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Microbial Growth & Nutrition
Applied Microbiology
Bath Spa University
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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
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):
- 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 H2O 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 O 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
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 B 12
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 CO 2 o CO 2 not a source of H or electrons or energy · Photoautotrophs: energy obtained from light · Chemoautotrophs: energy obtained from inorganic sources eg sulphur, nitrite .. electrons · Lithotrophs · Organotrophs
Nomenclature: trophy Carbon source for biomass · Auto : CO 2 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
ATP synthase 2 components: F 0 and F 1 Functions like a rotary engine Flow of protons causes F 0 and stalk to rotate Conformational changes in F 1
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+ +2e- NADH NADP+ + H+ +2e- 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. · C 6 H 12 O 6 + 6O 2 6CO 2 + 6H 2 O · 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 CO 2 and H 2 O 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
Microbial Growth & Nutrition
Module: Applied Microbiology
University: Bath Spa University
