Microbial Growth & Nutrition PDF

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Summary

Information regarding the growth of microbial colonies, and what they need to survive. Taught by: Dr. Ian Haysom...

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Microbial Growth Why are we interested?? - study, research, metabolite production, healthy biosystems Control of growth - hygiene and infection control, growth of industrial and biotech organisms, food productions Increase in number of cells (population) rather than cell size is key. One bacterium becomes a colony of millions of bacteria Growth takes place on 2 levels, 1) individual bacterial synthesise new cell components and increase in size, and 2) the number of bacteria in the population The basis of population growth is binary fission

Cell growth by binary fission Bacteria divide by binary fission Alternative means - budding (some env. Bacteria and yeasts), conidiospores (filamentous bacteria and moulds), fragmentation (moulds)

Chromosome replication Bacterial chromosomes are circular Replication occurs in 3 steps - initiation, elongation, and termination

Fungi - yeasts and moulds

Standard Growth Curve

Lag phase - relatively flat period - Newly inoculated cells require a period of adjustment, enlargement and synthesis - The cells are not yet multiplying at their maximum rate - The population is so sparse that the sampling misses them - Length of lag period varies from one pop to another Exponential growth phase - When growth curve increases geometrically - Cells reach the max rate of cell division - Will continue as long as cells have the adequate nutrients and env. Is favourable - Number of cells growing greatly outnumbers the number of cells dying Stationary phase - Rate of growth=rate of death - Depleted nutrients and oxygen - Excretion of organic acids and other biochemical pollutants into the growth medium - Endospores begin to form in this phase Rapidly declining phase - The curve dips downward - Cells begin to die at an exponential rate - The amount of cells dying out numbers the amount of cells growing - Dead cells become nutrients for growing cells Death phase - The curve continues to dip downwards - Most cellular activity stops - Endospores are formed and released from parent cells

Importance of the growth curve Implications in microbial control, infection, food microbiology, and culture technology Growth patterns in microorganisms can account for the stages of infection Understanding the stages of cell growth is crucial for working with cultures In some applications, closed batch culturing is inefficient, and instead, a chemostat or continuous culture system is used

The rate of population growth Generation or doubling time: the time required for a complete binary fission cycle Each new cycle or generation increases the population by a factor of 2 (doubles) As long as the env is favourable, the doubling effect continues at a constant rate The length of the generation time - a measure of the growth rate of an organism (average generation time is 30-60 mins under optimum conditions, but can be as short as 10-12 mins) This growth pattern is termed exponential

Graphing bacterial growth The data from growing bacterial populations are graphed by plotting the number of cells as a function of time - if plotted logarithmically, a straight line, if plotted arithmetically, a constantly curved slope

To calculate the size of a pop over time (Nf=(Ni)2^g Nf is the total number of the cells in the population at some point in the growth phase Ni is the starting number G denoted the generation number

Microbial Kinetics

Generation Time If 100 cells (Ni) growing for 5 hours produced 1,720,320 cells (Nf):

Temperature and Microbial growth

Danger zone for bacterial growth is between 15 and 50 degrees celsius - rapid growth but some may produce toxins

Physical Requirements pH Refers to the concentration of H+ ions pH is a logarithmic scale - a change of 1 unit corresponds to a 10-fold change in H+ ions Most organisms show a growth range of 2-3 pH units Most bacteria grow between 6.5 and 7.5 pH Moulds and yeasts grow between 4 and 6 pH Acidohoiles grow from 1-4 pH Alkalophiles grow from 8.5 - 11 pH

Osmotic Pressure - isotonic : balanced osmitic movement of water in and out the cell

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Hypotonic solution leads to water ingress Hypertonic envs: increased salt or sugar (solute) cause plasmosis (water loss) extreme halophiles tolerate high osmotic pressure, facultative halophiles tolerate high osmotic pressure

Oxygen and MIcrobial Growth Aerobes - Obligate - require oxygen to grow - facultative - grow better with, but can live without - Microaerophiles - require reduced level of oxygen (lower than atmospheric) Anaerobes - Aerotolerant anaerobes - can tolerate oxygen but grow better without oxygen - Facultative - can live and generate ATP by aerobic respiration when oxygen is present, but can switch to fermentation under anaerobic conditions - Obligate - do not require oxygen, often killed by oxygen Classification of organisms based on O2 utilisation

Utilisation of O2 during metabolism yields toxic by-products including O2-, Singlet oxygen (^1O2), and/or H2O2 Toxic O2 products can be converted to harmless substances if the organism has catalase (or peroxidase) and superoxide dismutase (SOD)

Catalase breaks H2O2 into H2O and O2, SOD converts O2- into H2O2 and O2 Any organism that can live in or requires O2 has SOD and catalase (or peroxidase)

Chemical Requirements Primary - Water!!! Elements - C (50% of cell’s dry weight) H O N P S, Trace elements Organic - Source of energy (glucose), vitamins (coenzymes), some amino acids, purines, and pyrimidines

Artificial culture of microbes Development of microbiology was driven by ability to grow pure culture in a lab Media must supply all essential nutrients Categories of media (defined or complex, liquid/semi-solid/solid, supportive/enriched/selective/differential)

Culture Media Supply the nutritional needs of microorganisms (C, N, Phosphorus, trace elements, etc) Defined medium - precise amounts of highly purified chemicals Complex mediums - highly nutritious substances Selective - contains compounds that selectively inhibit Differential - contains indicator -terms that describe media used for the isolation of a particular species or for comparative

studies of microorganisms

Agar Nutrient Agar - Complex agar VRBGA - Differential and selective Differential agars - Blood Agar (Streptococcus pyogenes), Baird Parker Agar (Staphylococcus aureus) Culturing Microorganisms

Learning Outcomes Requirements for growth - Physical requirements - Chemical requirements Reproduction in prokaryotes - Binary fission - Chromosome replication Microbial kinetics - Generation time - Growth dynamics Directed study - Minerva, Hogg Chapter 5.

Microbial Nutrition Learning Outcomes Last week introduced major macro and micronutrients required by bacteria This week: · Brief review of nutrition · Energy generation and storage · Aerobic respiration · Nutrient uptake Macronutrients 95% dry weight of a cell is C, O, H, N, S, P, K, Ca, Mg and Fe First 6 are components of proteins, carbohydrates, lipids and nucleic acids Other 4 are cations K+ required for enzyme activity Ca2+ contributes to heat resistance of spores, regulatory signalling molecule. Mg2+ is an enzyme cofactor, stabilises ribosomes and cell membrane

Fe2+ involved in ATP synthesis Micronutrients (trace elements) Mn, Zn, Co, Mo, Ni, Cu All metal ions - Regarded as ubiquitous contaminants in environment Normally part of enzymes and cofactors Mn2+ cofactor in enzymes that catalyse transfer of phosphate Mo2+ required for nitrogen fixation Co2+ component of vitamin B12 Growth factors Some microbes have additional specific requirements (growth factors) that reflect their metabolic capabilities. Streptococcus pyogenes Amino acids glutamic acid and alanine are readily available in normal environment Lost genes required to synthesise these nutrients Rickettsia prowazekii ~: Obligate intracellular parasite of eukaryotes Carbon, energy and electron sources Bacteria classified according to how they obtain C and energy… · Heterotrophs: obtain carbon as organic molecules from other organisms o Also obtain H, O and electrons from same source Autotrophs: obtain C from CO2 · o CO2 not a source of H or electrons or energy · Photoautotrophs: energy obtained from light · Chemoautotrophs: energy obtained from inorganic sources eg sulphur, nitrite …and electrons · Lithotrophs · Organotrophs Nomenclature: ____trophy Carbon source for biomass · Auto___ : CO2 is fixed and assembled into organic molecules · Hetero___ : Preformed organic molecules are acquired from outside cell and assembled Energy source · Photo___ : Light absorption excites electron to high energy state · Chemo___ : Chemical electron donors are oxidised Electron source · Litho___: inorganic molecules donate electrons · Organo___: organic molecules donate electrons Question: In a mixed ecosystem of autotrophs and heterotrophs, what happens when the autotrophs grow rapidly and produce excess carbon? Rhodospirillum rubrum example of an organism that can utilise more than one system

Nutrient uptake Nutrients must be moved into the cytoplasm across plasma membrane – a considerable barrier Membrane must be selectively permeable to nutrients cell can use. · Simple diffusion · Facilitated diffusion · Active transport 3 general classes of transport systems: 1. Uniport 2. Symport 3. Antiport Nutrient uptake - diffusion Simple diffusion · Small molecules move along concentration gradient until reach equilibrium o H2O, Na+, Cl-, O2 (soluble in membrane lipids) Facilitated diffusion – requires transport protein Aquaporins - Transport water and small polar molecules such as glycerol Active transport – requires energy ABC Transporters - Largest family of energy-driven transport systems · ATP-binding cassette / ABC transporters · Found in bacteria, archaea and eukaryotes · E.coli has 70 different varieties of ABC transporters (5% of genome dedicated to them) Uptake transporters critical for nutrient transport Efflux transporters critical for eliminating hazardous chemicals ABC importer Energy Storage Adenosine tri-phosphate Membrane potential ATP – Cell’s energy cycle Membrane potential The difference in electrical potential across the plasma membrane. · Generated when chemical energy used to pump protons (H+) outside of the cell This electrical gradient used to: · Move nutrients into the cell · Rotate flagella · Synthesise ATP ATP Synthesis ATP synthase

2 components: F0 and F1 Functions like a rotary engine Flow of protons causes F0 and stalk to rotate Conformational changes in F1 Oxidation-reduction reactions Many metabolic reactions involve the transfer of electrons from one molecule to another; these are called oxidation-reduction or redox reactions. NAD and NADP Coenzymes: · Nicotinamide adenine dinucleotide · Nicotinamide adenine dinucleotide phosphate Found in redox reactions as carrier molecules for the transfer of electrons NAD+ + H+ +2eNADP+ + H+ +2e-

NADH NADPH

Electron transport chain - Substances vary in their affinity for binding electrons as measured by redox potential. Chemoheterotrophic nutrition Used by majority of microorganisms Glucose is carbohydrate most widely used as an energy source. · C6H12O6 + 6O2 6CO2 + 6H2O · In microbes, results in the release of 38 molecules of ATP · 38ADP + 38Pi 38ATP Glycolysis (Embden-Meyerhof pathway) Can occur with or without oxygen Converts a molecule of glucose into two molecules of pyruvate · Glucose is phosphorylated · 6-carbon structure rearranged, then cleaved into two three-carbon molecules · Each three-carbon molecule is oxidised to pyruvate. Process uses 2 molecules of ATP and generates 4 molecules of ATP. Net gain of two ATP Entner-Doudoroff pathway Used by some Gram negative bacteria, predominantly the pseudomonads Pentose phosphate pathway Can operate in tandem with glycolysis or the Entner-Doudoroff pathway. mainly anabolic function, acting as a source of precursor molecules for other metabolic pathways Aerobic respiration Pyruvate from glycolysis completely oxidised to CO2 and H2O Tricarboxylic acid (TCA cycle) · Also known as Krebs or citric acid cycle · Series of redox reactions, transferring energy from pyruvate to coenzymes (mainly

NADH) · Energy conserved in ATP via oxidative phosphorylation Pyruvate does not participate directly in TCA cycle How does transfer of electrons lead to formation of energy? Chemiosmotic theory proposed in 1961. Fermentation “a microbial process by which an organic substrate (usually carbohydrate) is broken down without the involvement of oxygen or an electron transport chain, generating energy by substrate-level phosphorylation” Hogg (2013, p141) Two common pathways result in production of lactic acid and ethanol - very important in food industry and we will be returning to these later in the module. Summary Ensure you understand the following: Nutrient requirements of bacteria Mechanisms of nutrient uptake Energy storage Energy generation Directed Study Hogg chapter 4 and 6...