gen (t) number log 2n log 10 n - unipd.it frontali stbc... · figure 9.1 electrons released during...

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)

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

8

Le conte vitali e il concetto di CFU

9


10


11

Fattori ambientali determinanti per la crescita: soluti ed attività dell’acqua, pH, pressione, temperatura, ..

Applicazioni industriali di enzimi termoresistenti………...

12

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

13

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


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


18

Electron acceptors for chemotrophic processes

Figure 9.2 exogenous electron acceptors


19

Chemoorganotrophic metabolism• fermentation

– energy source oxidized and degraded using endogenous electron acceptor

– often occurs under anaerobic conditions

– limited energy made available


20

Chemoorganotrophic metabolism

• aerobic respiration– energy source degraded using oxygen

as exogenous electron acceptor– yields large amount of energy,

primarily by electron transport activity


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


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)


23

Figure 9.3

manydifferentenergysources are funneledinto commondegradativepathways

ATP madeprimarilybyoxidativephosphory-lation


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


25

The Breakdown of Glucose to Pyruvate

• Three common routes– glycolysis– pentose phosphate pathway– Entner-Doudoroff pathway


26

The Glycolytic Pathway

• also called Embden-Meyerhof pathway

• occurs in cytoplasmic matrix of both procaryotes and eucaryotes


27

Figure 9.5

addition of phosphates“primes the pump”

oxidation step –generates NADH

high-energy molecules –used to synthesize ATPby substrate-levelphosphorylation


28

Summary of glycolysis

glucose + 2ADP + 2Pi + 2NAD+

↓↓↓↓

2 pyruvate + 2ATP + 2NADH + 2H+


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


30

Figure 9.6

oxidationsteps

produceNADPH,which isneeded forbiosynthesis

sugartrans-formationreactions

producesugarsneededforbiosynthesis

sugars canalso befurtherdegraded


31

Figure 9.7


32

Summary of pentose phosphate pathway

glucose-6-P + 12NADP+ + 7H2O

↓↓↓↓

6CO2 + 12NADPH + 12H+ Pi


33

The Entner-Doudoroff Pathway• yield per

glucose molecule:– 1 ATP

– 1 NADPH

– 1 NADH

Figure 9.8

reactions ofglycolyticpathway

reactions ofpentosephosphatepathway


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


35

Figure 9.10

homolacticfermenters

heterolacticfermenters

foodspoilage

yogurt,sauerkraut,pickles, etc.

alcoholicfermentation

alcoholicbeverages,bread, etc.


36

methyl red test – detects pH change in media caused bymixed acid fermentation


37

Butanediol fermentation

Voges-Proskauer test –detects intermediate acetoin

Methyl red test and Voges-Proskauer test important fordistinguishing pathogenicmembers ofEnterobacteriaceae


38

Fermentations of amino acids• Strickland

reaction– oxidation of

one amino acid with use of second amino acid as electron acceptor

Figure 9.11


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


40 Figure 9.12

high-energymolecule

oxidation anddecarbox-ylation steps

completeoxidation anddegradation

also formNADH

energy drivescondensationof acetylgroup withoxaloacetate

substrate-levelphosphory-lation

oxidationsteps – formNADH andFADH2


41

Summary

• for each acetyl-CoA molecule oxidized, TCA cycle generates:– 2 molecules of CO2

– 3 molecules of NADH– one FADH2

– one GTP


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)


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


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


45

Figure 9.13

large difference inE0 of NADH andE0 of O2

large amount ofenergy released


46

Mitochondrial ETC

Figure 9.14 electron transfer accompanied byproton movement across innermitochondrial membrane


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


48

ETC of E. coli

Figure 9.15

branched pathway

upper branch –stationary phase andlow aeration

lower branch – log phase and highaeration


49

ETC of Paracoccus denitrificans - aerobic

Figure 9.16a


50

ETC of P. denitrificans -anaerobic

Figure 9.16b example of anaerobic respiration


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)


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


53

Figure 9.17

movement of protonsestablishesPMF

ATP synthaseuses protonflow downgradient to make ATP


54

Figure 9.19a


55

Figure 9.19b


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


57

Importance of PMF

Figure 9.18


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


59


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


61

Anaerobic Respiration

• uses electron carriers other than O2

• generally yields less energy because E0of electron acceptor is less positive than E0 of O2


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


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)


64

Carbohydrates• monosaccharides

– converted to other sugars that enter glycolytic pathway

• disaccharides and polysaccharides– cleaved by

hydrolases or phosphorylases

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