Post on 14-Feb-2018
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Gen(t) number log2N log10Nof bacteria
0(0’) 1 or 20 0 0
1(20’)2 or 21 1 .301
2(40’)4 or 22 2 .602
3(60’)8 or 23 3 .903
4 ... 16 or 24 4 ...
5 ... 32 or 25 5 …
n (t) 2n n ...
Gen(t) number of bacteria
0(0) 1 N0
n (t) 2n N0 2n
Nt = N0 2n
Vedi dip. lin.f(t)
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3
4
5
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1 mm
Conta cellulare totale con la camera di Petroff-Haussero con il Coulter Counter (pref. dimens.eucar.)
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Il concetto di crescita bilanciata
Cel
lule
b
iom
assa
prot
eine
D
NA
ecc
Nota: Le misure di assorbanza riflettono la massa, ma anche il numero, la forma, la complessitàdelle cellule
Dry weight - Cell mass determination. Sensitivity: ~ 109
cells/mg; tedious; time-consuming.
* Filter cells from a knownvolume of culture.
* Wash to remove medium components.
* Dry. * Weigh. time
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Le conte vitali e il concetto di CFU
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Le conte vitali e il concetto di CFU
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Le conte vitali e il concetto di CFU
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Fattori ambientali determinanti per la crescita: soluti ed attività dell’acqua, pH, pressione, temperatura, ..
Applicazioni industriali di enzimi termoresistenti………...
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I microbi si alimentano sull' acqua libera e non possono accedere all' acqua segregata da altre molecole. I gruppi idrossilici dei polisaccaridi, carbossilici e aminici delle proteine ad esempio legano l’acqua
L' attività dell' acqua (aw) è la misura di quanto l' acqua è legata strutturalmente o chimicamente, in una sostanza o cellula.
aw = P/P0
P=pressione vapore del campioneP0=press. Vap. di acqua puraMoltiplicando la attività dell’acqua per 100 abbiamo l’umiditàrelativa dell’atmosfera in equilibrio col campione.R.H. (%) = 100 x aw
Salando, essiccando e zuccherando un alimento ne diminuiamo P e quindiaw (aw e pressione osmotica sono inversamente correlati)
Fattori ambientali e crescita1-acqua
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La pressione osmotica
I batteri resistono a notevoli press osmotiche grazie alla forzameccanica della parete ( si contrappone alla pressione idrostatica in un ambiente ipotonico)
I protozoi contraggono un vacuolo che convoglia l’acquaattirata per osmosi espellendola dall cellula
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Gli ambienti iperosmotici
sintesi di soluti compatibili con le attività cellulari:colina, betaina, prolina, glicerolo, glutamico ecc
possibilità di fare selezioni per osmotolleranti come gli stafilococchi (crescono sulla cute)->terreni con 7-8% sali
-->gli alofili, richiedono alto salepossono accumulare enormi quantità di sali intracellulari (es. potassio) e hanno modificazioni strutturali di mbr pareti e proteine(archea)
Il pH: i batteri di solito sono neutrofili, i funghi acidofili moderati.Meccanismi: antiporti ioni/H+,H+ATPasi,nuove proteineTerreni di selezione; uso di tamponi
pH in
tca.
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[O2], ecc..
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An Overview of Metabolism• metabolism
– total of all chemical reactions occurring in cell
• catabolism– breakdown of larger, more complex
molecules into smaller, simpler ones– energy is released and some is trapped and
made available for work
• anabolism– synthesis of complex molecules from simpler
ones with the input of energy
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Sources of energy
Figure 9.1
electrons releasedduring oxidation of chemical energy sources must be accepted by an electron acceptor
microorganisms vary in terms of the acceptors they use
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Electron acceptors for chemotrophic processes
Figure 9.2 exogenous electron acceptors
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Chemoorganotrophic metabolism• fermentation
– energy source oxidized and degraded using endogenous electron acceptor
– often occurs under anaerobic conditions
– limited energy made available
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Chemoorganotrophic metabolism
• aerobic respiration– energy source degraded using oxygen
as exogenous electron acceptor– yields large amount of energy,
primarily by electron transport activity
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Chemoorganotrophic metabolism• anaerobic respiration
– energy source oxidized and degraded using molecules other than oxygen as exogenous electron acceptors
– can yield large amount of energy (depending on reduction potential of energy source and electron acceptor), primarily by electron transport activity
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Overview of aerobic catabolism• three-stage process
– large molecules (polymers) →→→→ small molecules (monomers)
– initial oxidation and degradation to pyruvate
– oxidation and degradation of pyruvate by the tricarboxylic acid cycle (TCA cycle)
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Figure 9.3
manydifferentenergysources are funneledinto commondegradativepathways
ATP madeprimarilybyoxidativephosphory-lation
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Two functions of organic energy sources• oxidized to release
energy• supply carbon and
building blocks for anabolism– amphibolic pathways
• function both as catabolic and anabolic pathways
Figure 9.4
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The Breakdown of Glucose to Pyruvate
• Three common routes– glycolysis– pentose phosphate pathway– Entner-Doudoroff pathway
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The Glycolytic Pathway
• also called Embden-Meyerhof pathway
• occurs in cytoplasmic matrix of both procaryotes and eucaryotes
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Figure 9.5
addition of phosphates“primes the pump”
oxidation step –generates NADH
high-energy molecules –used to synthesize ATPby substrate-levelphosphorylation
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Summary of glycolysis
glucose + 2ADP + 2Pi + 2NAD+
↓↓↓↓
2 pyruvate + 2ATP + 2NADH + 2H+
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The Pentose Phosphate Pathway• also called hexose monophosphate
pathway• can operate at same time as glycolytic or
Entner-Doudoroff pathways• can operate aerobically or anaerobically• an amphibolic pathway
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Figure 9.6
oxidationsteps
produceNADPH,which isneeded forbiosynthesis
sugartrans-formationreactions
producesugarsneededforbiosynthesis
sugars canalso befurtherdegraded
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Figure 9.7
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Summary of pentose phosphate pathway
glucose-6-P + 12NADP+ + 7H2O
↓↓↓↓
6CO2 + 12NADPH + 12H+ Pi
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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The Entner-Doudoroff Pathway• yield per
glucose molecule:– 1 ATP
– 1 NADPH
– 1 NADH
Figure 9.8
reactions ofglycolyticpathway
reactions ofpentosephosphatepathway
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Fermentations• oxidation of
NADH produced by glycolysis
• pyruvate or derivative used as endogenous electron acceptor
• ATP formed by substrate-level phosphorylation Figure 9.9
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Figure 9.10
homolacticfermenters
heterolacticfermenters
foodspoilage
yogurt,sauerkraut,pickles, etc.
alcoholicfermentation
alcoholicbeverages,bread, etc.
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methyl red test – detects pH change in media caused bymixed acid fermentation
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Butanediol fermentation
Voges-Proskauer test –detects intermediate acetoin
Methyl red test and Voges-Proskauer test important fordistinguishing pathogenicmembers ofEnterobacteriaceae
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Fermentations of amino acids• Strickland
reaction– oxidation of
one amino acid with use of second amino acid as electron acceptor
Figure 9.11
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The Tricarboxylic Acid Cycle
• also called citric acid cycle and Kreb’s cycle
• completes oxidation and degradation of glucose and other molecules
• common in aerobic bacteria, free-living protozoa, most algae, and fungi
• amphibolic– provides carbon skeletons for biosynthesis
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40 Figure 9.12
high-energymolecule
oxidation anddecarbox-ylation steps
completeoxidation anddegradation
also formNADH
energy drivescondensationof acetylgroup withoxaloacetate
substrate-levelphosphory-lation
oxidationsteps – formNADH andFADH2
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Summary
• for each acetyl-CoA molecule oxidized, TCA cycle generates:– 2 molecules of CO2
– 3 molecules of NADH– one FADH2
– one GTP
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Electron Transport and Oxidative Phosphorylation
• only 4 ATP molecules synthesized directly from oxidation of glucose to CO2
• most ATP made when NADH and FADH2 (formed as glucose degraded) are oxidized in electron transport chain (ETC)
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The Electron Transport Chain
• series of electron carriers that operate together to transfer electrons from NADH and FADH2 to a terminal electron acceptor
• electrons flow from carriers with more negative E0 to carriers with more positive E0
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Electron transport chain…
• as electrons transferred, energy released• some released energy used to make ATP
by oxidative phosphorylation– as many as 3 ATP molecules made per
NADH using oxygen as acceptor• P/O ratio = 3
– P/O ratio for FADH2 is 2• i.e., 2 ATP molecules made
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Figure 9.13
large difference inE0 of NADH andE0 of O2
large amount ofenergy released
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Mitochondrial ETC
Figure 9.14 electron transfer accompanied byproton movement across innermitochondrial membrane
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Procaryotic ETCs• located in plasma membrane• some resemble mitochondrial ETC,
but many are different– different electron carriers– may be branched– may be shorter– may have lower P/O ratio
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ETC of E. coli
Figure 9.15
branched pathway
upper branch –stationary phase andlow aeration
lower branch – log phase and highaeration
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ETC of Paracoccus denitrificans - aerobic
Figure 9.16a
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ETC of P. denitrificans -anaerobic
Figure 9.16b example of anaerobic respiration
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Oxidative Phosphorylation
• chemiosmotic hypothesis– most widely accepted explanation of
oxidative phosphorylation– postulates that energy released during
electron transport used to establish a proton gradient and charge difference across membrane• called proton motive force (PMF)
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PMF drives ATP synthesis• diffusion of protons back across
membrane (down gradient) drives formation of ATP
• ATP synthase– enzyme that uses proton movement
down gradient to catalyze ATP synthesis
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Figure 9.17
movement of protonsestablishesPMF
ATP synthaseuses protonflow downgradient to make ATP
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Figure 9.19a
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Figure 9.19b
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Inhibitors of ATP synthesis• blockers
– inhibit flow of electrons through ETC
• uncouplers– allow electron flow, but disconnect it from
oxidative phosphorylation– many allow movement of ions, including
protons, across membrane without activating ATP synthase
• destroys pH and ion gradients
– some may bind ATP synthase and inhibit its activity directly
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Importance of PMF
Figure 9.18
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The Yield of ATP in Glycolysis and Aerobic Respiration• aerobic respiration provides much more
ATP than fermentation• Pasteur effect
– decrease in rate of sugar metabolism when microbe shifted from anaerobic to aerobic conditions
– occurs because aerobic process generates greater ATP per sugar molecule
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ATP yield…
• amount of ATP produced during aerobic respiration varies depending on growth conditions and nature of ETC
• under anaerobic conditions, glycolysis only yields 2 ATP molecules
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Anaerobic Respiration
• uses electron carriers other than O2
• generally yields less energy because E0of electron acceptor is less positive than E0 of O2
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An example
• dissimilatory nitrate reduction– use of nitrate as terminal electron
acceptor– denitrification
• reduction of nitrate to nitrogen gas
• in soil, causes loss of soil fertility
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Catabolism of Carbohydrates and
Intracellular Reserves
• many different carbohydrates can serve as energy source
• carbohydrates can be supplied externally or internally (from internal reserves)
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Carbohydrates• monosaccharides
– converted to other sugars that enter glycolytic pathway
• disaccharides and polysaccharides– cleaved by
hydrolases or phosphorylases