Industrial Biotechnology

Cheatsheet Content

### Effect of Temperature #### Effects on Growth - Higher temperatures speed up chemical reactions (approx. double rate for every 10°C). - Cells grow faster as temperature rises, up to an optimal point. - Too high temperatures cause **denaturation of proteins and nucleic acids**, leading to loss of critical enzymes and metabolism. #### Cardinal Temperatures Every organism is characterized by three temperatures: - **Minimum temperature:** Below this, no growth occurs. - **Optimum temperature:** Fastest growth occurs. - **Maximum temperature:** Above this, no growth occurs. #### Microbial Temperature Adaptations - **Psychrophiles:** Optimum temp. typically 15°C or lower. - **Psychrotolerant:** Optimum 20-40°C, but can grow as low as 0°C. - **Physiological adaptations:** Produce enzymes with lower temperature optima, often denature at room temp. Higher **unsaturated fatty acids** in membrane lipids keep membranes fluid at lower temperatures. - **Mesophiles:** Optima from 20-45°C, minimum around 15-20°C. - **Thermophiles:** Optima 55°C or higher. - **Hyperthermophiles:** Optima 80°C or higher (mostly Archaea), found in hot springs, deep-sea hydrothermal vents. - **Physiological adaptations:** Enzymes are heat stable, ribosomes work at higher temps. Membranes have many long-chain, **saturated fatty acids** which "freeze" at warm temperatures, but function well at very high temperatures. ### Effect of Oxygen #### Oxygen Requirement - Above a critical oxygen concentration, growth rate is independent of dissolved oxygen (DO). - **Bacteria/Yeast:** 5-10% oxygen. - **Molds:** 10-50% oxygen (pellet size dependent). #### Classification of Microbes by Oxygen Response - **Obligate aerobes:** Grow only when oxygen is present. - **Facultative anaerobes:** Grow with or without oxygen, but better with oxygen (respire). - **Aerotolerant anaerobes:** Ignore oxygen, grow equally well with or without. - **Obligate anaerobes:** Die in the presence of oxygen. - **Microaerophiles:** Won't grow at normal atmospheric oxygen (20%), but require some oxygen for growth (2-10%). #### Why Oxygen is Lethal for Obligate Anaerobes - Oxygen is a **reactive (oxidizing) agent** and can degrade organic molecules. - It can generate **toxic byproducts** (superoxide, peroxide, hydroxyl radical) which are strong oxidizing agents, reacting indiscriminately with organic molecules like DNA and proteins. - Aerobes (and all oxygen-tolerant cells) have enzymes like **Superoxide dismutase** and **Catalase** to neutralize these radicals. - A mutated *E. coli* (facultative anaerobe) that loses these enzymes behaves like an obligate anaerobe. #### Culture Techniques - **For aerobes:** Shake or rotate culture to add more oxygen, or bubble filtered air through culture. - **For anaerobes:** Use media with reducing agents, pump out air, flush with pure nitrogen gas, seal plates in jars, or use catalyst + hydrogen gas to remove oxygen. ### Effect of pH #### Influence on Microbial Growth - Hydrogen ion concentration affects enzyme activity, thus affecting microbial growth rate. - pH measures acidity: $pH = -\log_{10}[H^+]$. - Pure water has a pH of 7. #### Microbial pH Optima - **Acidophiles:** Acidic pH optimal (1 to 5.5). - **Neutrophiles:** pH 5.5 to 8 optimal. - **Alkaliphiles:** pH 8.5 to 11.5. - **Extreme alkaliphiles:** Optimum pH 10 or greater. - Most bacteria are neutrophiles (exceptions include some hot springs bacteria with optimum pH 1-3). Most fungi prefer slightly acidic conditions (pH 4-6). #### pH in Fermentation - Organisms maintain intracellular pH at a constant level. - Different organisms require different pH ranges. - Supply of CO2 can alter medium pH (e.g., in animal cell culture/seawater). - Selection of medium components affects pH profile during fermentation: - **Ammonium** as N source: consumption reduces pH. - **Nitrate** as N source: reduced to ammonium, increases pH. #### Optimal pH for Growth (Examples) | Organism | Optimal pH Range | |---------------|------------------| | Bacteria | 3 - 8 | | Yeast | 3 - 6 | | Molds | 3 - 7 | | Plant Cells | 5 - 6 | | Animal Cells | 6.5 - 7.5 | - pH can be controlled using buffers. ### Effect of Osmotic Pressure #### Water Availability (Osmotic Effects) - Cells require a certain amount of free water for metabolism. - In hypertonic environments (high solute concentration), many cells stop growing. - **Water activity** refers to the concentration of ions or solutes in available water affecting its utilization. - Some organisms can deal with high salt/solute concentrations and may even require them to survive. #### Classification by Osmotic Tolerance - **Halophiles:** Require salt for growth. - Optimal growth in 1-15% NaCl. - **Extreme Halophiles:** Require very high salt concentrations. - Optimal growth in 15-30% NaCl. - Example: *Halobacterium halobium* grows in the Dead Sea; won't grow if salt concentration is less than 3M. - **Halotolerant:** Can survive in higher salt concentrations but do not prefer it. - **Nonhalophiles:** Grow best without salt. Example: *Escherichia coli*. - Osmotic pressure of seawater is approximately 3% NaCl. ### Effect of Pressure - **Barophile:** Organisms that grow optimally at high hydrostatic pressure. - **Barotolerant:** Organisms that can tolerate high hydrostatic pressure but grow optimally at normal atmospheric pressure. - **Extreme Barophile:** Organisms that require very high pressure for growth. ### Effect of Radiation #### Types of Radiation - **Light and UV:** Parts of the electromagnetic (EM) spectrum. - Very strong radiation (gamma rays), very weak radiation (heat, radio). - **Visible light:** Especially energetic violet and blue light, can kill bacteria. - Pigmented bacteria (common in air) adsorb radiation, preventing cell damage. Pigment-less mutants are more sensitive to light. #### Mechanisms of Damage - **Light adsorption:** Pigments (e.g., cytochrome, flavin, chlorophyll) absorb light, transferring energy to oxygen to generate singlet oxygen (a strong oxidizing agent), causing damage. - **UV light:** Causes specific damage to DNA, with maximum effect at 260 nm, leading to **thymine dimers**. - **Ionizing radiation:** Causes many types of damage, including breaking H-bonds, oxidizing groups, and breaking DNA strands (most vulnerable target). ### Effect of Solutes - Cells require a certain amount of free water for metabolism. In hypertonic environments, many cells stop growing. - Some organisms can synthesize **compatible solutes** (molecules that balance osmotic strength) to compensate. - *Staphylococci* are halotolerant; they grow on skin (salty environment) and can tolerate up to 10% salt. Culture media can be designed with 7.5% salt to select for *Staph* by suppressing other bacteria. #### Cell Membrane Function The cell membrane acts as a major barrier governing differential passage of chemical components, with functions including: 1. Keeping essential nutrients and macromolecules inside the cell. 2. Pumping certain nutrients inside the cell **against** a concentration gradient. 3. Permitting free flow of nutrients across the membrane. 4. Excluding some solutes from the environment from entering the cell. ### Product Formation #### Growth-Associated Product Formation - Product expression occurs as a consequence of growth. - Product formation is linked to energy metabolism. - **Example:** Alcohol fermentation. Ethanol is produced as a byproduct when cells metabolize sugar for energy. #### Non-Growth Associated Product Formation - Product expression occurs as a product of secondary metabolism. - Product formation is **not** linked to energy metabolism. ### Preparing Media - **Media:** The feed solution for microbial growth. - Must contain all essential nutrients for microbe growth (e.g., cane molasses, beet molasses, cereal grains). #### Factors for Consideration - **Quality, consistency, and availability** of media components. - Ensure no problems with media preparation or other aspects of the production process. ### Sterilization - Sterilization is a **first-order process**. - $n = n_0 e^{-k_d t}$, where $n$ is number concentration of organisms, $k_d$ is death rate constant ($k_d = k_0 \exp(-E_a/RT)$). - High activation energy means $k_d$ rises much faster with temperature than the rate constant for protein denaturation. - **Pasteurization:** Sterilization by short exposure to very high temperature, preserving food taste. - Plug flow reactors are suitable for first-order sterilization processes. #### Methods of Sterilization 1. **Sterilization by Heat (Most widely used)** - **Dry heat:** Requires longer exposure, greater intensity. Used for glassware. - **Moist heat (Autoclaving):** Absolutely required for heat-stable liquid solutions. Performed under pressure, achieving higher temperatures to kill bacterial spores effectively. - **Batch sterilization:** Heating and cooling in a batch. - **Continuous sterilization:** Feed is heated (e.g., in a steam trim heater to 140°C), held in a "trombone" tube for residence time (e.g., 10 minutes), then cooled. Uses heat exchangers (e.g., shell & tube). 2. **Sterilization by Chemical Treatment** - For compounds not destroyed by heat/pressure. - Requires volatile and toxic sterilizing agents (e.g., ethylene oxide). 3. **Sterilization by Filtration** - For heat-labile solutions. - Passage through filters capable of retaining microorganisms (e.g., pore size 0.45 µm). ### Inoculum - **Inoculum:** The starter culture injected into the fermenter. - Must be of sufficient size and quality for optimal growth kinetics. - Industrial fermenters are large, so inoculum volume must be substantial. - A **seed fermenter** is typically used to produce the required inoculum volume. - The purpose of a seed fermenter is to prepare inoculum, not to produce the final product.