Mp s6

Session 6

Microbial Cycling of the ElementsThe sulfur cycleThe iron cycle

Nutrient-limited growthThe chemostat Kinetics of nutrient-limited growth; affinityMixed-substrate utilisationMixotrophic growth

Microbial Physiology LB2762

1

The sulfur cycle


reservoir

flux

2

The sulfur cycle

3

SO42

-

H2S

sulfate reduction:anaerobic respiration

oxic

anoxic

Dissimilatory sulfate reduction

4

Wadlopen:a Dutch pastime

Sulfate reduction at work:Iron sulfide in mud from Waddenzee tidal flats

5

SO42

-

oxic

anoxic

SH-groupsof proteins


Assimilatory sulfate reduction

many aerobicmicroorganisms

many anaerobicmicroorganisms

6

PAPS = phosphoadenosine5’-phosphosulfate

Assimilatory sulfate reduction

• Investment of 3 ATP equivalents for sulfate activation• Expensive process (ATP, reducing equivalents)

7

oxic

anoxic



Desulfurylation of proteins/amino acids

many aerobicmicroorganisms

many anaerobicmicroorganisms

H2S

e.g. D-cysteine + H2O → sulfide + NH3 + pyruvate

8

SO42

-

H2S

oxic

anoxic

Chemolithoautotrophic sulfide & sulfur oxidationS0

S0

aerobicoxygen as e-acceptor

anaerobicnitrate or Fe3+

as e-acceptor9

Sulfur-rich acidic hot spring containing hyperthermophilic

H2S and S0 oxidizing Sulfolobus

Symbiotic sulfide-oxidizing bacteria living in tube worms

10

The iron cycle


reservoir

flux

11

12

The iron reservoirs

Natural forms of iron: Fe2+ & Fe3+ (Fe0 mainly anthropogenic)

pyrite (FeS2) magnetite (Fe3O4) hematite (Fe2O3)

jadeite (Na(Al, Fe)Si2O6) goethite (FeO(OH)) jarosite (HFe3(SO4)2(OH)6)

1313

14

Bacterial iron reduction

chemoorganotrophic iron-reducing bacteria chemolithotrophic iron-reducing bacteria

Geobacter metallireducens:

Acetate- + 8 Fe3+ + 4 H2O 2 HCO3- + 8 Fe2+ + 9H+

Geobacter can also use H2 as the electron donor (lithotrophic)

15

Bacterial iron oxidation by A. ferrooxidans

16

Iron oxidation: bacterial vs chemical

17

Iron oxidation: bacterial activity and deposits

18

Iron oxidation: biofilms

Rio Tinto, Spain

19

Iron oxidation: acid mine drainage

Acidic mine runoff: pH < 1Environmental problems (leaching of additional metals)

20

AlsoCuS covelliteCuFeS2 chalcopyrite

copper mining

2121

Nutrient-limited growth


22

All nutrients in excess: batch

Nutrient-limited growth: the chemostat

Kinetics of nutrient-limited growth; affinity

Mixed-substrate utilisation

Mixotrophic growth


23

All nutrients in excess: batch

dCx/dt = rx = ‧Cx (assuming constant volume)

Cx = Cx0 ‧et

Mass balance biomass: change = in - out + production


24

Batch fermentation

Doubling time: td = ln(2)/ (h)

Maximum specific growth rate: max (h-1)


25

Nutrient depletion/starvation

absence/exhaustion of an essential nutrient leads to cessation of growth ( = 0 h-1)


26

Nutrient depletion in an industrial process: citric acid production by the yeast Yarrowia

lipolytica

Time (h)

glucose

ammonia

citrate

biomass

Klasson et al. (1989) Appl. Biochem. Biotechnol. 20:491


27

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 2 4 6 8 10

Batch: growth rate profile

=0 h-1

= max

time

a rarity in nature:

often at least one growth-limiting nutrient


28

Kinetics of nutrient-limited growth

= maxCSKS + CS

original Monod:

q = qmaxCS

KS + CS

q1 = specific substrate consumption rateKS = substrate saturation constantCS1 = growth-limiting nutrient concentation

q

maximumrate

Ks

50 %maximum rate

S


29


30

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 2 4 6 8 10

=0 h-1

= max

time


31

Nutrient-limited growth: the chemostat

chemostat cultivation requiresa perfectly mixed culture vessel

Pump (in and out)

Medium reservoir(one single growth limiting

substrate)

Chemostat

Receiving bottle

Chemostat – experimental set-up


32


33

Chemostat: mathematics

Mass balance biomass: change = in - out + production

D(VCx)/dt = - v‧Cx + ‧Cx‧V

dCx/dt = ( - D)‧Cx (at constant volume and with D = v/V)

dCx/dt = ( - D)‧Cx = 0 = D (at steady state)

With chemostat cultivation the specific growth rate can be set!

dilution rate in h-1


34

Kinetics of nutrient-limited growth and the chemostat

= maxCSKS + CS

original Monod:

q

maximumrate

Ks

50 %maximum rate

S

What is the highest dilution rate at which a steady state can be obtained?

Dhighest = highest = max

CS,inKS + CS,in


35

Can chemostat cultivation be used as a tool to determine the substrate saturation constant KS?

Kinetics of nutrient-limited growth and the chemostat

= maxCSKS + CS

original Monod:

Yes, by accurately measuring the concentration of the limiting nutrient (CS)

at varying dilution rates (D=).

0

1

2

3

4

5

6

7

8

9

10

0 0.2 0.4 0.6 0.8 1

D

CSKS and max can be estimated:

KS

max

= 0.1 g l-1

= 1.0 h-1

D = 1.0CS

0.1 + CS

This would also work if you plot Cs versus qs.


36

The yeast Saccharomyces jansenii is cultivated in a glucose-limited chemostat at a dilution rate of 0.1 h-1. The glucose concentration in the medium vessel is 10 g/l. From previous studies it is known that the max of this organism is 0.4 h-1 and that the KS of this yeast for glucose is 30 mg/l (assuming Monod kinetics of growth). After 5 days a steady state is obtained.

a. Calculate the residual glucose concentration in steady state (CS in g/l).

b. Calculate the volumetric glucose consumption rate (rS in g/l‧h).

Accurately measuring the limiting nutrient?

D = maxCSKS + CS

0.1 = 0.4CS

30 + CS

CS = 10 mg/l = 0.01 g/l

rs= glucosein - glucoseout = DCS,in – DCS = 0.1(10-0.01) = 0.999 g/l‧h

This means that 0.28 mg glucose per liter per second is consumed.Many organisms even demonstrate a much lower KS for limiting nutrients!


37

Measuring the limiting nutrient requires quenching

Accurately measuring residual nutrient concentration requires instantaneously stopping the metabolism of that nutrient.

Methods for this include:

• Liquid nitrogen• Cold inert coolant (e.g. steel balls)

How do micro-organisms adapt to growthat limiting concentrations of a nutrient?

• kinetic adaptation- reduce Ks

- increase µmax

• adaptation of biomass composition• induction of systems for alternative nutrients

38

Strategies at low substrate concentration: kinetics

decrease Ks

µ

increase µmax

Cs

Affinity

µmax/KS

39

Glutamate + NADP

NH3 NH3

Glutamate

2-oxoglutarate

out in

2-OG + NH3 + NAD(P)H

Net Reaction:

NADPH

NADP

N excess

GDH

Decrease Ks: the ammonia assimilation paradigm

Km = 1 – 10 mM40

2-OG + NH3 + NAD(P)H + ATP

NH3 NH3

Glutamine

glutamate

out in

Net Reaction:

ATP

ADP

2-oxoglutarate

NADH

NAD

2 glutamate

Glutamate + NAD + ADP

GOGAT

GS

N limitation

Km = ca. 0.1 mM

Decrease Ks: potassium uptake by Escherichia coli

out in

K+ K+

Km ~1 mM

K+ excess

41

TrK

outin

K+ K+

ATP

ADP

Km ~ 1 µM

K+ limitation

Kdp

NADH

NAD

O2

H2O

NUO/NDH

cytbo

Q

2 H+

2 H+

e-

e-

O2 excess

Decrease Ks: respiration in Escherichia coli

NADH

NAD

O2

H2O

NUO/NDH

cytbd

Q

1 H+

2 H+

e-

e-

O2 limitation

Cytochrome bd oxidase:Low Km for oxygen, but lower H+ pumping efficiency 42

Adaptive strategies at low substrate concentration

decrease Ks

µ

increase µmax

Cs

Affinity

µmax/KS

43

Increase capacity: methanol-limited chemostat cultures of Hansenula polymorpha

Methanol oxidase: methanol + O2 formaldehyde + H2O2

First step in methanol metabolism by methylotrophic yeasts

methanol oxidasecrystalloids

44

Alternative response to nutrient limitationchange biomass composition

Reduce content of growth limiting nutrient/elementin biomass

=

Increase biomass yield on growth limiting nutrient/element

45

Example: cell wall composition in Bacillus subtilis

O

OC

C

CC

C

COOH

OHH

H OC

C

CC

C

CH2OH

NHCOCH3H

H

O

OHH

O

P excess

P limitation Teichuronic acid

O

O

O

O

OCH2

C

C

C

Ala POHR

O

O

O

O

CH2

C

C

C

AlaR

OP

OH

Teichoic acid

n

Ellwood & Tempest, 1972. Adv. Microbial Physiol. 7:83

46

Transcription of pyruvate decarboxylase genes in Saccharomyces cerevisiae

PDC's

0

500

1000

1500

2000

2500

3000

C-lim N-lim P-lim S-lim

leve

l o

f ex

pre

ssio

n

PDC1

PDC5

PDC6

mRNA levels (Affymetrix GeneChips) from aerobic,nutrient-limited chemostat cultures (D = 0.10 h-1)

Viktor Boer et al. (2003) J. Biol. Chem. 278:3265

47

PDC6 encodes a ‘low-sulfur’ pyruvate decarboxylase

PDC6 encodes a ‘low sulfur’ pyruvate decarboxylasethat is specifically expressed during S-limited growth

Number of sulfur-containing amino acids in the 3 pyruvatedecarboxylases of S. cerevisiae

Gene Total amino acidsCys Met

PDC1 563 4 13PDC5 563 4 14PDC6 563 1 5

48


‘Sulfur economy’ in Saccharomyces cerevisiae

• transcriptome data: shift to ‘low sulfur’ proteins in sulfur- limited cultures• ‘sulfur economy’ is also observed in other microorganisms

49


Responses of micro-organisms to nutrient limitationinduction of genes for alternative pathways

Oxygen limitation in facultative anaerobes:induction of pathways for alternative respirationpathways (other electron acceptors) or fermentation:

fnr (fumarate-nitrate respiration) in E. coli alcoholic fermentation in bakers’ yeast

lactate fermentation in Rhizopus

50

Responses of micro-organisms to nutrient limitationinduction of genes for alternative pathways

Induction of systems for uptake and metabolism of less-preferred sources of an element

Examples

- Induction of amino acid transporters during ammonium- limited growth

- Induction of phosphatases during phosphate-limited growth

- Induction of sulfatases and systems for cysteine-methionine uptake during sulfate-limited growth

51

transportSUL2, AGP3, MMP1, MUP1, MUP3, SAM3, GNP1, HGT1, ATM1, COT1,sulfate assimilationSER33, MET1, MET8, MET2, MET3, MET10, MET16, MET22, MHT1, CYS3, STR3other metabolismARN1, PDC6, DLD3regulationMET28, MET32, YOR302Wdetoxification/stress responseGTT2, YHR176W, OYE3, CTT1, RAD59, FLR1, YLL057Cother (cell cycle and structure)CSM2, KIC1, ASE1, CWP1poorly defined/unclassifiedTIS11, CBP1, SOH1, CHL4, YOL163W, MCH5, YBR293W, YOR378W, YIL166C, YLL055W, BNA3, YJL060W, YFL057C, YLL058W, ICY2, PCL5, YLR364W, YBR281C, YBR292C, YEL072W, YFL067W, YGR154C, YIR042C, YKL071W, YLL056C, YML018C, YNL095C, YNL191W, YOL162W, YOL164W, YPL052W

68 transcripts specifically up-regulated in sulfate-limitedcultures of bakers’ yeast (Saccharomyces cerevisiae)

low Km sulfate transporter

‘Sulfur Economy’ pyruvate decarboxylase

Cys, Met transporterslow Km sulfate transporter

Capacity of sulfate assimilation

52

Strategies for nutrient-limited growth: no free lunch…

• Increased Y by changed biomass composition at the expense of decreased functionality

(?)

• Decreased Ks

at the cost of ATP equivalents (energy efficiency)

• Increased enzyme synthesis resulting in overcapacity/’metabolic burden’

53


54

So far:

Growth on single substrates

- one carbon source- one nitrogen source- one sulfur source,

etc.

How do micro-organisms deal withsubstrate mixtures?


55

Mixed substrate utilization in batch cultures:diauxic growth

Mechanism:

• Repression by favoured substrate

• Induction by less favoured substrate

Often at level of transcription,but post-transcriptionalmechanisms may also contribute


56

Co-consumption is possible at very low concentrations

Batch cultivation of E. coli on glucose and galactose at non-Repressing glucose concentration (2 mg/l)

glu

gal


57

Nutrient-limited chemostat cultivation: mixed-substrate utilizationat low to intermediate specific growth rates

Aerobic chemostat cultivation of Hansenula polymorpha on glucose and methanol at different dilution rates

glu

biomass methanol • repression at high µ due to ‘Monod’ kinetics


58

Mixed-substrate utilization enables growth at lowersubstrate concentration than on pure substrates

Sugar-limited growth of E. coli in chemostat cultures at differentglucose-galactose ratios: effect on residual sugar concentrations

• important in natural environments

• breakdown of pollutants

glu gal

59

Facultative chemolithoautotrophs:Organic substrates suppress utilizationof inorganic electron donor andCO2 fixation

Example:Growth of Thiobacillus versutus onacetate and thiosulfate (S2O3

2-) inaerobic batch cultures

Biomass yield

Sum of autotrophicand heterotrophic biomass yields Rubisco


60

Mixotrophic growth: simultaneous utilization oforganic and inorganic carbon sources

Chemostat cultivation of T. versutus on thiosulfate-acetate mixtures

AUT HET


61

Aerobic, respiratory, heterotrophic growth

organicsubstrate

organicsubstrate

biomass

CO2

CO2

assimilation dissimilation


62

Aerobic, respiratory, heterotrophic growth withadditional energy source (e.g. thiosulfate)

organicsubstrate

organicsubstrate

biomass

CO2

CO2


inorganicsubstrate

e.g. sulfate

dissimilation


63

Aerobic, respiratory, chemolithoheterotrophic growth

organicsubstrate

organicsubstrate

biomass

CO2

CO2


inorganicsubstrate

e.g. sulfate

dissimilation

Higher biomass yield than sum of autotrophic and heterotrophic yields!


64

Aerobic, respiratory, mixotrophic growth

organicsubstrate

biomass

CO2

CO2


inorganicsubstrate

e.g. sulfate

assimilation

biomass

Biomass yield

Sum of autotrophicand heterotrophic biomass yields Rubisco


65

Mixotrophic growth: simultaneous utilization oforganic and inorganic carbon sources

Chemostat cultivation of T. versutus on thiosulfate-acetate mixtures

AUT HET


Next lectures:

Tuesday June 6

66

Technology

Mp s6