruit
• Alters target cell proton pumps and ATP production
• Induces cell elongation
• Suppresses lateral bud growth
• Stimulates adventitious roots
• Example: 2-4-dichorophenoxyacetic acid (2-4D) - used as a herbicide

### Cytokinins

• Promote cell division
• Produced in young leaves, fruits, and seeds
• Stimulate cell division
• Induce shoot formation
• Promote axillary shoot proliferation
• Inhibit root formation
• Delays cell aging
• Increases fruit blooming and growth
• Can increase fruit size when mixed with gibberellins
• Ratio of Auxin and cytokinin control root formation
  • More auxin than cytokinin: promotes root growth
  • More cytokinin than auxin: promotes shoot growth

### Gibberellins & Abscisic Acid

• Regulate cell elongation and determine plant height
• Gibberellins
  • Increase growth of low-density cultures
  • Enhance callus growth
  • Elongate dwarf plants
• Abscisic Acid
  • Alters callus growth
  • Enhances bud and shoot formation
  • Inhibits cell division
  • Commonly used in somatic embryogenesis

### Ethylene

• Volatile gas produced during ripening, stress, mechanical damage, or infection
• Produced from methyl group of methionine
• Almost all plant tissues produce ethylene
• Induces fruit ripening and flower blooming
• Commercially used to initiate flowering and ripen tomatoes, citrus, and bananas
• Specific protein receptors for ethylene act as transcription factors
• Can be a problem in culture without proper air circulation

### Design of Experiments

• Focus on identifying the most important factors that impact a process
• Experiment types:
  • Fractional Factorial Designs
  • Plackett-Burman
  • Box-Behnken

### Fractional Factorial Designs

• Help study multiple factors (k) in a limited number of runs (N)
• Assume high-order interactions are negligible
• Desirable properties:
  • Balanced (same number of observations in each run)
  • Orthogonal (effects of factors sum to zero across other factors)

### Plackett-Burman Designs

• Fractional factorial designs used to study k = N – 1 variables in N runs (where N is a multiple of 4)
• Focus on main effects only
• Standard orthogonal arrays
• No defining relation since interactions are not equal to main effects
• No degrees of freedom for calculating error term

When to Use Plackett-Burman Designs

• Screening experiments
• When higher-order interactions are negligible
• To efficiently identify large main effects

### Box-Behnken Designs

• Three-level multi-factor experiments
• Combine two-level factorial designs with incomplete block designs
• Complex confounding of interactions
• Efficient for detecting large main effects
• Can test linear and quadratic (non-linear) effects for each factor
• Rotatable

When to Use Box-Behnken Designs

• Three-level multi-factor experiments
• To detect large main effects when resources are limited
• To determine linear and quadratic effects of variables
• To avoid extreme factor combinations

### Number of Runs Required for Central Composite and Box-Behnken Designs:

• As the number of factors increases, the required number of runs increases