mp s6
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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
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The sulfur cycle
Microbial Physiology LB2762
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
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SO42
-
oxic
anoxic
SH-groupsof proteins
SH-groupsof proteins
Assimilatory sulfate reduction
many aerobicmicroorganisms
many anaerobicmicroorganisms
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PAPS = phosphoadenosine5’-phosphosulfate
Assimilatory sulfate reduction
• Investment of 3 ATP equivalents for sulfate activation• Expensive process (ATP, reducing equivalents)
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oxic
anoxic
SH-groupsof proteins
SH-groupsof proteins
Desulfurylation of proteins/amino acids
many aerobicmicroorganisms
many anaerobicmicroorganisms
H2S
e.g. D-cysteine + H2O → sulfide + NH3 + pyruvate
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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
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The iron cycle
Microbial Physiology LB2762
reservoir
flux
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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)
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Bacterial iron oxidation by A. ferrooxidans
16
Iron oxidation: bacterial vs chemical
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Iron oxidation: bacterial activity and deposits
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Iron oxidation: biofilms
Rio Tinto, Spain
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Iron oxidation: acid mine drainage
Acidic mine runoff: pH < 1Environmental problems (leaching of additional metals)
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AlsoCuS covelliteCuFeS2 chalcopyrite
copper mining
2121
Nutrient-limited growth
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All nutrients in excess: batch
Nutrient-limited growth: the chemostat
Kinetics of nutrient-limited growth; affinity
Mixed-substrate utilisation
Mixotrophic growth
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All nutrients in excess: batch
dCx/dt = rx = ‧Cx (assuming constant volume)
Cx = Cx0 ‧et
Mass balance biomass: change = in - out + production
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Batch fermentation
Doubling time: td = ln(2)/ (h)
Maximum specific growth rate: max (h-1)
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Nutrient depletion/starvation
absence/exhaustion of an essential nutrient leads to cessation of growth ( = 0 h-1)
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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
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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
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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
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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
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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
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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
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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
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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.
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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!
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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
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Strategies at low substrate concentration: kinetics
decrease Ks
µ
increase µmax
Cs
Affinity
µmax/KS
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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
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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
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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
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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
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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
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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
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Viktor Boer et al. (2003) J. Biol. Chem. 278:3265
‘Sulfur economy’ in Saccharomyces cerevisiae
• transcriptome data: shift to ‘low sulfur’ proteins in sulfur- limited cultures• ‘sulfur economy’ is also observed in other microorganisms
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Viktor Boer et al. (2003) J. Biol. Chem. 278:3265
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
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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’
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So far:
Growth on single substrates
- one carbon source- one nitrogen source- one sulfur source,
etc.
How do micro-organisms deal withsubstrate mixtures?
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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
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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
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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
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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
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Mixotrophic growth: simultaneous utilization oforganic and inorganic carbon sources
Chemostat cultivation of T. versutus on thiosulfate-acetate mixtures
AUT HET
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Aerobic, respiratory, heterotrophic growth
organicsubstrate
organicsubstrate
biomass
CO2
CO2
assimilation dissimilation
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Aerobic, respiratory, heterotrophic growth withadditional energy source (e.g. thiosulfate)
organicsubstrate
organicsubstrate
biomass
CO2
CO2
assimilation dissimilation
inorganicsubstrate
e.g. sulfate
dissimilation
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Aerobic, respiratory, chemolithoheterotrophic growth
organicsubstrate
organicsubstrate
biomass
CO2
CO2
assimilation dissimilation
inorganicsubstrate
e.g. sulfate
dissimilation
Higher biomass yield than sum of autotrophic and heterotrophic yields!
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Aerobic, respiratory, mixotrophic growth
organicsubstrate
biomass
CO2
CO2
assimilation dissimilation
inorganicsubstrate
e.g. sulfate
assimilation
biomass
Biomass yield
Sum of autotrophicand heterotrophic biomass yields Rubisco
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Mixotrophic growth: simultaneous utilization oforganic and inorganic carbon sources
Chemostat cultivation of T. versutus on thiosulfate-acetate mixtures
AUT HET
Microbial Physiology LB2762
Next lectures:
Tuesday June 6
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