5BY543 Growth and Metabolism of Microbes (5).pdf

5BY543 MICROBIOLOGY Bacterial Growth and Metabolism; Aseptic Techniques Dr Isaac Thom Shawa [email protected] derby.ac.uk  Learning Outcomes At the end of this lesson, learners should be able to: Recapitulate on the Structure a...

5BY543 MICROBIOLOGY Bacterial Growth and Metabolism; Aseptic Techniques Dr Isaac Thom Shawa [email protected] derby.ac.uk  Learning Outcomes At the end of this lesson, learners should be able to: Recapitulate on the Structure and function of prokaryotic cells. List the microbial growth requirements. Explain determinants of bacterial growth curve. Discuss bacterial growth requirements and metabolism. derby.ac.uk  Microbial cultivation Why is microbial cultivation important? Diagnosis of infectious diseases. Selection of antibiotic of choice. Preparation of vaccine. Research tool in molecular genetics. Molecular cloning derby.ac.uk  Culture Media Liquid Semi-solid Solid Images: Bacterial culture media preparation materials. derby.ac.uk  Culture Media Blood Agar MacConkey Nutrient Agar Etc derby.ac.uk  Culture Media Basal media. Enriched media. Selective media. Enrichment media. Chocolate Agar Indicator media or differential media. Transport media. Storage media. derby.ac.uk What is a Bacterial colony? Colony - visible growth on a culture plate. Group of bacteria derived from the same mother cell. Colony Forming Unit- CFU ? a unit that estimates the number of viable cells in a sample. derby.ac.uk  Microbial cultivation Growth Chemical growth requirements: factors. Physical growth Other factors factors. derby.ac.uk  Microbial growth requirements Chemical growth factors Carbon Nitrogen Required for isolation of microbes Organic source – Glucose Organic source – Protein in vitro. Inorganic source – CO2 Inorganic source – atmospheric nitrogen derby.ac.uk  Microbial growth requirements Chemical growth factors Autotrophic microbes – using inorganic carbon and nitrogen. Heterotrophic microbes – using organic carbon and nitrogen. Other Chemicals: Hydrogen, Oxygen, Phosphorous, and Sulphur. derby.ac.uk  Microbial growth requirements Physical growth factors pH and buffer requirements Pathogenic microbes grow best at neutral pH (6.8-7.4). Acidophilic – survive with acidic pH. H.pylori (pH 7) derby.ac.uk  Microbial growth requirements Physical growth factors Salt concentration 0.9% NaCl Halophiles – salt loving – resist high salt concentrations. Are extremophiles – thrive in high salt concentrations. Halophiles require NaCl for growth. Halotolerant organisms - do not require salt but can grow under saline conditions. Few organisms adapt and survive in high salinity. derby.ac.uk  Microbial growth requirements Physical growth factors Salt concentration How halophilic and halotolerant organisms survive? Expend energy to exclude salt from their cytoplasm to avoid protein aggregation (salting-out). Two strategies employed to prevent desiccation through osmotic movement of water out of their cytoplasm (increase the internal osmolarity of the cell). Accumulation of organic compounds in their cytoplasm. Osmoprotectants known as compatible solutes (aa, sugars etc). Absorbing K+ ions into the cytoplasm. derby.ac.uk  Microbial growth requirements Physical growth factors Temperature requirement The majority of microbes grow at near human body temp. of 37oC. Mesophiles – grow and thrive under moderate temp. 15oC – 45oC. Grow at optimum of 37oC. Staphylococcus, Salmonella, Listeria derby.ac.uk  Microbial growth requirements Physical growth factors Temperature requirement Psycrophiles – Grow best in cooler temps (between 4oC - 25oC). Thermophiles Grow best in hot temps (50oC – 80oC). Hyperthermophiles Favour extremely hot temps (80oC - 110oC. derby.ac.uk  Microbial growth requirements Physical growth factors Light requirement Some bacteria have light capturing pigments and gather light energy at certain wavelengths and convert it to chemical energy. Photoautotrophs – contain chlorophyll Require light for photosynthesis E.g. Cyanobacteria Bacteriochlorophyll – A pigment capable of absorbing shorter wavelengths of light than chlorophyll derby.ac.uk  Microbial growth requirements Physical growth factors Gaseous and Humidity Based on their O2 requirements, microbes divisions: Strict, Obligate Aerobe Grow better in the presence of O2 Mycobacterium Image: Mycobacterium tuberculosis derby.ac.uk  Microbial growth requirements Physical growth factors Gaseous and Humidity Based on their O2 requirements, microbes divisions: Strict, or Obligate Anaerobe Grow in absence of O2 Clostridium Image: Clostridium difficile derby.ac.uk  Microbial growth requirements Physical growth factors Gaseous and Humidity Based on their O2 requirements, microbes divisions: Facultative Anaerobes Can grow with or without O2. Switch to anaerobic pathways in low O2. utilize either fermentation or anaerobic respiration for energy production. Aerotolerant anerobes utilize anaerobic respiration but Image: Escherichia coli are not harmed in the presence of O2. Escherichia derby.ac.uk  Microbial growth requirements Physical growth factors Gaseous and Humidity Based on their O2 requirements, microbes divisions: Microaerophilic Grow in low conc of O2 (5%). Helicobacter Image: Helicobacter pylori Campylobacter jejuni derby.ac.uk  Bacterial growth curve There are four distinct phases: Represents the number of live cells in a bacterial Lag phase: bacteria are metabolically population over a period of time. active but not dividing. Exponential (log)phase: Exponential Relationship between microbial quantity and time growth of incubation. Stationary phase: Growth reaches a plateau Death phase: Exponential decrease. derby.ac.uk  Bacterial growth curve Lag phase Characterised by cell activity but not growth. Bacteria initially adjust to the new environment. They synthesize proteins, enzymes, RNA, and various molecules necessary for multiplication. Cell maturation and increase Diagram: The bacterial growth curve represents the number of living cells in a population over time. in size but no cell division. derby.ac.uk  Bacterial growth curve Log (exponential) phase Time of rapid cell doubling. Cells start dividing regularly by binary fission (20 min). Metabolic activity is high as DNA, RNA, cell wall components, & other substances necessary for growth are generated for division. Antibiotics & disinfectants are most effective. they typically target bacteria cell walls or the protein synthesis processes of Diagram: The bacterial growth curve represents the DNA transcription & RNA translation. number of living cells in a population over time. derby.ac.uk  Bacterial growth curve Stationary phase Growth reaches a plateau. Depletion of nutrients and accumulation of waste products. Cells become less metabolically active. Diving cells = Dead cells No overall population growth Some cells continue to divide as others die. Production of endospores. Diagram: The bacterial growth curve represents the number of living cells in a population over time. Generation of virulence factors help bacteria survive harsh conditions & cause dse. derby.ac.uk  Bacterial growth curve Death phase Characterized by an exponential ↓ in the ♯ of living cells. ↓nutrients, ↑waste products, ↑dying cells. Dead cells become nutrients for other bacteria to survive long enough for spore production. Spores survive the harsh conditions of the death phase and become growing bacteria when placed in an environment that supports life. Diagram: The bacterial growth curve represents the number of living cells in a population over time. derby.ac.uk  Maintenance of cells in exponential phase Continuous culture Done by repeatedly transferring bacterial cells into fresh medium of identical composition. Transfer is done while they are multiplying in exponential phase. Two techniques are used: Chemostat device Turbidostat device derby.ac.uk  Microbial Metabolism According to biochemical pathway in energy production, microbial Aerobic respiration Anaerobic respiration Fermentation metabolism can be divided into three categories: Complete breakdown of Yield > 2 ATP Yield = 2 ATP Glu to CO2 & H2O. End products: Final electron Absence of O2. Lactic receptor O2. acid/Alcohol Final electron receptor is Yield = 38 ATP organic molecule. derby.ac.uk Energy Generating Process Microbes that use aerobic respiration, detoxify generated waste: Catalase: H2O2 -------→ H2O and O2 Superoxide dismutase : oxygen radical -----→ H20 and O2 Catalase is produced by bacteria that respire using O2 protects them from the toxic by-products of oxygen metabolism. include strict aerobes as well as facultative anaerobes. they respire using oxygen as a terminal electron acceptor. derby.ac.uk  Microbial Metabolism Aerobic respiration Molecular O2 serves as the final electron acceptor. 38 ATP molecules will be produced by oxidation of one glucose molecule. Used by obligatory aerobic bacteria for energy production; such as: Mycobacterium. derby.ac.uk  Microbial Metabolism Anaerobic respiration Inorganic sulfate or nitrate act as the final electron acceptor. The net yield of ATP molecules is less than it is with aerobic respiration. Used by obligatory anaerobic bacteria such as: Clostridium. derby.ac.uk  Microbial Metabolism Fermentation Lactic acid (produced by bacteria) or ethanol (production of yeast) serves as final electron acceptor. Only 2 ATP molecules will be produced by fermentation of glucose molecule. Used by facultative anaerobic bacteria such as: E.coli. derby.ac.uk  Eukaryotes Eukaryote – any cell or organism that possesses a clearly defined nucleus. Has a nuclear membrane that surrounds the nucleus, in which the well-defined chromosomes are located. Also contain organelles, including mitochondria, Golgi apparatus, endoplasmic reticulum, and lysosomes. derby.ac.uk  Prokaryotes Prokaryote – lacks a distinct nucleus and other organelles due to the absence of internal membranes. The cell membrane is made up of phospholipids and constitutes the cell’s primary osmotic career. The cytoplasm contains ribosomes, which carry out protein synthesis, and a dsDNA chromosome, which is circular. derby.ac.uk  Microbial classification based on energy and carbon sources Based on energy source Phototrophs Use light as an energy source – photosynthesize. Chemotrophs Use inorganic and organic chemicals. derby.ac.uk  Microbial classification based on energy and carbon sources Based on carbon source Autotrophs Use carbon dioxide. Synthesize their own food. They derive energy from light or chemical reactions. They utilize simple inorganic compounds like CO2, H2O, H2S, etc. and convert them into organic compounds like carbohydrates, proteins, etc. to supplement their energy requirements. derby.ac.uk  Microbial classification based on energy and carbon sources The two different types of autotrophic bacteria are: Photoautotrophs – or photosynthetic. They derive energy from sunlight. Chemoautotrophs – or chemosynthetic. They use chemical energy to prepare their food. derby.ac.uk  Microbial classification based on energy and carbon sources Based on carbon source Heterotrophs Do not use carbon dioxide as their carbon source. Use carbon from organic sources. Salmonella derby.ac.uk  Photoautotrophic Bacteria Photoautotrophics trap light energy and convert it into chemical energy. They make their own food like plants. May perform oxygenic photosynthesis or anoxygenic photosynthesis. Are used as biofertilizers, for bioremediation, waste water treatment and purification of polluted water. derby.ac.uk  Photoautotrophic Bacteria Oxygenic Photosynthetic Bacteria Cyanobacteria (blue-green algae) perform oxygenic photosynthesis. They use H2O as an electron donor and O2 is produced in the reaction. They do not possess chloroplasts but photosynthetic pigments like chlorophyll are present in the cytosol. 6CO2 + 12H2O + light energy → C6H12O6 + 6O2 + 6H2O derby.ac.uk  Photoautotrophic Bacteria Anoxygenic Photosynthetic Bacteria They do not utilize H2O as an electron donor, instead, they use H2S, H2 or thiosulphate as reducing agent. They contain a photosynthetic pigment known as bacteriochlorophyll. E.g. green-sulphur, purple-sulphur bacteria, purple non- sulphur bacteria, acidobacteria and heliobacteria. derby.ac.uk  Photoautotrophic Bacteria Purple sulphur bacteria (PSB) They are found in hot sulphur springs and stagnant water. Thrive in anaerobic or O2 poor environments. They utilize H2S or thiosulphates as a reducing agent and release sulphur. The main pigments are bacteriochlorophyll ‘a’ and ‘b’ located in the plasma membrane. derby.ac.uk  Photoautotrophic Bacteria Purple non-sulphur bacteria (PNSB) They mainly use H2 as a reducing agent. They belong to the order Rhodospirillales. Purple photosynthetic bacteria produce various beneficial substances such as polyphosphates, vitamins, pigments, hydrogen, extracellular nucleic acids and growth promoting substances for plants. They can increase the plants yield, resistance to environmental stress and improve biomass quality. derby.ac.uk  Photoautotrophic Bacteria Green sulphur bacteria (GSB) They are obligate anaerobe and generally non-motile. They are found deep in the ocean in extremely low light and anoxic environment and near thermal vents. The electron donor is sulphide, H2 or ferrous ion. They contain bacteriochlorophyll ‘c’, ‘d’ and ‘e’ along with bacteriochlorophyll ‘a’. Pigments are present in the plasma membrane and chlorosomes. derby.ac.uk  Chemoautotrophic Bacteria They perform chemosynthesis, which utilizes chemical energy. They lack photosynthetic pigments. Carbon sources can be CO2, H2S, methane (CH4), etc. The chemical energy is produced from oxidation of inorganic compounds such as H2, H2S, CO, NH3, CH4, iron salts, NO2-, etc. The energy liberated from oxidation is trapped in ATP for the synthesis of organic compounds. derby.ac.uk  Chemoautotrophic Bacteria Sulphur bacteria They oxidise, hydrogen sulphide or thiosulphates to molecular sulphur or sulphates. E.g. Beggiatoa, Thiobacillus, Thiothrix, Sulfolobus, etc. 2H2S + O2 → 2H2O + 2S + Energy derby.ac.uk  Chemoautotrophic Bacteria Nitrogen bacteria Nitrifying bacteria convert ammonia to nitrite and then to nitrate. In this oxidation process, energy is released. Nitrate is utilized by plants. E.g. Nitrosomonas, Nitrobacter NH3 + O2 → NO2- + H2O + Energy (Nitrosomonas).. 2 NO2- + O2 → NO3 + Energy (Nitrobacter). derby.ac.uk  Chemoautotrophic Bacteria Hydrogen bacteria They oxidise molecular hydrogen. Aerobic hydrogen-oxidizing bacteria use O2 as an electron acceptor, whereas anaerobic H2 bacteria use NO2- or sulphate as an electron acceptor. E.g. Helicobacter pylori, Hydrogenobacter thermophilus, Hydrogenovibrio marinus, etc. 2H2 + O2 → 2H2O + Energy derby.ac.uk  Chemoautotrophic Bacteria Methanotrophs They use CH4 as a carbon source to derive energy. They can be aerobic or anaerobic. Aerobic methanotrophs oxidize CH4 to formaldehyde, which is then utilized in various pathways to form organic compounds. Anaerobic methanotrophs utilize other compounds as electron acceptors. E.g. Methylomonas, Methylococcus capsulatus, etc. It assimilates, formaldehyde by the RuMP pathway. It is used to produce animal feed. derby.ac.uk  Chemoautotrophic Bacteria Iron bacteria They oxidise ferrous ions to ferric ions. They are present in iron-rich environments like hot lava bed, hydrothermal vents. E.g. Thiobacillus ferrooxidans, Geobacter metallireducens, Zetaproteobacteria, Gallionella, Ferrobacillus, etc. 4FeCO3 + O2 + 6H2O → 4Fe(OH)3 + 4CO2 + Energy derby.ac.uk SUMMARY: energy & carbon sources derby.ac.uk  Bacterial growth Cells are the most fundamental units of life. All living organisms are made of one or more cells. Cells reproduce by copying their genetic material and then dividing. A parent cell gives rise to daughter cells. Types of cell division Binary fission (simply cloning) Mitosis and Meiosis derby.ac.uk Bacterial growth derby.ac.uk  Bacterial growth Budding Fragmentation Reproduces by budding. The body breaks into distinct pieces. A small bud forms at one Each fragment develops into a mature clone end of the mother cell or genetically and morphologically identical to its parent. on filaments Seen in organisms such as filamentous cyanobacteria, called prosthecae. molds, lichens, sponges, some annelid worms, sea stars, etc. derby.ac.uk Bacterial growth factors: SUMMARY derby.ac.uk  Factors affecting bacterial growth Water Oxygen Carbon dioxide Temperature Hydrogen ion concentration Light Osmotic pressure Symbiosis and antagonism derby.ac.uk  Factors affecting growth Water Processes taking place in bacterial cell are in a H20 base. 80% of bacterial cell consists of H20. Dehydration is detrimental for most bacteria e.g. Treponema pallidum. Staphylococcus can resist drying for months. Spores are particularly resistant to desiccation and may survive in the dry state for several decades. derby.ac.uk  Factors affecting growth Oxygen Most life forms depend on O2 for survival and growth. Microbes requires O2 to act as terminal electron acceptor in their respiratory chain. Aerobe – Mycobacterium Anaerobes – Fusobacterium Obligate aerobes – Pseudomonas Facultative anaerobes – Streptococcus derby.ac.uk  Factors affecting growth Carbon dioxide Approximately half of dry weight. CO2 is provided by cellular metabolism and from environment. Autotrophic organisms are able to use CO2 as a source of carbon. Heterotrophic bacteria require some amount of CO2 from exogenous sources. 5 – 10% CO2 is supplied for them in culture. Capnophilic = requiring excess amount of CO2 e.g. Brucella abortus (10% CO2). derby.ac.uk  Factors affecting growth Carbon dioxide The CO2 available in the carbohydrate sugar molecules is cycled further by microorganisms in tricarboxylic acid (or TCA) cycle. The breakdown of the carbohydrate serves to supply energy to the microorganism. Respiration. In anaerobic environments, microorganisms can cycle the carbon compounds to yield energy. Fermentation. derby.ac.uk  Factors affecting growth Temperature Psychrophiles – grow below 20oC. Mesophiles – grow between 20 - 40oC. Thermophiles – grow to higher temps 60 - 80oC. Bacillus stearothermophillus – up to 250oC. Thermal death point – lowest temp that kills bacterium. 50 - 65oC – vegetative 100 -120oC – spores derby.ac.uk  Factors affecting growth Hydrogen Ion Concentration The pH requirement is also variable. Most bacteria have an average pH requirement of 7.2 – 7.6 (matches with pH in human environment). Some bacteria grow in acidic pH e.g. Lactobacilli (pH = 3). Some bacteria grow in alkaline pH e.g. alkaligenes (pH =10.5) derby.ac.uk  Factors affecting growth Light Most bacteria prefer darkness for growth. Cultured microbes die if exposed to sunlight. Bacteria that require sunlight are called phototropic. Exposure to light may influence pigment production. Photochromogenic mycobacteria form a pigment only on exposure to light and not when incubated in the dark. derby.ac.uk  Factors affecting growth Osmotic pressure There is a wide range of osmotic tolerance found in bacteria. 0.5% NaCl is added in culture media to provide suitable osmolarity. Plasmolysis = sudden exposure to hypertonic solutions. may cause osmotic withdrawal of H2O and shrinkage of protoplasm. Plasmoptysis = excessive osmotic inhibition leading to swelling and rupture of the cell. sudden transfer from a concentrated solution to distilled H2O. derby.ac.uk  Factors affecting growth Symbiosis and antagonism Fungal interactions with the human host: exploring the spectrum of symbiosis - To be discussed in the next lesson ScienceDirect derby.ac.uk  Factors affecting growth Nitrogen – found in all amino acids, nitrogenous bases of nucleic acids. Hydrogen – found in all biological molecules, carbs, fats, proteins, nucleic acids etc. Phosphorous – found in nucleic acids, ATP, and phospholipids of membranes. Sulphur – found in 2 or 3 amino acids of microbes. Trace elements – inorganic elements needed in very tiny concentrations (manganese, cobalt, Zinc etc). derby.ac.uk  Bacterial Metabolism Metabolism The series of changes of a substance (carbohydrate, protein, fat) that take place within the bacterial cell from absorption to elimination. Help us in identifying bacteria by their end products. Help us in knowing how to inhibit bacteria. derby.ac.uk  Bacterial Metabolism Catabolism Breakdown of macromolecules into smaller micromolecules, absorption into cell, conversion into basic blocks including interconversion of ADP to ATP. Anabolism A process by which the basic building blocks are utilized in synthesis of various cellular structures such as monomers and polymers. derby.ac.uk  Bacterial Metabolism Image: Summary of anabolism and catabolism derby.ac.uk  Components Of Bacterial Metabolism Components Functions Enzymes Biological catalyst, facilitates each step of metabolic reaction by lowering the activation of energy reaction. Adenosine Serves as energy currency of the cell. Triphosphate Energy source Compound that is oxidized to release energy, also called as electron donor. Electron Carry electrons that are removed during oxidation of energy carriers source. Precursor Intermediate metabolites that link anabolic and catabolic metabolites pathway. derby.ac.uk  Glucose breakdown Glucose is a key energy-storing molecule. Nearly all cells metabolize glucose for energy. Glucose metabolism is fairly simple. Other organic molecules are converted to glucose for energy harvesting. derby.ac.uk  Glucose breakdown Glycolysis occurs in cytosol. Does not require O2. Breaks glucose to pyruvate. Yields two molecules of ATP per molecule of Glucose. If oxygen is absent, fermentation occurs. Pyruvate is converted into either lactate, or into ethanol and CO2. If O2 is present cellular respiration occurs. derby.ac.uk  Pathways alternative to Glycolysis Many bacteria have another pathway in addition to glycolysis for degradation of glucose. Pentose Phosphate Pathway Entner Doudoroff Pathway derby.ac.uk  Pentose Phosphate Pathway Hexose monophosphate shunt Occurs simultaneously with glycolysis and provide breakdown of both pentose sugar and glucose. Intermediate pentoses are used for nucleic acid synthesis, aa synthesis. Important producers of reduced co- enzyme i.e. NADPH used for biosynthetic reaction. derby.ac.uk  Entner-Doudoroff Pathway Uses 6-phosphogluconate dehydratase and 2-keto-3- deoxyphosphogluconate aldolase to create pyruvate from glucose. Most of gram negative bacteria like Pseudomonas, Rhizobium, Agrobacterium. Produces 1 molecule NADH, 1 molecule NADPH, and 1 molecule of ATP. derby.ac.uk  Glucose Breakdown overview Cellular respiration – pyruvate obtained from glucose breakdown are channelled either to respiration or fermentation. Requires oxygen Breaks down pyruvate into carbon dioxide and water. Image: Kreb’s cycle derby.ac.uk  Electron Transport Chain Last phase of respiration which generates ATP from reduced substrates. Consists of a sequence of carrier molecules through which electron passes. Occurs in plasma membrane. Electron transport chain is different in different bacteria. derby.ac.uk  Factors affecting bacterial growth Water Oxygen Carbon dioxide Temperature Hydrogen ion concentration Light Osmotic pressure Symbiosis and antagonism derby.ac.uk Aseptic techniques and Health and Safety (Microbiology Laboratory) derby.ac.uk  Rules of aseptic techniques Ensuring that the environment is free from sources of contamination. All sterilized equipment should be easy to reach. Working beside a Bunsen burner creates an upward flow of air through convection. Test tubes containing sensitive biological samples should be flamed around the cap and neck. Agar plates should be opened facing away from the user, and preferably only just enough to complete the work. derby.ac.uk  Microbiology/Immunology Lab Streak plating Asceptic technique derby.ac.uk Most popular piece of equipment derby.ac.uk  Independent study Draw a chart classifying bacteria into two broad terms (i.e. gram positive and gram negative). In each category, indicate their growth requirements, type of culture media. Submission deadline: Tuesday, 27th Feb 2024 at 9:00am. derby.ac.uk University of Derby, Kedleston Road, Derby, DE22 1GB T +44 (0)1332 594010 E [email protected] derby.ac.uk