julia vorholt lecture 6 - eth zn.ethz.ch/~nielssi/download/3. semester/mikrobiologie...copyright ©...

Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Brock Biology of Microorganisms, Twelfth Edition – Madigan / Martinko / Dunlap / Clark

752-4001-00L Mikrobiologie

Julia Vorholt Lecture 6: Nutrients, microbial growth and introduction to principles of metabolism Oct 29, 2012


Brock Biology of Microorganisms, Twelfth Edition – Madigan / Martinko / Dunlap / Clark

1) Nutrients and microbial growth 2) Introduction to principles of metabolism 3) Chemoorganotrophy 4) Chemolithotrophy 5) Phototrophy 6) Autotrophy, nitrogen fixation 7) Global carbon, nitrogen, sulfur cycles


Microbial Nutrition: Microbial Periodic Table of Elements

Fig. 4.1 Chap. 4.1

Group

Period 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

1

2

3

4

5

6

Essential for all microorganisms Essential cations and anions for most microorganisms Trace metals, some essential for some microorganisms Used for special functions Unessential, but metabolized Unessential, not metabolized

Essential elements as a percent of cell dry weight Macromolecular composition of a cell

Protein Lipid Polysaccharide Lipopolysaccharide DNA RNA

Macromolecule Percent of dry weight

50% 17% 13% 8.2%

2.5% 1.8%

55 9.1 5.0 3.4 3.1

20.5


Microbial Nutrition

Carbon Typical bacterial cell ~50% carbon (by dry weight) Major element in all classes of macromolecules

Nitrogen Typical bacterial cell ~13% nitrogen (by dry weight) Key element in proteins, nucleic acids, and many more cell

constituents

Chap. 4.1

Heterotrophs use carbon from reduced (organic) carbon compounds Autotrophs use CO2 as main carbon source


Microbial Nutrients

Some examples of macroelements:

Phosphorus (P) Required by cell for synthesis of nucleic acids and

phospholipids

Sulfur (S) Plays structural role in S-containing amino acids (cysteine

and methionine) Present in several vitamins

Iron (Fe) Plays major role in cellular respiration; key component of

cytochromes and FeS proteins involved in electron transport

Chap. 4.1


Culture Media

Culture Media

Nutrient solutions used to grow microbes in the laboratory

Two broad classes

Defined media: precise chemical composition is known

Complex media: composed of digests of chemically undefined

substances (e.g., yeast and meat extracts)

Chap. 4.2

For successful cultivation of a microorganism it is important to know the nutritional requirements and supply them in proper form and proportions in a culture medium


Culture Media


Cell Growth and Binary Fission

Growth: increase in the number of cells OR increase in biomass

Binary fission: cell elongation following enlargement of a cell to twice its minimum size

Generation time: time required for a population of microbial cells to double

During cell division each daughter cell receives a chromosome and sufficient copies of all other cell constituents to exist as an independent cell

Fig. 5.1 Chap. 5.1

Septum

Cellelongation

Septumformation

Completionof septum;formation ofwalls; cellseparation

One

gen

erat

ion


The Microbial Growth Cycle

Batch culture: a closed-system microbial culture of fixed volume

Typical growth curve for population of cells grown in a closed system is characterized by four phases Lag phase Exponential phase Stationary phase Death phase

Chap. 5.2-5.3


Typical Growth Curve for a Bacterial Population

Fig. 6.10 Chap. 5.7


Measuring Microbial Growth

Measurements of Total Cell Numbers: Microscopic Counts

Viable Cell Counting

Measurements of Microbial Mass: Turbidimetric Methods

Chap. 5.9-5.11


Direct Microscopic Counting Procedure

Fig. 5.14

Microbial cells can be enumerated by microscopic observations

Chap. 5.9

Limitations of microscopic counts Live/dead cells Small cells difficult to see Low density hard to count Motile cells Debris (mistaken for cells)


Viable Cell Counting

Viable cell counts (plate counts): measurement of living, reproducing population To obtain the appropriate colony number, the sample to be counted needs to be diluted

Fig. 5.15a, 5.16 Chap. 5.10

Plate counts can be highly unreliable when used to assess total cell numbers of natural samples (e.g., soil and water)


Turbidity Measurements of Microbial Growth

Turbidity measurements are an indirect but very rapid and useful method of measuring microbial growth (referred to as optical density (O.D.))

Most often measured with a spectrophotometer

Fig. 5.17 Chap. 5.11

Light

Prism

Incident light, I0

Filter

Sample containingcells ( )Unscattered light, I

Photocell (measuresunscattered light, I )

Spectrophotometer

Optical density (OD)

Log I0I

Sometimes problematic (e.g., microbes that form clumps or biofilms in liquid medium)


The Rate of Growth of a Microbial Culture

Fig. 5.8 Chap. 5.5

Exponential growth: Growth of a microbial population in which cell numbers double within a specific time interval


The Rate of Growth of a Microbial Culture

Nt

N0

t

Nt = No2n

log Nt = log N0 + n log 2

log Nt - log N0 = n log 2

n = log Nt - log N0

log 2

log Nt - log N0

0.301 =

3.3 (log Nt - log N0) =

g = t

n

Nt or Nt = cell number at time t or 0 n = number of divisions (generations)

g = generation time t = time elapsed

Increase in cell number in an exponentially growing bacterial culture is a geometric progression of the number 2

Chap. 5.5-5.6


Introduction into Principles of Metabolism

Metabolism

The sum total of all of the chemical reactions that occur in a cell

Catabolic reactions (catabolism)

Energy-releasing metabolic reactions

Anabolic reactions (anabolism)

Energy-requiring metabolic reactions


Bioenergetics

Energy is defined in units of kilojoules (kJ)

Free energy (G): energy released that is available to do work

The change in free energy during a reaction under standard conditions (pH 7, 25°C, 1 atm, all reactants and products at 1 M concentration) is referred to as Go′

Exergonic reactions: Reactions with a negative Go′value release free energy

Endergonic reactions: Reactions with a positive Go′ value require energy

Chap. 4.4


ATP as prime “energy currency”

ATP

ADP + Pi

exergonic

endergonic

Fig. 4.12 Chap. 4.7


Introduction into Principles of Metabolism

Catabolism/ Dissimilation

Anabolism/ Assimilation

ATP

ADP + Pi

Biomass

Free energy of chemical reactions

Free energy of light Maintenance

energy Heat


Introduction into Principles of Metabolism: Important Terms

• Chemotrophy (energy from chemical substrates) > Lithotrophy (anorganic electron acceptor and electron donor) > Organotrophy (organic electron donor or electron acceptor)

• Phototrophy (energy from light)

Energy sources:

• Autotrophy (more than 50% of carbon from CO2) • Heterotrophy (less than 50% of carbon from CO2)

Carbon sources:


Catalysis and Enzymes

Activation energy: Energy required to bring molecules in a chemical reaction into the reactive state

Fig. 4.6 Chap. 4.5

Enzymes lower the activation energy barrier Free energy calculations do not provide information on reaction rates



Catalyst: substance that Lowers the activation energy of a reaction Increases reaction rate Does not affect energetics or equilibrium of a reaction

Enzymes Biological catalysts Typically proteins Highly specific Active site: region of enzyme that binds substrate Increase the rate of chemical reactions by 108-1020 times the

spontaneous rate Enzyme catalysis: E + S E - S E + P

Chap. 4.5



Many enzymes contain small nonprotein molecules that participate in catalysis but are not substrates

Prosthetic groups

Bind tightly to enzymes Usually covalently and permanently (e.g., heme group in

cytochromes) Coenzymes

Loosely bound to enzymes Important coenzyme: NAD+/NADH (redox electron carrier)

Chap. 4.5


Oxidation-Reduction

Energy is conserved in cells from oxidation-reduction (redox) reactions This energy is conserved in the synthesis of energy-rich compounds

(in particular ATP) Redox reactions occur in pairs (two half reactions) Electron donor: the substance oxidized in a redox reaction Electron acceptor: the substance reduced in a redox reaction

Fig. 4.8 Chap. 4.6


Oxidation-Reduction

Reduction potential (Eo′): tendency of a compound to donate electrons (expressed as volts (V)

The redox tower represents the range of possible reduction potentials for redox couples in nature

The reduced substance in the redox couple at the top of the tower has the greatest tendency to donate electrons

The oxidized substance in the redox couple at the bottom of the tower has the greatest tendency to accept electrons

The farther the electrons “drop” from donor to acceptor, the greater the amount of energy released

G = - n F E (kJ/mol)

Fig. 4.9 Chap. 4.6


NAD as a Redox Electron Carrier

NAD+ and NADH facilitate redox reactions without being consumed; they are recycled

Fig. 4.10, 4.11 Chap. 4.6

NAD NADH H

Nicotinamide

AdenineNAD/ NADHE0 0.32V

NAD reduction

NAD

bindingsite

Activesite

Enzyme–substratecomplex

Enzyme I

NAD Electron donor(substrate)

NADH

Electrondonoroxidized(product)

Electronacceptorreduced(product) NADH

bindingsite

Activesite

Electronacceptor(substrate)Enzyme–

substratecomplex

NADH oxidationEnzyme II

Enzyme I reacts with electron donor andoxidized form of coenzyme, NAD

Enzyme II reacts with electronacceptor and reduced formof coenzyme, NADH


Energy Conservation

Substrate-level phosphorylation (SLP)

Electron-transport-coupled phosphorylation (ETP)

Fig. 4.13 Chap. 4.8

IntermediatesEnergy-richintermediates

Energizedmembrane

Less energizedmembrane


Substrate-level phosphorylation within glycolysis

Fig. 4.14 Chap. 4.8

Glucose

Pyruvate

2 Pyruvate 2 lactate

2 ethanol 2 CO2

Energetics Yeast

Lactic acid bacteria

Intermediates Glucose 6-P Fructose 6-P Fructose 1,6-P Dihydroxyacetone-P

Glyceraldehyde-3-P

1,3-Bisphosphoglycerate 3-P-Glycerate

2-P-Glycerate Phosphoenolpyruvate

Enzymes Hexokinase Isomerase Phosphofructokinase Aldolase

Triosephosphate isomerase Glyceraldehyde-3-P

dehydrogenase

Phosphoglycerokinase Phosphoglyceromutase Enolase Pyruvate kinase

Glucose + 2 ADP + 2 NAD -> 2 pyruvate + 2 ATP + 2 NADH


Electron –transport-coupled phosphorylation

Membrane associated

Mediates transfer of electrons from primary donor to terminal acceptor

Conserves some of the energy released during transfer, ATP synthesis via ATP synthase

Many oxidation-reduction en-zymes are involved in electron transport

Results in generation of pH gradient and an electrochemical potential across the membrane (the proton motive force)

Electrontransport

chain

ATP synthase

Dred + Aox

Dox + Ared

ADP + Pi

ATP

nH+ • H+

3 H+

Chap. 4.10


Structure and Function of ATP Synthase (ATPase)

ATP synthase (ATPase): complex that converts proton motive force into ATP; two major parts:

F1: multiprotein extramembrane complex, faces cytoplasm

Fo: proton-conducting intramembrane channel

Reversible; dissipates proton motive force

Fig. 4.20 Chap. 4.10


Principles of Electron Transport Systems

Electron transport system oriented in cytoplasmic membrane so that as electrons are transported, protons are separated

Carriers in electron transport chain arranged in membrane in order of their increasingly positive reduction potential

The final carrier in the chain donates the electrons and protons to the terminal electron acceptor

Chap. 4.6

NAD(P)+/NAD(P)H E°’= - 0.32 V

Oxygen E°’= + 0.81 V

1.13 V 218 kJ/mol

G = - n F E (kJ/mol)


The Citric Acid Cycle

Citric Acid Cycle (plus pyruvate dehydrogenase: pathway through which pyruvate is completely oxidized to CO2

Plays a key role in catabolism and biosynthesis

Fig. 4.21a Chap. 4.11

Pyruvate (three carbons)

Acetyl-CoA

Oxalacetate2

Malate2

Fumarate2

Succinate2

Succinyl-CoA

Citrate3

Aconitate3

Isocitrate3

-Ketoglutarate2

C2

C4

C5

C6

Pyruvate + GDP + 4 NAD + FAD -> 3 CO2 + GTP + 4 NADH + FADH2


Catabolic Diversity

Fig. 4.22 Chap. 4.12

Fermentation Carbon flowOrganic compound

Carbon flow inrespirations Electron transport/

generation of pmf

Aerobic respiration

Biosynthesis

Biosynthesis

BiosynthesisBiosynthesis

Organiccompound

Electronacceptors

Anaerobic respirationChemoorganotrophy

Chemolithotrophy

Phototrophy

Electron transport/generation of pmf

Anaerobic respiration

Electronacceptors

Aerobic respiration

LightPhotoheterotrophy Photoautotrophy

Electrontransport

Generation of pmfand reducing power

e

donor

Che

mot

roph

sPh

otot

roph

s

julia vorholt lecture 6 - eth zn.ethz.ch/~nielssi/download/3. semester/mikrobiologie...copyright ©...

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