Download N = N 2n

Gen(t)
number
of bacteria
log2N
log10N
Gen(t)
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
...
n (t)
2n
number of bacteria
0(0) 1
N0
n (t) 2n N0 2n
Nt = N0 2n
Vedi dip. lin.
f(t)
1
2
3
4
5
Conta cellulare totale con la camera di Petroff-Hausser
o con il Coulter Counter (pref. dimens.eucar.)
1 mm
6
Cellule biomassa
proteine DNA ecc
Il concetto di crescita bilanciata
time
Dry weight - Cell mass
determination. Sensitivity: ~ 109
cells/mg; tedious; timeconsuming.
* Filter cells from a known
volume of culture.
* Wash to remove medium
components.
* Dry.
* Weigh.
Nota: Le misure di assorbanza riflettono la massa, ma anche
il numero, la forma, la complessità
delle cellule
7
Le conte vitali e il concetto di CFU
8
Le conte vitali e il concetto di CFU
9
Le conte vitali e il concetto di CFU
10
Fattori ambientali determinanti per la crescita: soluti ed attività dell’acqua, pH,
pressione, temperatura, ..
Applicazioni industriali di enzimi termoresistenti………...
11
Fattori ambientali e crescita
1-acqua
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 campione
P0=press. Vap. di acqua pura
Moltiplicando la attività dell’acqua per 100 abbiamo l’umidità
relativa dell’atmosfera in equilibrio col campione.
R.H. (%) = 100 x aw
Salando, essiccando e zuccherando un alimento ne diminuiamo P e quindi
aw (aw e pressione osmotica sono inversamente correlati)
12
La pressione osmotica
I batteri resistono a notevoli press osmotiche grazie alla forza
meccanica della parete ( si contrappone alla pressione
idrostatica in un ambiente ipotonico)
I protozoi contraggono un vacuolo che convoglia l’acqua
attirata per osmosi espellendola dall cellula
13
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 sale
possono 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 proteine
Terreni di selezione; uso di tamponi
pH int ca.7
Gli ambienti iperosmotici
14
[O2], ecc..
15
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
16
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Sources of energy
electrons released
during oxidation
of chemical
energy sources
must be accepted
by an electron
acceptor
microorganisms
vary in terms of the
acceptors they use
Figure 9.1
17
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Electron acceptors for
chemotrophic processes
Figure 9.2
18
exogenous electron acceptors
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Chemoorganotrophic
metabolism
• fermentation
– energy source oxidized and degraded
using endogenous electron acceptor
– often occurs under anaerobic
conditions
– limited energy made available
19
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Chemoorganotrophic
metabolism
• aerobic respiration
– energy source degraded using oxygen
as exogenous electron acceptor
– yields large amount of energy,
primarily by electron transport activity
20
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
21
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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)
22
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
many
different
energy
sources
are
funneled
into
common
degradative
pathways
ATP made
primarily
by
oxidative
phosphorylation
Figure 9.3
23
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
24
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
The Breakdown of Glucose
to Pyruvate
• Three common routes
– glycolysis
– pentose phosphate pathway
– Entner-Doudoroff pathway
25
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
The Glycolytic Pathway
• also called Embden-Meyerhof
pathway
• occurs in cytoplasmic matrix of both
procaryotes and eucaryotes
26
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addition of phosphates
“primes the pump”
oxidation step –
generates NADH
high-energy molecules –
used to synthesize ATP
by substrate-level
phosphorylation
Figure 9.5
27
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Summary of glycolysis
glucose + 2ADP + 2Pi + 2NAD+
2 pyruvate + 2ATP + 2NADH + 2H+
28
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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
29
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
oxidation
steps
produce
NADPH,
which is
needed for
biosynthesis
Figure 9.6
30
sugar
transformation
reactions
produce
sugars
needed
for
biosynthesis
sugars can
also be
further
degraded
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Figure 9.7
31
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Summary of pentose
phosphate pathway
glucose-6-P + 12NADP+ + 7H2O
6CO2 + 12NADPH + 12H+ Pi
32
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
The Entner-Doudoroff
Pathway
• yield per
glucose
molecule:
– 1 ATP
– 1 NADPH
– 1 NADH
Figure 9.8
33
reactions of
glycolytic
pathway
reactions of
pentose
phosphate
pathway
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Fermentations
• oxidation of
NADH produced
by glycolysis
• pyruvate or
derivative used as
endogenous
electron acceptor
• ATP formed by
substrate-level
phosphorylation
34
Figure 9.9
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
homolactic
fermenters
heterolactic
fermenters
food
spoilage
yogurt,
sauerkraut,
pickles, etc.
Figure 9.10
35
alcoholic
fermentation
alcoholic
beverages,
bread, etc.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
methyl red test – detects pH change in media caused by
mixed acid fermentation
36
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Butanediol fermentation
Voges-Proskauer test –
detects intermediate acetoin
Methyl red test and VogesProskauer test important for
distinguishing pathogenic
members of
Enterobacteriaceae
37
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Fermentations of amino
acids
• Strickland
reaction
– oxidation of
one amino
acid with use
of second
amino acid
as electron
acceptor
Figure 9.11
38
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
39
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energy drives
condensation
high-energy of acetyl
molecule
group with
oxaloacetate
oxidation
steps – form
NADH and
FADH2
oxidation and
decarboxylation steps
complete
oxidation and
degradation
substratelevel
phosphorylation
40
also form
NADH
Figure 9.12
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Summary
• for each acetyl-CoA molecule
oxidized, TCA cycle generates:
– 2 molecules of CO2
– 3 molecules of NADH
– one FADH2
– one GTP
41
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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)
42
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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
43
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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
44
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
large difference in
E0 of NADH and
E0 of O2
large amount of
energy released
Figure 9.13
45
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Mitochondrial ETC
Figure 9.14
46
electron transfer accompanied by
proton movement across inner
mitochondrial membrane
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
47
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
ETC of E. coli
branched pathway
upper branch –
stationary phase and
low aeration
lower branch – log
phase and high
aeration
Figure 9.15
48
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ETC of Paracoccus
denitrificans - aerobic
Figure 9.16a
49
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ETC of P. denitrificans anaerobic
Figure 9.16b
50
example of anaerobic respiration
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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)
51
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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
52
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movement of
protons
establishes
PMF
Figure 9.17
53
ATP synthase
uses proton
flow down
gradient to
make ATP
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Figure 9.19a
54
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Figure 9.19b
55
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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
56
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Importance of PMF
Figure 9.18
57
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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
58
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59
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
60
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Anaerobic Respiration
• uses
electron
carriers
other than
O2
• generally
yields less
energy
because E0
of electron
acceptor is
less positive
than E0 of
O2
61
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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
62
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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)
63
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Carbohydrates
• monosaccharides
– converted to
other sugars that
enter glycolytic
pathway
• disaccharides and
polysaccharides
– cleaved by
hydrolases or
phosphorylases
64