1

Growth and Cultivation of bacteria

2

GGrowthrowth requirements requirements

• oxygen (or absence)oxygen (or absence)• energy energy • nutrientsnutrients• optimal temperatureoptimal temperature• optimal pHoptimal pH

Oxygen requirements

4

Obligate aerobesObligate aerobes

• grow in presence of oxygengrow in presence of oxygen• no fermentationno fermentation• oxidative phosphorylationoxidative phosphorylation

5

• killed by oxygen • fermentation • no oxidative phosphorylation

• lack certain enzymes:superoxide dismutase O2

-+2H+ => H2O2

catalase H2O2 => H20 + O2

peroxidase H2O2 + NADH + H+ => 2H20 + NAD

Obligate anaerobesObligate anaerobes

6

Aerotolerant anaerobesAerotolerant anaerobesnot killed by oxygennot killed by oxygen

• respire anaerobicallyrespire anaerobically

7

Facultative anaerobesFacultative anaerobes• fermentation fermentation • aerobic respirationaerobic respiration• survive in oxygensurvive in oxygen

8

Microaerophilic bacteriaMicroaerophilic bacteria

• grow grow – low oxygenlow oxygen

• killed killed – high oxygenhigh oxygen

Temperature

10

Optimal growth temperature Optimal growth temperature • Mesophiles: Mesophiles:

– human body temperaturehuman body temperature* pathogens pathogens * opportunistsopportunists

• pyschrophilepyschrophile– close to freezing close to freezing

• thermophilethermophile– close to boilingclose to boiling

11

pH

• Many grow best at neutral pH

• Some can survive/grow

- acid

- alkali

12

Nutrient RequirementsNutrient Requirements• Carbon Carbon • NitrogenNitrogen• PhosphorusPhosphorus• SulfurSulfur• Metal ions (e.g. iron)Metal ions (e.g. iron)

13

Siderophores (S)Siderophores (S)

Fe Fe 2+2+//SS

Fe Fe 2+2+//SS

ReceptorReceptorTransport of ironTransport of iron

14

Measuring bacterial mass (live + dead) Measuring bacterial mass (live + dead) in liquid culturein liquid culture

TurbidityTurbidity(Cloudiness)(Cloudiness)

15

Measuring viable bacteria

colonycolony

Colony forming unitsColony forming units

16

Growth Curve

COLONY FORMING UNITS

TIME

Lag

Log

Stationary

Death

17

Growth Curve

TURBIDITY(cloudiness)

TIME

Lag

Log

Stationary

Autolysis

18

Generation timeGeneration time

• time for bacterial mass to doubletime for bacterial mass to double

• Example Example 100 bacteria present at time 0 100 bacteria present at time 0 If generation time is 2 hrIf generation time is 2 hr After 8 hr mass = 100 x 2After 8 hr mass = 100 x 244

19

SUGAR CATABOLISM

• Glycolysis

– Embden Meyerhof Parnas Pathway– most bacteria– also animals and plants

20

Other pathways for catabolizing sugars

• Pentose phosphate pathway (hexose monophosphate shunt)– generates NADPH– common in plants and animals

• Entner Doudoroff Pathway – a few bacterial species

21

•

GlycolysisGlycolysisNADNAD NADHNADH

GlucoseGlucose PyruvatePyruvateC6C6 C3C3

ADP ADP ATPATP

22

FermentationFermentation

PyruvatePyruvate

(C3)(C3)

NADHNADH NADNAD

Short chain alcoholsShort chain alcohols, , fatty acidsfatty acids(C2-C4)(C2-C4)

23

Anaerobic Respiration = Anaerobic Respiration = Glycolysis + FermentationGlycolysis + Fermentation

NADNAD NADHNADH

NADHNADH NADNAD

ATPATP

24

Krebs Cycle (C4-C6 intermediate compoundsKrebs Cycle (C4-C6 intermediate compounds)

PyruvatePyruvate 3CO3CO22

(C3)(C3)

NADNAD NADHNADH

NADHNADH NADNAD

Oxidative phosphorylationOxidative phosphorylation

OO22 HH22OO

ADPADP ATPATP

(C1)(C1)

25

Aerobic Respiration =Glycolysis +

Krebs Cycle/oxidative phosphorylation

• Pyruvate to COPyruvate to CO22

– NADNAD toto NADHNADH

– glycolysis glycolysis

– Krebs cycleKrebs cycle

• Oxidative phosphorylationOxidative phosphorylation

– NADHNADH to to NAD NAD

– ADPADP to to ATP ATP

26

Oxidative phosphorylationOxidative phosphorylation

• converts Oconverts O22 to H to H220 0 (oxidative)(oxidative)

• converts ADP to ATP converts ADP to ATP (phosphorylation)(phosphorylation)

• electron transport chainelectron transport chain

• ubiquinones/cytochrome intermediates ubiquinones/cytochrome intermediates

27

The Krebs cycle

Citrate

Isocitrate

Alpha-keto glutarate

Succinate

Fumarate

Malate

OxaloacetatePyruvate

-CO-CO22

Acetate

+-CO-CO2 2 NADHNADH

-CO-CO2 2 NADHNADH

C2

C

C

C4

X

x

C6

28

Krebs Cycle - sugar as sole Krebs Cycle - sugar as sole carbon sourcecarbon source

PyruvatePyruvate

AcetateAcetate-CO-CO22

C4C4

PyruvatePyruvate+ CO+ CO22

+Citrate

CCC3C3

Oxaloacetate

Oxaloacetate

-2CO-2CO22

Aspartic acidAspartic acid

Krebs Krebs cyclecycle

ENERGYSTORAGE

BIOSYNTHESISC3C3

CCC2C2

C6C6C4C4

OxaloacetateX

29

Krebs Cycle – fatty acids as Krebs Cycle – fatty acids as sole carbon sourcesole carbon source

Fatty acidsFatty acids AcetateAcetate

+ CitrateOxaloacetate

-2CO-2CO22

Aspartic acidAspartic acid

Krebs Krebs cyclecycle

ENERGY

BIOSYNTHESIS

Isocitrate Succinate Glyoxylate+

AcetateAcetate+

MalateMalate

Oxaloacetatex

C4

C2

C2

C4C6

-2CO-2CO22

Krebs cycle

30

The Glyoxylate and Krebs cycles

Citrate

Isocitrate

Alpha-keto glutarate

Succinate

Fumarate

Glyoxylate

++ AcetateMalate

Oxaloacetate

1

2

Krebs cycle only Glyoxylate cycle onlyKrebs and Glyoxylate cycles

31

Krebs CycleKrebs Cycle

– biosyntheticbiosynthetic

– energy storage energy storage

• Removal of intermediatesRemoval of intermediates

– must be replenishedmust be replenished

• Unique enzymatic replenishment pathwaysUnique enzymatic replenishment pathways

– sugars sugars

– fatty acidsfatty acids

Major nutritional types of procaryotes 

Nutritional Type Energy Source Carbon Source Examples

Photoautotrophs Light CO2

Cyanobacteria, some Purple and Green Bacteria

Photoheterotrophs Light Organic compounds Some Purple and Green Bacteria

Chemoautotrophsor Lithotrophs(Lithoautotrophs)

Inorganic compounds, e.g. H2, NH3, NO2, H2S

CO2A few Bacteria and

many Archaea

Chemoheterotrophs or Heterotrophs Organic compounds Organic compounds Most Bacteria,

some Archaea