Growth of microbial cultures
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1 Growth of microbial cultures 1 The dream of a bacterium is becoming two bacteria. Microorganisms proliferate mostly by binary division. Hereby they are potentially immortal. By rapid and exponential growth ressources are consumed and starvation arises. 2 1
2 What is your metabolism at this moment?! Growth rate = 0! Catabolism 100 % (net)! Maintenance metabolism! Main substrates metabolised: Glucose + O 2 (the only substrates used by the brain!) 3 Cell division Primitive scheme! Biomass and length increase Replication of the chromosom, segregation of the daughter molecules Formation of new membranes and cell walls 4 2
3 5 Cell division Brock 10th ed. Pictures Peter Graumann group 6 FtsZ: Tubulin-like protein filamenting temperature-sensitive mutant Z MreB: Actin-like protein Forms helical structures along the membrane 3
4 7 8 4
5 9 Cell numbers in a culture o To keep in mind: Z = Z 0 * 2 g " o o with " o o o o " o o o o o o o o " o o o o o o o o o o o o o o o o 2 10 = 1024 # # 1 million 2 30 # 1 billion (dt. Milliarde) Z 0 = initial cell number g = number of generations " oooooooooooooooooooooooooooooooo " oooooooooooooooooooooooooooooooo oooooooooooooooooooooooooooooooo " Growth phases A: Lag phase B: Exponential or log phase C: Stationary phase D: Death phase logarithmic 10 5
6 11 Some terms Generation time: time required for a bacterial cell to double (h) Division rate:1/generation time (v, h -1 ) Growth rate: Increase per time per amount present (!, h -1 ) Doubling time (t d ): Time required for a growth parameter as dry mass, protein and even cell numbers to double (h) Maximum growth rate! max : Growth rate during the exponential phase (h -1 ) 12 6
7 13 Growth kinetics Exponential growth: 2 0! 2 1! 2 2! 2 3! 2 n N = N 0 2 n => lgn = lgn 0 + n lg2 n = lgn - lgn 0 / lg2 Division rate (v): v = n/t [ h -1 ] v = lgn - lgn 0 / lg2 (t - t 0 ) Generation time (g): g = t/n [ h ] 14 7
8 15 Growth kinetics Exponential growth: 2 0! 2 1! 2 2! 2 3! 2 n Kinetic follows 1st order reaction:!x = dx / dt (= change of biomass to every time point) Growth rate! is constant x = x 0 e! t For doubling of x 0 to 2 x 0 2 x 0 = x 0 e! td => ln2 =! t d! = ln2 / t d ( = / t d ) 16 8
9 Growth kinetics Exponential growth: 2 0! 2 1! 2 2! 2 3! 2 n Kinetic follows 1st order reaction:!x = dx / dt (= change of biomass to every time point) Growth rate! is constant x = x 0 e! t For doubling of x 0 to 2 x 0 2 x 0 = x 0 e! td => ln2 =! t d V = 1/ td If td = g! = ln2 v! = ln2 / t d ( = / t d ) 17 Direct conting 18 9
10 Turbidity measurements of microbial growth 19 Growth yield (Y) The growth yield depends on - Catabolic pathway or other possibilities of energy conservation - Type of available carbon sources - Energy demand for maintenance The growth yield is better predictable than the growth rate. Often the specific growth yield (e.g. per mol of glucose consumed) is used
11 21 Essentials of successful cultivation Scientific question/ hypothesis Medium choice Carbon and energy source Other media components Gelling agent Inoculum and interaction Growth conditions, temperature, ph, atmosphere Incubation time 22 11
12 What do I need for successful cultivation Organism source Media Culture vessel Incubator Detection system Creativity
13 Chemical composition of a prokaryotic cell Molecule Percent of dry weight Protein 55 Polysaccharide 5 Lipid 9 Lipopolysaccharide 4 DNA 3 RNA 19 Amino acids and precursors 1 Sugars and precursors 2 Nucleotides and precursors 1 Inorganic ions 1 25 Macro elements of a prokaryotic cell Macro element Percent of dry weight Carbon (C) 50 Hydrogen (H) 8 Oxygen (O) 20 Nitrogen (N) 14 Phosphorus (P) 3 Sulfur (S) 1 Potassium (K) 1 Magnesium (Mg) 0.5 Calcium (Ca) 0.5 Iron (Fe)
14 Trace elements of prokaryotic cell Trace element Cellular function (example) Cobalt (Co) Copper (Cu) Molybdenum (Mo) Nickel (Ni) Selenium (Se) Tungsten (W) Vanadium (V) Zinc Iron (Fe) Vitamin B12 respiration, photosynthesis nitrogenase, nitrate reductase hydrogenase Hydrogenase, formate dehydrogenase Formate dehydrogenase Vanadium nitrogenase Alcohol dehydrogenase, RNA and DNA polymerases, DNA-binding protein Cytochromes, catalases, oxygenases 27 General requirements in microbiological media Energy source Source of macro elements (including carbon and nitrogen) Source of trace elements Buffer Growth factors (including Vitamins or amino acids) 28 14
15 Chemically defined versus undefined (complex) media Defined medium for E. coli Undefined medium for E. coli K 2 HPO 4 7 g KH 2 PO 4 2 g (NH 4 )SO 4 1 g MgSO g CaCl g Glucose 5-10 g Trace element solution Destilled water 1000 ml Glucose 15 g Yest extract 5 g Peptone 5 g KH 2 PO 4 2 g Destilled water 1000 ml 29 Isolation of microorganisms into pure cultures A culture containing only a single kind of microorganism, originate from a single cell (monoclonal). Most common is the isolation of microbes by the use of solid media. Alternatives: serial agar dilution, serial liquid dilution Highest priority: Avoid contaminants! 30 15
16 Why do we need pure cultures? Precise physiology Biochemistry and structure Taxonomy Genetics Reproducibility of experiments 31 The majority of microbes present in nature have no counterpart among previously cultured organism validly described species versus about species in 1L sea water about species in 1g soil total of 10 millions (estimations) 32 16
17 How to apply cultivation? Estimation of bacterial numbers using MPN Selective enrichment and isolation of members belonging to one physiological group Culturing an abundant phylotype Cultivation of all microorganisms from a marine environment 33 Estimation of bacteria numbers by tenfold dilution series MPN - most probable number Estimation of viable microorganisms Obtained by the statistical method of maximum likelihood Many variations in cultivation conditions possible (complex - defined medium) Detection of growth essential 34 17
18 35 Quelle: Brock Biology of Microorganisms Chemostat In a chemostat cells can be cultivated under contant conditions in an exponential phase with! <! max 36 18
19
20 Essentials of successful cultivation Scientific question/ hypothesis Medium choice Carbon and energy source Other media components Gelling agent Inoculum and interaction Growth conditions, temperature, ph, atmosphere Incubation time
21
22 43 Culturing anaerobes Oxygen free media.! Remove oxygen! Keep it away Low redox potential! Addition of reducing agents Optional: oxygen (redox) indicator 44 22
23 Culturing anaerobes Flush headspace (Hungate-technique) Cultivation in sealed anaerobic jars or chambers Cultivation without gaseous headspace Co-culture with oxygen consuming bacteria
24 The Widdel-flask 47 24
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