Microbial Nutrition Past Paper 2024 PDF

TH W R O G N D A N IA I O R I T TE T R C...

TH W R O G N D A N IA I O R I T TE T R C U B A N F O D Y O O D A N O I 4 F 2 0 2 LEARNING OUTCOMES Be able to; Describe the role of macronutrients in microbial nutrition. Describe the role of micronutrients and growth factors in microbial nutrition. Be able to describe the four nutritional categories of microorganisms. Describe the different types of culture media used in the laboratory and describe the main types of selective and differential media employed to grow bacteria. Be able to describe the physical and chemical growth requirements of bacteria. Be able to describe the methods used to exclude Oxygen when culturing anaerobic bacteria in the laboratory. Be able to describe the four stages of bacteria growth in batch culture. NUTRITION AND GROWTH OF BACTERIA  GENERAL MICROBIAL REQUIREMENTS FOR GROWTH Every microorganism has a minimum set of nutrient requirements for growth – referred to as Nutritional requirements/nutrient requirements. When these organisms are cultured in the laboratory the growth medium must meet the nutritional requirements of the particular m/organism. At an elemental level the cell’s requirements are reflected by its composition. Each of the elements plays a different role in the cell. Some elements are usually present as a part of organic molecules or macromolecules (e.g. carbon, nitrogen, phosphorous) while other are present as ions (e.g. magnesium, zinc). NUTRITIONAL REQUIREMENTS Nutrients are substances required by micro-organisms as raw materials for biosynthesis of their cellular macromolecules, for energy production (ATP production) and for growth. Essential nutrients are those that they absolutely require and cannot make themselves. These include the following: TWO categories of nutrients 1. Macroelements: Usually the products of living things. Containing C and H. Carbohydrates, proteins, fats, nucleic acids. Needed in large quantities. Essential role as components in cell structure and metabolism. C, H, N, O, P, S and K, Mg, Fe and Ca. For example: Nitrogen required to synthesise nucleotides. C to synthesise organic molecules. 2. Micronutrients : (Inorganic nutrients: trace elements). (But just as important as macronutrients). 3. Involved in enzyme function (Micronutrients play a structural role in various enzymes, the cells catalysts) and maintenance of protein structure. – Mn, Zn, Co, Mo, Ni, and Cu. a). Required in trace amounts as an essential component of certain enzymes (protein) as an inorganic cofactors, Macromolecule Functions Energy storage, receptors, food, structural Carbohydrates role in plants, fungal cell walls, exoskeletons of insects Energy storage, membrane structure, Lipids insulation, hormones, pigments Nucleic acids Storage and transfer of genetic information Enzymes, structure, receptors, transport, Proteins structural role in the cytoskeleton of a cell and the extracellular matrix BuildingBlocks Macromolecules Cellular Structure Fatty acids Lipid Envelope Lipopolysaccharide Envelope Sugars Glycogen Inclusions Peptidoglycan Envelope Amino acids Protein Envelope Flagella Pili Cytosol Polyribosomes Nucleotides RNA Cytosol Polyribosomes DNA Nucleoid NUTRITIONAL REQUIREMENTS Carbohydrates Carbohydrates are polymers of carbon, hydrogen and oxygen. They can be classified as monosaccharides, disaccharides and polysaccharides Nucleic Acids The nucleic acids include DNA and RNA that are the polymers of nucleotides. Nucleotides comprise a pentose group, a phosphate group, and a nitrogenous base group. All the hereditary information is stored in the DNA. The DNA synthesised into RNA and proteins. Proteins Proteins are the polymers of amino acids. These include the carboxylic and the amino group. Lipids Lipids are a hydrophobic set of macromolecules, i.e., they do not dissolve in water. These involve triglycerides, carotenoids, phospholipids, and steroids. They help in the formation of the cell membrane, formation of hormones and in the and as stored fuel.  MACROMOLECULES Most macromolecules are made from single subunits, or building blocks, called monomers. The monomers combine with each other using covalent bonds to form larger molecules known as polymers. In doing so, monomers release water molecules as byproducts. This type of reaction is known as dehydration synthesis, which means “to put together while losing water.” Polymers are broken down into monomers in a process known as hydrolysis, which means “to split water,” a reaction in which a water molecule is used during the breakdown Dehydration and hydrolysis reactions are catalyzed, or “sped up,” by specific enzymes; dehydration reactions involve the formation of new bonds, requiring energy, while hydrolysis reactions break bonds and release energy. Each macromolecule is broken down by a specific enzyme. For instance, carbohydrates are broken down by amylase, sucrase, lactase, or maltase. Proteins are broken down by the enzymes pepsin and peptidase, and by hydrochloric acid. Lipids are broken down by lipases. Breakdown of these macromolecules provides energy for cellular activities. ROLE OF CARBON The carbon atom has unique properties that allow it to form covalent bonds to as many as four different atoms, making this versatile element ideal to serve as the basic structural component, or “backbone,” of the macromolecules. Carbon atoms can form up to four covalent bonds with other atoms to satisfy the octet rule. The methane molecule provides an example: it has the chemical formula CH4. Each of its four hydrogen atoms forms a single covalent bond with the carbon. ROLE OF CARBON Organic molecules contain carbon; inorganic compounds do not Hydrocarbons can exist as chains or rings (Purines, Pyrimidines). MACROMOLECULES Nitrogen is a crucially important component for all life. It is an important part of many cells and processes such as amino acids, proteins and even our DNA. It is also needed to make chlorophyll in plants, which is used in photosynthesis to make their food. Phosphorus, like nitrogen, is a critical nutrient required for all life. The most common form of phosphorus used by biological organisms is phosphate (PO4), which plays major roles in the formation of DNA, cellular energy, and cell membranes (and plant cell walls) Without nitrogen and phosphorus, there would be no DNA, no energy to power our cells, no proteins MACROMOLECULES Sulfur is a structural component of protein disulfide bonds, amino acids, vitamins, and cofactors potassium is needed for enzymes Potassium ion homeostasis is essential for bacterial survival, playing roles in osmoregulation, pH homeostasis, regulation of protein synthesis, enzyme activation, membrane potential adjustment and electrical signaling magnesium is used to stabilize ribosomes and membrane association of the small and large ribosomal subunits to form intact ribosomes depends strongly on Mg2+ ion concentration, Mg++ ions act as co-factors for joining two subunits of ribosomes Magnesium helps to maintain the ribosome structure NUTRITIONAL REQUIREMENTS Iron is an important micronutrient for virtually all living organisms except lactic acid bacteria where manganese and cobalt are used in place of iron. Siderophilic bacteria are bacteria that require or are facilitated by free iron. They may include Listeria monocytogenes, Salmonella enterica, Klebsiella pneumoniae and Escherichia coli. calcium ions are involved in the maintenance of cell structure, motility, transport and cell differentiation processes such as sporulation Up to 20% of the dry weight of the endospore consists of calcium dipicolinate within the core, which is thought to stabilize the DNA. Dipicolinic acid could be responsible for the heat resistance of the spore, and calcium may aid in resistance to heat and oxidizing agents. NUTRITIONAL REQUIREMENTS GROWTH FACTORS Most of the organisms are capable of producing enzymes required for biochemical pathways by the presence of nutrients; however, there are several organisms that lack specific enzymes required by the microbes. Therefore, they must obtain these constituents or their precursors from the environment. Organic compounds that are essential cell components or precursors of such components but cannot be synthesized by the organism are called growth factors Some bacteria like E. coli can make all their cell constituents from the macro and micronutrients Others like Borrelia burgdorferi require organic supplements (nutrients) called Growth factors. Some bacteria are fastidious: Only grow if CERTAIN nutrients are added to the growth medium. These bacteria require GROWTH FACTORS in addition to the Macronutrients and micronutrients. Growth factors are organic compounds that are essential cell components that the cell cannot synthesise and must be added to growth medias. Ex: Lactobacillus species require vitamins added to their media. These compounds are not used by the bacteria for energy or carbon but instead are assimilated by cells for a very specific role in cell metabolism. Bacteria that CAN synthesise growth factors are used in industrial fermentations to make large quantities of growth factors for human use. Examples: Vit B12 by Streptomyces, Vitamin C by Corynebacteria. GROWTH FACTORS a. Organic compounds. b. Essential cell components that the cell cannot synthesise. c. Must be supplied by environment if cell is to survive and reproduce. Types of growth factors. 1. Amino acids – needed for protein synthesis. 2. Purines and pyrimidines – needed for nucleic acid synthesis. 3. Vitamins (organic cofactors): Non protein organic molecules that bind loosely to enzymes. Vitamins function as coenzymes, organic molecules that help enzyme bind to substrate. EXAMPLE: Streptococcus pyogenes requires the amino acid alanine in it’s laboratory medium. NUTRITIONAL REQURIEMENTS A cofactor is a non-protein chemical compound or metallic ion that is required for an enzyme's role as a catalyst (a catalyst is a substance that increases the rate of a chemical reaction). Cofactors can be considered "helper molecules" that assist in biochemical transformations. Cofactors can be divided into two major groups: 1. organic cofactors (coenzymes), such as flavin or heme; 2. inorganic cofactors, such as the metal ions Mg2+, Cu+, Mn2+ and iron–sulfur clusters Organic cofactors are small organic molecules that can be either loosely or tightly bound to the enzyme and directly participate in the reaction Some cofactors are permanently attached, these are referred to as Prosthetic cofactors. Other cofactors are only temporarily attached ENZYME-SUBSTRATE REACTION COENZYMES FAD- (flavin adenine dinucleotide) NAD (nicotinamide adenine dinucleotide COFACTORS inorganic cofactors, such as the metal ions Mg2+, Cu+, Mn2+ and iron–sulfur clusters, directly participate in catalysis. DNA polymerase, structure is negatively charged with phosphate groups, Mg2+ binds to the negative charge of the phosphate groups, stabilising the charge and structure. Ion Enzyme containing this Ion Magnesium DNA Polymerase Zinc DNA Polymerase COMPETITIVE, NON COMPETITIVE ENZYME INHIBITION Competitive Inhibition: Inhibitor and enzyme are competing for the same active site on the enzyme If inhibitor binds to active site, substrate cannot bind, enzyme is Lovastatin is a potent competitive inactive inhibitor of the rate-limiting enzyme of cholesterol synthesis, 3-hydroxy-3- methylglutaryl-coenzyme A reductase. Non competitive inhibition Inhibitor binds to a different site of enzyme, not competing with substrate, if inhibitor binds to a different site of enzyme it alters the shape of the active site, substrate cannot bind, enzyme inactive. Non-competitive inhibition cannot be reversed by increasing the substrate concentration. Examples of non-competitive inhibitors include cyanide, mercury and silver Cyanide interacts with over 40 metalloenzymes, but its lethal action is non-competitive inhibition of cytochrome c oxidase, halting cellular respiration and causing hypoxic anoxia  ROLE OF TRACE ELEMENTS- ENZYME 1. FUNCTION Cofactors and Coenzymes: Cofactors: Inorganic ions or small molecules that bind to enzymes and are necessary for their activity. Examples include zinc (Zn), copper (Cu), iron (Fe), and manganese (Mn). Coenzymes: Organic molecules, often derived from vitamins, that assist enzymes in carrying out catalysis. Examples include NAD+ (nicotinamide adenine dinucleotide), FAD (flavin adenine dinucleotide), and coenzyme Q. 2. Catalysis and Reaction Facilitation: Many enzymes require metal ions as cofactors to catalyse specific reactions. For instance, zinc is essential for the activity of various metalloenzymes involved in DNA repair, protein digestion, and immune function. 3. Stabilization of Enzyme Structure: Certain trace elements help stabilize the three-dimensional structure of enzymes, ensuring their proper folding and functionality. For instance, disulfide bonds formed by sulfur-containing amino acids contribute to the stability of some enzymes. 4. Electron Transfer: Trace elements such as iron and copper play critical roles in electron transfer reactions. They are involved in redox reactions, where electrons are transferred between molecules during metabolic processes. Enzymes with iron- sulfur clusters or copper centers participate in these electron transfer reactions.  ROLE OF TRACE ELEMENTS- ENZYME FUNCTION 5. Oxygen Binding and Transport: Iron is a crucial component of heme groups found in hemoglobin and myoglobin. These proteins are involved in binding and transporting oxygen in the blood and tissues. Copper is also involved in oxygen transport in some organisms. 6. Maintenance of pH and Ionic Balance: Trace elements help maintain the proper pH and ionic balance in the active site of enzymes, creating an optimal environment for catalysis. For example, zinc is involved in regulating the pH of certain enzymes. 7. DNA Synthesis and Repair: Trace elements like zinc and manganese are essential for enzymes involved in DNA synthesis and repair processes. They contribute to the stability and fidelity of these crucial cellular functions. NUTRITIONAL REQUIREMENTS NUTRITIONAL CATEGORIES OF MICROORGANISMS Questions we ask with regard to microbial nutrition is 1. where do they get their Carbon from, to make biological molecules in the cell 2. where do they get their energy from 3. Where do they get their electrons from  AUTOTROPHIC BACTERIA Autotrophic bacteria are organisms that can synthesize organic molecules, including carbohydrates, from inorganic sources of carbon. They use carbon dioxide (CO2) as their primary carbon source for the synthesis of organic compounds. Autotrophic bacteria obtain energy through various means, such as sunlight (photoautotrophs) or inorganic chemical reactions (chemoautotrophs). Examples: Photoautotrophs: Cyanobacteria, purple sulfur bacteria, and green sulfur bacteria use sunlight for photosynthesis. Chemoautotrophs: Hydrogen bacteria, nitrifying bacteria, and sulfur bacteria obtain energy from inorganic chemical reactions. Role in Ecosystems: Photoautotrophic bacteria contribute to oxygen production through photosynthesis, playing a vital role in the global carbon cycle.  HETEROTROPHIC BACTERIA Heterotrophic bacteria are organisms that cannot synthesize organic compounds from inorganic sources and, therefore, depend on organic compounds produced by other organisms. They utilize organic carbon compounds (such as carbohydrates, proteins, and lipids) as their carbon source. Heterotrophic bacteria obtain energy by breaking down complex organic molecules through processes like fermentation, respiration, or other metabolic pathways. Examples: Many common bacteria fall into the heterotrophic category, including most pathogenic bacteria, decomposers, and symbiotic bacteria. Role in Ecosystems: Heterotrophic bacteria play crucial roles in nutrient cycling and decomposition. They break down complex organic matter into simpler compounds, contributing to the recycling of nutrients in ecosystems. BACTERIAL DIVERSITY 3 main categories of bacterial classification based on nutritional requiremetns Autotrophs 1. Carbon source Auto Self Heterotrophs Hetero: Other Photo: Light Phototrophs Chemo: Chemical (organic/inorganic) Litho: stone (rock i.e. minerals) 2. Energy source Organo : organic compound Chemotrophs Lithotrophs 3. Electron source Organotrophs BACTERIA DIVERSITY BACTERIAL DIVERSITY Photoautotrophs Photosynthetic Capture light from light rays and transform it to chemical energy Produce organic molecules using CO2 Chemoautotrophs 2 types 1. chemo organic autotrophs: use organic compound for energy use inorganic compound for carbon source 2. lithoautotrophs: rely on inorganic minerals require neither sunlight or organic nutrients unique methods of getting energy (remove electrons from inorganic substrates like H2, Fe, H2S) BACTERIAL DIVERSITY Chemoheterotrophs Derive carbon and energy from organic compounds Get energy from molecules through processes respiration fermentation 2 types 1. Saprobes: free living organisms that feed on dead organisms Act as decomposers of dead organisms and recycle organic nutrients 2. Parasites: derive nutrients from the cells and tissues of a living host, can either be Ectoparasites: live on the body Endoparasites: live in the body in organs or tissues LITHOTROPHS V ORGANOTROPHS Classified based on their electron donor 1. Lithotroph: use reduced inorganic substances as their electon donor e.g. H, Ammonia, Iron, sulfur 2. Organotrophs: extract electrons from reduced organic compounds What is carbon, energy and electron source of : Photolithotrophic Autotrophs: Chemo organotrophic heterotrophs NUTRITIONAL CLASSIFICATION Photolithotrophic Autotrophs: Carbon from CO2 Energy from sunlight Electron from reduced inorganic molecules Chemo organotrophic heterotroph Carbon from reduced organic molecules Energy from chemical reactions Electron from organic molecules  Physical/Environmental Requirements for Microbial Growth Prokaryotes exist in nature under an enormous range of physical conditions such as 1. O2 concentration, *Different levels of O2 in different environments. 2. Hydrogen ion concentration (pH)* 3. Temperature* 4. Water availability. (Osmotic pressure). 5. Barometric pressure.* Hydrostatic pressure-a physical parameter. Pressure exerted by a column of water. Barophiles 400-1035 atm. (land 1 atm) 1. OXYGEN Microorganisms display a wide range of different responses to molecular oxygen (O2). Microorganismsm can be categorised based on their tolerance to oxygen. Oxygen is not toxic but can be converted into highly toxic substances which can be toxic to some bacteria. Inside the cell oxygen can form free radicals like: 1. Superoxide (O3-) 2. Hydrogen peroxide (H2O2) 3. Hydroxyl radical (OH-) To survive in an oxygen environment cells must have the enzymes to convert toxic oxygen by products to a non toxic form. These by-products can damage protein and DNA, killing bacteria. Radicals (often referred to as free radicals) are atoms, molecules, or ions with unpaired electrons or an open shell configuration. Free radicals may have positive, negative, or zero charge. With some exceptions, the unpaired electrons cause radicals to be highly chemically reactive. OXYGEN 3 main enzymes involved in converting toxic to non toxic oxygen by products 1. Superoxide dismutase 2. Catalase 3. Peroxidase Superoxide dismutase degrades superoxide to produce hydrogen peroxide O2- H2O2 Catalase converts hydrogen peroxide to water and oxygen H2O2 H2O + O2 OXYGEN Classification based on oxygen requirements/tolerence 1. Obligate anaerobes 2. Facultative anaerobes 3. Obligatory aerobes 4. Microaerophilic organisms 5. Capnophilic organisms  OXYGEN REQUIREMENTS 1. Obligate anaerobes (strict anaerobes) Cannot grow in presence of oxygen (killed by traces of oxygen because of its toxic derivitives) They carry out fermentation or anaerobic respiration Lack enzymes requried to convert toxic radicals into non toxic form Example: Clostridium botulinun 2. Facultative anaerobes (facultative means flexible) Can grow under aerobic and anaerobic conditions Grow best aerobically but can grow anaerobically In the presence of oxygen, they use aerobic respiration; in its absence, they switch to fermentation or anaerobic respiration. Example: E. coli, Salmonella, S. aureus 3. Aerotolerant anaerobes Exclusively anaerobic (fermentative) type of metabolism but are insensitive to the presence of oxygen. Have superoxide dismutase but no catalase) Example Streptococcus pyogenes OXYGEN REQUIREMENTS 4. Obligatory aerobes (strict aerobes) Mandatory requirement for oxygen Use it to transform energy in process of respiration Have enzymes required to convert toxic to non toxic by products Aerobic bacteria thrive in oxygenated environments and utilise oxygen for their metabolic energy production process, this process is known as cellular respiration. - a metabolic pathway that oxidises nutrients, like sugars, to produce energy. Example: Pseudomonas, Mycobacterium tuberculosis 5. Microaerophilic Can grow under conditions with low oxygen Require small amounts of oxygen (2-10%), high concentration are inhibitory Have small amount of enzyme Example: H. pylori Growth patterns in these tubes can give a lot of information about the metabolism of the bacteria. How?. GROWTH PATTERNS Tube A: all the growth is seen at the top of the tube. The bacteria are obligate (strict) aerobes that cannot grow without an abundant supply of oxygen Tube B: the opposite of tube A. Bacteria grow at the bottom of tube B. Those are obligate anaerobes, which are killed by oxygen. Tube C: heavy growth at the top of the tube and growth throughout the tube, a typical result with facultative anaerobes. Facultative anaerobes are organisms that thrive in the presence of oxygen but also grow in its absence by relying on fermentation or anaerobic respiration, if there is a suitable electron acceptor other than oxygen and the organism is able to perform anaerobic respiration. Tube D:indifferent to the presence of oxygen. They do not use oxygen because they usually have a fermentative metabolism, but they are not harmed by the presence of oxygen as obligate anaerobes are. Tube E: The oxygen level has to be just right for growth, not too much and not too little. These microaerophiles are bacteria that require a minimum level of oxygen for growth, about 1%–10%, well below the 21% found in the atmosphere. EXCLUSION OF OXYGEN Obviously in growing certain cultures of bacteria O2 must be excluded. Exclusion of O2: Anaerobic culture. Special methods are used. 1. Bottles or tubes filled completely to the top with culture medium, provided with tightly fitting stoppers are sufficient to provide anoxic (without oxygen) conditions for organisms that are not too sensitive to small amounts of oxygen EXCLUSION OF OXYGEN EXCLUSION OF OXYGEN  EXCLUSION OF OXYGEN To remove all traces of oxygen in order to grow obligate anaerobes, place an O2 consuming gas in a jar holding the tubes or plates. The air in the jar is replaced with a mixture of H2 and CO2, and in the presence of a chemical catalyst the traces of oxygen left in the vessel is consumed. EXCLUSION OF OXYGEN Components of Gas jar: In the presence of water, chemicals present inside the sachet i.e. sodium bicarbonate (NaHCO3) and sodium borohydride (NaBH4) react chemically producing hydrogen and carbon dioxide gas. The hydrogen thus produced reacts with oxygen present inside the jar producing water (which forms as condensation on the inside of the jar). 2H2 + O₂ + catalyst = 2H2 This reaction is catalyzed by the element palladium, which is attached to the underside of the lid of the jar. The carbon dioxide replaces the removed oxygen, creating a completely anaerobic environment Anaerobic indicator strips: Impregnated with methylene blue: remains colorless in anaerobic conditions, but turns blue on exposure to oxygen. EXCLUSION OF OXYGEN DIFFERENTIATION TESTS This test demonstrate the presence of catalase, an enzyme that catalyses the release of oxygen from hydrogen peroxide (H2O2). It is used to differentiate those bacteria that produces an enzyme catalase, such as staphylococci, from non-catalase producing bacteria such as streptococci. CATALASE TEST The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and water. The presence of the enzyme in a bacterial isolate is evident when a small inoculum is introduced into hydrogen peroxide, and the rapid elaboration of oxygen bubbles occurs. The lack of catalase is evident by a lack of or weak bubble production. The culture should not be more than 24 hours old. Bacteria thereby protect themselves from the lethal effect of Hydrogen peroxide which is accumulated as an end product of aerobic The morphologically carbohydrate similar Enterococcusor Streptococcus (catalase metabolism. negative) and Staphylococcus (catalase positive) can be differentiated using the catalase test, both gram positive cocci. It is used to differentiate aerotolerant strains of Clostridium, which are catalase negative, from Bacillus species, which are positive.  CAPNOPHILES Capnophiles Those organisms that grow best at higher than normal levels of CO2, rather than less O2. Unlike many organisms that are harmed by increased concentrations of carbon dioxide, capnophiles have adapted to and actually require higher levels of CO2 for optimal growth. These bacteria are often encountered in the human body, particularly in the respiratory and gastrointestinal tracts, where conditions can be rich in carbon dioxide. Examples Neisseria, Brucella, Streptococcus pneumonia, Haemophilus influenzae. Table 1: Aerobic bacteria vs Anaerobic bacteria Aerobic bacteria Anaerobic bacteria Define aerobic bacteria: Define anaerobic bacteria: Aerobic bacteria are bacteria that thrive and Anaerobic bacteria are bacteria that thrive and grow in an aerobic environment (with oxygen). grow in the absence of oxygen. Such bacteria A single bacterium that needs oxygen for can not tolerate the presence of oxygen in their survival is referred to as an aerobic bacterium. environment Bacterial cellular respiration – Aerobically Bacterial cellular respiration – Anaerobically Molecular oxygen serves as the terminal Varies; carbon dioxide, sulfur, ferric, nitrate, or electron acceptor fumarate may serve as an electron acceptor Survive only in the presence of molecular Anaerobes cannot survive in the presence of oxygen molecular oxygen. In addition, molecular oxygen is toxic for such bacteria. Such bacteria possess catalase, peroxidase, and Nitrate, acetate, methane, and sulfide, like superoxide dismutase enzymes to neutralize products, are generated. the reactive oxygen species generated due to aerobic respiration The amount of energy produced by aerobes is The amount of energy produced by anaerobes greater than in anaerobes could be less than in aerobes Aerobes can be found in different areas like Anaerobes are located in regions that are water, soil, etc. deficient in oxygen or oxygen-depleted areas. Aerobes get localized at the surface of the liquid Aerobes localize themselves at the bottom of growth medium (Fig 1) the liquid growth medium (Fig 1) Lactobacillus, Nocardia, Mycobacterium Clostridium, Bacteroides, etc. are examples of tuberculosis, etc. are examples of aerobic anaerobic bacteria bacteria 2. pH potential of hydrogen( measure of the concentration of hydrogen ions in a substance) pH Extreme pH affects the structure of all macromolecules. The hydrogen bonds holding together strands of DNA break up at high pH. Lipids are hydrolyzed by an extremely basic pH. The proton motive force responsible for production of ATP in cellular respiration depends on the concentration gradient of H+ across the plasma membrane. If H+ ions are neutralized by hydroxide ions, the concentration gradient collapses and impairs energy production. The component most sensitive to pH in the cell is its workhorse, the protein. Moderate changes in pH modify the ionization of amino-acid functional groups and disrupt hydrogen bonding, which, in turn, promotes changes in the folding of the molecule, promoting denaturation and destroying activity.  EFFECT OF PH 1.. Protein Function and Enzyme Activity: pH influences the ionization state of amino acid side chains in proteins. Changes in pH can affect the structure and function of proteins, including enzymes that play crucial roles in bacterial metabolism. Most enzymes have an optimal pH at which they exhibit maximum activity. 2. Cell Membrane Integrity: The bacterial cell membrane is sensitive to changes in pH. Extreme pH conditions can disrupt the integrity of the cell membrane, leading to changes in membrane permeability and potential loss of cellular components. 3. DNA Stability: pH can influence the stability of DNA molecules. Extreme pH levels can lead to denaturation or changes in the secondary structure of DNA. This can affect processes such as DNA replication and transcription. 4. Acid Stress and Alkaline Stress: Bacteria that encounter environments with pH levels outside their optimal range may experience stress. Acid stress occurs in acidic environments, while alkaline stress occurs in alkaline environments. Bacteria have evolved mechanisms to cope with these stresses, such as proton pumps and pH-regulating systems.  EFFECT OF PH 4. Nutrient Availability: The solubility and availability of nutrients in the environment are pH- dependent. Some nutrients may become less soluble or more available under certain pH conditions, impacting bacterial growth. 5. Microbial Communities and Ecosystems: pH is a key factor shaping microbial communities in various ecosystems. Soil pH, for example, affects the types of bacteria that dominate in a particular habitat, influencing nutrient cycling and ecosystem functions. 6. Human Health and Disease: pH plays a role in bacterial infections within the human body. Different body sites have varying pH levels, and pathogenic bacteria may exploit or be hindered by these conditions. For example, the stomach's acidic pH serves as a defense against ingested pathogens.  pH The optimum growth pH is the most favorable pH for the growth of an organism. The lowest pH value that an organism can tolerate is called the minimum growth pH and the highest pH is the maximum growth pH. These values can cover a wide range, which is important for the preservation of food and to microorganisms’ survival in the stomach. For example, the optimum growth pH of Salmonella spp. is 7.0–7.5, but the minimum growth pH is closer to 4.2. Most bacteria are neutrophiles, meaning they grow optimally at a pH within one or two pH units of the neutral pH of 7. Most familiar bacteria, like Escherichia coli, staphylococci, and Salmonella spp. are neutrophiles and do not fare well in the acidic pH of the stomach. However, there are pathogenic strains of E. coli, S. typhi, and other species of intestinal pathogens that are much more resistant to stomach acid. In comparison, fungi thrive at slightly acidic pH values of 5.0–6.0. pH Microorganisms that grow optimally at pH less than 5.55 are called acidophiles. For example, the sulfur-oxidizing Sulfolobus spp. isolated from sulfur mud fields and hot springs in Yellowstone National Park are extreme acidophiles. These archaea survive at pH values of 2.5–3.5. Lactobacillus bacteria, which are an important part of the normal microbiota of the vagina, can tolerate acidic environments at pH values 3.5–6.8 and also contribute to the acidity of the vagina At the other end of the spectrum are alkaliphiles, microorganisms that grow best at pH between 8.0 and 10.5. Vibrio cholerae, the pathogenic agent of cholera, grows best at the slightly basic pH of 8.0; it can survive pH values of 11.0 but is inactivated by the acid of the stomach. pH ROLE OF pH Acidic foods have been a mainstay of the human diet for centuries, partly because most microbes that cause food spoilage grow best at a near neutral pH and do not tolerate acidity well 2. TEMPERATURE Microbes can be roughly classified according to the range of temperature at which they can grow. The growth rates are the highest at the optimum growth temperature for the organism. The lowest temperature at which the organism can survive and replicate is its minimum growth temperature. The highest temperature at which growth can occur is its maximum growth temperature. Classified into the following groups: 1. Psychrophiles (cold-loving microorganisms). 2. Psychrotrophs: Grow slowly in the cold. 3. Mesophiles (moderate-temperature-loving bacteria) 4. Thermophiles (heat-loving microbes) 5. 5.Hyperthermophile/extreme thermophiles/obligate thermophiles TEMPERATURE Unlike other environmental conditions such as pH or osmolarity, microbes have no way to regulate their temperature: their internal temperature matches that of their environment. Changes in temperature have the biggest effect on enzymes and their activity, with an optimal temperature that leads to the fastest metabolism and resulting growth rate TEMPERATURE Life in extreme environments raises fascinating questions about the adaptation of macromolecules and metabolic processes. Very low temperatures affect cells in many ways. Membranes lose their fluidity and are damaged by ice crystal formation. Chemical reactions and diffusion slow considerably. Proteins become too rigid to catalyse At the opposite end of the temperature spectrum heat denatures proteins and nucleic acids. Increased fluidity impairs metabolic processes in membranes. TEMPERATURE 1. Psychrophiles (cold-loving microorganisms). Found in the depths of the oceans, in ice and snow and in the arctic regions, have an optimum growth temperature of 15°C or lower. psychrophiles produce enzymes with lower temperature optima. They often denature at room temperatures. psychrophiles have higher unsaturated fatty acids in membrane lipids, keeps membranes fluid at lower temperatures. Produce anti freeze proteins which protect the DNA and prevent ice formation. Examples: Pseudomonas, Flavobacterium  TEMPERATURE 2. Psychrotrophs: Grow slowly in the cold. Play a major role in the spoilage of refrigerated food –grow well between 0 to 7oC. The human pathogen Listeria monocytogenes is an example. It grows in the guts of cattle, can contaminate beef, milk and crops but unlike typical mesophilic human pathogens, it grows at refrigerated temperatures. Food-borne infections results from the consumption of ready-to-eat foods, including lettuce, unpasteurized cheeses and cold cuts. Because they are active at low temperature, psychrophiles and psychrotrophs are important decomposers in cold climates. While Listeria monocytogenes is psychrotrophic, it is important to note that it can also grow at higher temperatures, including those found in the human body. This adaptability to a wide range of temperatures contributes to its ability to cause foodborne illnesses. Therefore, proper food handling, storage, and hygiene practices are essential to prevent the growth and transmission of Listeria monocytogenes in food products. TEMPERATURE 3. Mesophiles (moderate-temperature-loving bacteria) found in water, soil and in higher organisms. Optimum growth temperature 37oC. Some notable mesophiles include, Staphylococcus aureus, and Escherichia coli 4. Thermophiles (heat-loving microbes) are capable of growth at high temperatures with an optimum 50°C. Bacillus geostearothermophilus. Membranes high in long chained fatty acids, heat stable proteins with more H bonds and other bonds to strengthen their structure. The enzymes in thermophiles function at high temperatures. Some of these enzymes are used in molecular biology, for example the Taq polymerase used in PCR. Note of interest Thermophiles can be discriminated from mesophiles from genomic features. For example, the GC-content levels in the coding regions of some signature genes were consistently identified as correlated with the temperature range, more GC nucleotides, higher temperatures tolerated.  TEMPERTATURE Thermoduric -can survive exposure to high temperatures for a short time such as those used in pasteurization processes–but normally mesophilic. E. coli. In the context of food safety and industry, the ability of E. coli to resist heat treatments is a concern, and it underscores the importance of proper food processing techniques to eliminate or reduce bacterial contamination. Adequate cooking and pasteurization are effective methods for controlling E. coli and other pathogenic bacteria in food products. 5.Hyperthermophile/extreme thermophiles/obligate thermophiles have optima of 80 degrees or higher (mostly Archaea in this group). Thermus, Sulfolobus. Found in hot springs, deep-sea hydrothermal vents, other locations. Stability Have fatty acids with side chains and DNA with a high degree of G: C base pairs -raising the Melting Point.  TEMPERATURE Thermophiles can be classified in various ways. One classification sorts these organisms according to their optimal growth temperatures: 1. Simple thermophiles: 50–64 °C 2. Extreme thermophiles 65–79 °C Examples include bacteria of the genera Thermus and Aquifex. 3. Hyperthermophiles 80 °C and beyond, but not < 50 °C. example include bacteria belonging to the genera Thermococcus. In a related classification, thermophiles are sorted as follows: 4. Facultative thermophiles (also called moderate thermophiles) can thrive at high temperatures, but also at lower temperatures (below 50 °C), whereas 5. Obligate thermophiles (also called extreme thermophiles) require such high temperatures for growth. 6. Hyperthermophiles are particularly extreme thermophiles for which the optimal temperatures are above 80 °C. OSMOTIC EFFECTS Hypotonic Environments: In a hypotonic environment (lower solute concentration outside the bacterial cell compared to inside), water tends to move into the cell, leading to cell swelling and potential lysis (bursting). Bacteria have evolved mechanisms to cope with this, such as the synthesis of compatible solutes or osmoregulatory systems that actively pump out excess water to maintain cell integrity. Isotonic Environments: An isotonic environment has an equal concentration of solutes inside and outside the cell. Bacteria typically grow well in isotonic conditions, as there is no net movement of water into or out of the cell. Nutrient uptake and waste elimination can occur efficiently, supporting bacterial metabolism and growth. Hypertonic Environments: In a hypertonic environment (higher solute concentration outside the bacterial cell), water tends to move out of the cell, leading to dehydration and shrinkage. Bacteria in hypertonic environments may face challenges in obtaining sufficient water for metabolic processes. Some bacteria can counteract this by accumulating compatible solutes or increasing the internal osmotic pressure to prevent excessive water loss.  OSMOTIC EFFECTS ON MICROBIAL GROWTH Osmotic Pressure The force that drives the water diffusing across cell membranes in response to solute concentration. Microbes contain approximately 80-90% water and if placed in a solution with a higher solute concentration will lose water which causes shrinkage of the cell (plasmolysis). Some bacteria can adjust to hypertonic solutions by increasing their cytoplasmic solute concentration of amino acids, sodium ions and potassium ions. In a hypotonic environment some bacteria can adjust/reduce their solute concentration by storing cytoplasmic solutes in inclusion bodies. Osmoprotectants or compatible solutes, such as glycine betaine and proline, are synthesized or accumulated by bacteria to maintain osmotic balance. These compounds help stabilize cellular structures and enzymes, preventing damage caused by changes in osmotic pressure. OSMOTIC EFFECTS Water diffuses by the process of osmosis from an area of high water concentration to an area of low water concentration. In most cases, the cytoplasm of a cell has a higher solute concentration than the environment so water tends to diffuse into the cell and the cell is said to be in positive water balance. When a cell is in an environment of low water activity, there is a tendancy for water to flow out of the cell. In nature, osmotic effects are of interest mainly in habitats with high salt concentration. The only common solute in nature that occurs over a wide concentration range is salt [NaCl], and some microorganisms are named based on their growth response to salt. Microorganisms that require some NaCl for growth are halophiles. Mild halophiles require 1-6% salt, moderate halophiles require 6-15% salt; extreme halophiles that require 15-30% NaCl for growth are found among the Archaea and are found in salterns or in areas such as the Dead Sea. OSMOTIC EFFECTS Bacteria that are able to grow at moderate salt concentrations, even though they grow best in the absence of NaCl, are called halotolerant. Halophile V Osmophile Halophile" and "osmophile" are terms used to describe microorganisms based on their preferences and adaptations to different types of environments related to solute concentration. A halophile is an organism that thrives in environments with high salt concentrations. An osmophile is an organism that can tolerate high osmotic pressures. Osmotic pressure is influenced by solute concentration, and osmophiles can adapt to environments with high solute concentrations, which may include substances other than salts. These environments could be sugar-rich, nutrient-rich, or contain other osmotically active compounds. Examples of osmophiles include certain fungi like Saccharomyces cerevisiae, which is used in the fermentation of high-sugar content environments like those found in brewing and baking.  OSMOTIC EFFECT Staphylococci are good examples; grow on skin, where salts are common. Staph can tolerate up to 10% salt; can design culture media with 7.5% salt, suppress growth of most other bacteria, select for Staph (will do this in lab later on this semester) Some bacteria require very high osmotic strengths for growth = Halophiles; Ex. Halobacterium halobium grows in Dead Sea, Great Salt Lake, evaporating salt flats. Organisms which live in dry environments (made dry by lack of water) are called xerophiles. Most microorganisms are unable to cope with environments of very low water activity and either die or become dehydrated and dormant under such conditions. The concept of lowering water activity in order to prevent bacterial growth is the basis for preservation of foods by drying (in sunlight or by evaporation) or by addition of high concentrations of salt or sugar. LABORATORY CULTURE MEDIA Chemically Defined and Undefined (Complex) differ in the level of knowledge about the composition of the medium 1. Chemically Defined has known amounts of pure chemicals in carefully measured concentrations. Formulated using precisely known and specified chemical components. The exact types and amounts of each ingredient are explicitly defined. Applications: Commonly used in research and experimental settings where reproducibility, precision, and control over nutrient composition are crucial. Ideal for studying specific nutritional requirements of microorganisms. 2. Undefined (Complex) : Extracts and digests of yeasts, meat, or plant Nutrient broth, Nutrient agar: Exact chemical composition is not known. Nutrient broth, Plate count agar-basically SOUP. Applications: Widely used in routine laboratory work, general-purpose media, and situations where the specific nutritional requirements of microorganisms are not critical. Common examples include nutrient agar and tryptic soy agar. Complex or rich media provide the cells with the chemical building blocks that it would otherwise have to make itself, so complex media saves cellular energy  The Growth Cycle/phases of a bacterial population in batch culture LAG PHASE: When an organism is inoculated into a fresh medium, it needs to adapt to the new nutrients available, synthesize RNA, protein and finally replicate its DNA before starting division. These processes take time and there is no net increase in cell numbers, thus a lag phase is observed. A lag phase is observed when a population of cells is transferred from a nutritionally rich medium to a poorer one. This occurs because the cells need time to synthesis their complement of enzymes for the synthesis of essential metabolites not present in the new medium. If an exponentially growing culture is inoculated into the same medium under the same conditions a lag phase is not observed and exponential growth begins immediately  THE GROWTH CYCLE/PHASES OF A BACTERIAL POPULATION IN BATCH CULTURE EXPONENTIAL PHASE: Once the appropriate enzymes for growth in a particular medium have been expressed cells begin to multiply. Exponential growth is a consequence of the fact that each cell divides to form two new cells and so on. This period of maximal division can last for several hours or days, depending upon the organism, and is called the log or exponential growth phase. Cells dividing as rapidly as possible. Cells in mid-exponential growth are generally at their healthiest and often most desirable for studies on cell enzymes etc. Rate of exponential growth is influenced by temperature, composition of media as well as the genetic characteristics of the organisms. Many industrial bacteria produce useful products during this stage of growth  The Growth Cycle/phases of a bacterial population in batch culture STATIONARY PHASE: In a closed vessel, exponential growth cannot continue indefinitely. Eventually something must occur to limit microbial growth. What usually happens is that some limiting nutrient is depleted or some metabolite produced by the bacteria themselves builds up and inhibit their growth. Eventually the increase in cell number ceases, either because cells stop dividing or the rate of division equals the rate of cell death, resulting in a stationary phase (3 depending on the bacterium, stationary phase can last for several hours to many days. While there is no increase in cell numbers in the stationary phase, many cell functions continue including energy metabolism and biosynthesis. Some antibiotic producing bacteria produce antibiotics during this phase of growth. DEATH PHASE: The final stage of a growth curve is the death phase (4). An exponential decrease in the number of organisms due to cell death occurs during this phase. Some microorganisms never experience a death phase or it is greatly delayed due to their ability to survive for long periods without nutrients. The Growth Cycle/phases of a bacterial population in batch culture