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1
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)
2
3
4
5
6
1 mm
Conta cellulare totale con la camera di Petroff-Haussero con il Coulter Counter (pref. dimens.eucar.)
7
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
10
Le conte vitali e il concetto di CFU
11
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
14
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.
7
15
[O2], ecc..
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
16
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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17
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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18
Electron acceptors for chemotrophic processes
Figure 9.2 exogenous electron acceptors
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19
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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20
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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21
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
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
22
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)
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
23
Figure 9.3
manydifferentenergysources are funneledinto commondegradativepathways
ATP madeprimarilybyoxidativephosphory-lation
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24
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
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
25
The Breakdown of Glucose to Pyruvate
• Three common routes– glycolysis– pentose phosphate pathway– Entner-Doudoroff pathway
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
26
The Glycolytic Pathway
• also called Embden-Meyerhof pathway
• occurs in cytoplasmic matrix of both procaryotes and eucaryotes
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27
Figure 9.5
addition of phosphates“primes the pump”
oxidation step –generates NADH
high-energy molecules –used to synthesize ATPby substrate-levelphosphorylation
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
28
Summary of glycolysis
glucose + 2ADP + 2Pi + 2NAD+
↓↓↓↓
2 pyruvate + 2ATP + 2NADH + 2H+
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
29
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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30
Figure 9.6
oxidationsteps
produceNADPH,which isneeded forbiosynthesis
sugartrans-formationreactions
producesugarsneededforbiosynthesis
sugars canalso befurtherdegraded
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
31
Figure 9.7
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32
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.
33
The Entner-Doudoroff Pathway• yield per
glucose molecule:– 1 ATP
– 1 NADPH
– 1 NADH
Figure 9.8
reactions ofglycolyticpathway
reactions ofpentosephosphatepathway
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
34
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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35
Figure 9.10
homolacticfermenters
heterolacticfermenters
foodspoilage
yogurt,sauerkraut,pickles, etc.
alcoholicfermentation
alcoholicbeverages,bread, etc.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
36
methyl red test – detects pH change in media caused bymixed acid fermentation
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
37
Butanediol fermentation
Voges-Proskauer test –detects intermediate acetoin
Methyl red test and Voges-Proskauer test important fordistinguishing pathogenicmembers ofEnterobacteriaceae
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
38
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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39
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
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
40 Figure 9.12
high-energymolecule
oxidation anddecarbox-ylation steps
completeoxidation anddegradation
also formNADH
energy drivescondensationof acetylgroup withoxaloacetate
substrate-levelphosphory-lation
oxidationsteps – formNADH andFADH2
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
41
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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42
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)
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
43
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
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
44
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
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
45
Figure 9.13
large difference inE0 of NADH andE0 of O2
large amount ofenergy released
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
46
Mitochondrial ETC
Figure 9.14 electron transfer accompanied byproton movement across innermitochondrial membrane
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
47
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
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
48
ETC of E. coli
Figure 9.15
branched pathway
upper branch –stationary phase andlow aeration
lower branch – log phase and highaeration
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
49
ETC of Paracoccus denitrificans - aerobic
Figure 9.16a
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50
ETC of P. denitrificans -anaerobic
Figure 9.16b example of anaerobic respiration
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51
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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52
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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53
Figure 9.17
movement of protonsestablishesPMF
ATP synthaseuses protonflow downgradient to make ATP
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54
Figure 9.19a
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55
Figure 9.19b
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56
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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57
Importance of PMF
Figure 9.18
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58
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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59
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60
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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61
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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62
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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63
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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64
Carbohydrates• monosaccharides
– converted to other sugars that enter glycolytic pathway
• disaccharides and polysaccharides– cleaved by
hydrolases or phosphorylases