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Geobiology Week 3
How do microbes garner energy and carbon?
Review of redox couples, reaction potential and free energy
yields
Hydrogen as an energy currency for subsurface microbes.
Acknowledgements: Tori Hoehler
Redox structure of modern microbial ecosystems
Deep biosphere as an analogue of Early Earth Ecosystems
‡O2 as a driver of biological innovation
Readings : Brock Biology of Microorganisms. Hoehler et al., 1998.Thermodynamic
control on hydrogen concentration in anoxic sediments Geochim. Cosmochim.
Acta 62: 1745-1756. Hoehler TM, et al., 2002. Comparative ecology of H2 cycling
in sedimentary and phototrophic ecosystems Antonie von Leeuwenhoek 81: 575-
582. Hoehler et al., 2001. Apparent minimum free energy requirements for
methanogenic Archaea and Sulfate reducing bacteria in an anoxic marine
sediment. FEMS Microbial Ecol. 38; 33-41.
A staggering number of organism-organism and organism-environment interactions underlie global biogeochemistry
These can be studied at vastly different spatial and time scales
PRESS RELEASEDate Released: Thursday, February 21, 2002
Texas A&M UniversityRock-eating microbes survive in deep ocean off Peru
Rock-eating microbes survive in deep ocean off Peru Way
down deep in the ocean off the coast of Peru, in the rocks
that form the sea floor, live bacteria that don't need sunlight,
don't need carbon dioxide, don't need oxygen. These
microbes subsist by eating the very rocks they call home.
Researchers from the Ocean Drilling Program (ODP) have
embarked aboard the world's largest scientific drillship on a
voyage to understand the abundance and diversity of these
microbes and the environments in which they live.
Biogeochemical Redox CouplesWhat is the energy currency of metabolicreactions in cells ??
How do cells make it ?
What powers those reactions?
How do we measure the energy outputs orrequirements of metabolism?
How can we use this kind of information in anecological and biogeochemical sense?
Biogeochemical Redox CouplesD
CO2 + H2O ‡ CH2 O + O2 oxygenic photosynthesis
Interdependency?
CH2 O + O2 ‡ CO2 + H2O (+D) aerobic respiration
oxidative methanotrophy
D CH4 + 2O2 ‡ CO2 + 2H2O (+D)
anoxygenicCO2 + HS- + H2O ‡ biomass + SO4
2-photosynthesis
O6 ‡ 2CO2 + 2C2H6O (+D) fermentationC6H12
4H2 + SO42-‡ S2- + 4H2O (+D)
sulfate reduction
methanogenesisCO2 + 2H2 ‡ CH4 + 2H2O (+D)
pe(W)
–10
0
+10
–10
0
+10
E (V)oDGP680* P680+
OXIDATION
NO3
NO3
-0.5
& Energy Yields H
+H2 H
+ H2
NH4+ N2 NH4
+N2 The electron tower…….. CH4 CO2 CO2 CH4
100
+0.5
Fe3+ ‡ Fe2+ +0.76 V
Redox Potentials kJ/mol e-
CO2 CH2OCH2O CO2
S H2SH2S S Strongest reductants, or e donors, 2–SO4
2–SO4 H2SH2S on top LHS
Fe2+ Fe2+Fe(OH)3 Fe(OH)3 0 Electrons ‘fall’ until they are
‘caught’ by available acceptors
REDUCTION The further they fall before being
NH4+ 50 caught, the greater the differenceNO2–
32–+NH4
in reduction potential and energy2–NO3
2–NO2 NO2 released by the coupled reactionsMnO2 Mn2+Mn2+ MnO2
CO CO2 CO2 CO
2–N NO3 2–NO3 N22
0H2O O2 H2OO2 (Last Common Ances
P680+ P680 +1.0
pe(W)
OXIDATION
0
+10
0
+10
–10 –10
E (V)oDGP680+
NO3
NO3
P680* kJ/mol e- -0.5 Redox Potentials CO2 CH2O
100
+0.5
Fe3+ ‡ Fe2+ +0.76 V
CH2O CO2
& Energy Yields + +HH2 H H2 +
NH4 N2 NH4+
N2 CH4 CO2 CO2 CH4 Reaction must be exergonic (-ve DG)
S H2SH2S S 2– SO2–
4SO4H2S H2S The energetically most favored
reaction proceeds first ieFe2+ Fe2+Fe(OH)3 Fe(OH)3 0
CH2O first degraded with O2 -CH2O degraded with NO3 nextREDUCTION
CH2O degraded with Mn4+ next2– + 50NO3
2–
2–
+NH4
NO2
NH4 followed by SO42-, 2–NO3 NO2
Mn2+Mn2+ MnO2MnO2 and CO2 last (methanogenesis)CO CO2 CO2 CO
2–N NO3 2–NO3 N22
0H2O O2 H2O (Last Common Ancestor) O2
P680+ P680 +1.0
Energy Calculations
aA +bB ‡ cC + cD
DG = Gf°’ (aA + bB) – Gf °’ (cC + dD)
Where Gfo’ is the free energy of formation of 1 mole
under ‘standard’ conditions (pH 7, 25C)
DG = DG° + RT·ln K (T) .
K = CcDd/AaBb R= 1.98cal.mol-1.°K-1
[C]c[D]d
DG = DG° + RT·ln (T) [A]a[B]b
How do microbes garner energy and carbon?
O2
CO2
Electron flow
Carbon flow
CO2
Carbon flow
Electron flow
NO3 -SO4
2- Fe3+
Inorganic compound
H2 H2S NH3 Fe2+
O2
CO2
Electron flow
Carbon flow
Biosynthesis
respiration
anaerobic respiration
lithotrophy
Organic compound
Organic compound
Other organic compound
Mechanisms and Balance Sheets
Electron Donor
Electron “Carrier”
CO2
Carbon flow
Electron flow
NO3 -SO4
2- Fe3+
Organic compound
Other organic compound
NAD + H2 ‡ NADH
(catab)
or
NADP + H2 ‡ NADPH
(anab) Terminal Electron Acceptor
Balance Sheet: pyruvic acid ‡ 3CO2 = 4 NADH + 1 FADH (Flavoproetein e carrier)
1NADH ‡ 3 ATP; 1FADH ‡ 2ATP therefore 1 TCA cycle ‡ 15ATP; 1 glucose ‡ 30ATP
1ATP ‡ 7kcal/mole so 1 molecule glucose ‡ 266 kcal
Glucose oxidation with O2 DG = 688kcal Therefore aerobic respiration ca. 39% efficient
In contrast, glucose fermentation ‡ lactate = 29 kcal/mol ca. 50% efficient
Reactions of the TCA Cycle
Pyruvate
The TCA cycle showing enzymes, substrates and products. The abbreviated enzymes are: IDH = isocitrate dehydrogenase and a-KGDH = a-ketoglutarate dehydrogenase. The
GTP generated during the succinate thiokinase (succinyl-CoA synthetase) reaction is
equivalent to a mole of ATP by virtue of the presence of nucleoside diphosphokinase.
The 3 moles of NADH and 1 mole of FADH2 generated during each round of the cycle feed
into the oxidative phosphorylation pathway. Each mole of NADH leads to 3 moles of ATP
and each mole of FADH2 leads to 2 moles of ATP. Therefore, for each mole of pyruvate
which enters the TCA cycle, 12 moles of ATP can be generated
Balance Sheet:
pyruvic acid ‡ 3CO2 = 4 NADH + 1 FADH (Flavoproetein e carrier)
1NADH ‡ 3 ATP; 1FADH ‡ 2ATP therefore 1 TCA cycle ‡ 15ATP; 1glucose ‡ 30ATP
1ATP ‡ 7kcal/mole so 1 molecule glucose ‡ 266 kcal
Glucose oxidation with O2 DG = 688kcal Therefore, in this case, aerobicrespiration ca. 39% efficient
In contrast, glucose fermentation ‡ lactate = 29 kcal/mol ca. 50%efficient
Multi-Step Organic Matter Remineralization in Anoxic Systems
Monomers
CO2
Biopolymers
(CH2O)n
e -Small Organics
- +NO3 Æ NH4
Mn4+ Æ Mn2+
Fe3+ Æ Fe2+
SO42- Æ H2S
CO2 Æ CH4
oxidation reduction
Requires numerous extracellular electron transfers
2H+ + 2e-H2
A nearly ubiquitous means of
extracellular electron transport in
microbial redox chemistry
Hydrogen
• Anaerobic metabolism strongly sensitive to pH2
• Fermentation frequently characterized by obligate (1-2 C’s) or facultative (>3 C’s) H2 production
• Reaction only energetically feasibly with H2 sink
• Obligate H2 producers don’t grow in ‘pure’ culture• Readily grown in co-culture
• H2 consuming reactions affected oppositely
Hydrogen• H2 consuming reactions affected oppositely
e.g. with mM SO42- SRB can maintain very
low pH2.
• In presence of active SRB, H2 too low for
methane production to be energetically
feasible
• Often see zonation between SR and MP
under thermodynamic control
Hydrogen• 2H2 + 2CO2 ‡ CH3COOH + O2 + DG
• CH3COOH + O2 ‡ 2H2 + 2CO2 + DG
Opposite biochemistry when methanogen present
Anaerobic oxidation of methane is energetically marginal
unless????
• 2CH4 + SO42- ‡ S2- + 2CO2 + 4H2
H2 has a High Relative Stoichiometry in Many
Anaerobic Remineralization Processes
Production
CH3CH2COOH + 2H2O Æ CH3COOH + CO2 + 3H2
Consumption
CO2 + 4H2 Æ CH4 + 2H2O
Free Energy Yield Depends Exponentially onStoichiometry in Reaction
CO2 + 4H2 Æ CH4 + 2H2O
PCH4
DG = DG° + RT·ln (T) . (PH )4PCO2 2
DG is much more sensitive to PH2 than to PCH4 or PCO2mp
Thermodynamics of Inter-Species H2 Transfer
consumer
producer
H2
Both Organisms Depend Highly
on H2 Partial Pressure:
Too High Alters Production Æ
Too Low Inhibits
Consumption
Pathway Shifts, Inhibition, Reversal
H2
consumer
producer
H 2
For constant or decreasing H2
production rate (e.g. sediments), PH2
in practice reflects control by H2
consumption
coupled to production; PH2 held at
very low steady-state levels;
less)
PH2 controlled by the balance
consumption
in the Environment
Consumption very efficiently
residence times short (seconds or
between production and
Free Energy Regulation in Methanogenesis
4H2 + CO2 CH4 + 2H2O
CH3COOH CH4 + CO2
Responsiveness D [X] and Dt required to change free energy yield by 10kJ/mole
Conc.( m
M)tres(s) D[x] Æ D D
D D(s)H2
Data for methanogenic sediments from Cape Lookout Bight at 22°C;
.0051.32.8 x3.5
2 Transfer
H2consumer 1 consumer 3
consumer 2
producer 1
producer 3
producer 2
Inter-Species H
in a Complex Microbial Ecosystem
2
Steady-state PH2 reflects efficiency
of consumption; constrained by
2
consumers
2 consumption to permit
PH2
by H2 consumers
consumer
producer
H 2
Controls on H in Anoxic Sediments
physiologic limitations of H
Ultimate physiologic limitation:
requirement to extract sufficient free
energy from H
continued metabolism
in sediments is controlled
[ ] n 1
rxnT
ox
red H
RT GG
exp X X
P 2 ˜
˜¯
ˆ ÁÁË
Ê ˜˜¯
ˆ ÁÁË
Ê D ⋅=
o
Steady State H2 Concentrations Sensitive To:
ox and Xred)
Specific Redox Couple (e.g. CO2/CH4 -vs- SO4 2-/S2-)
Temperature
DGrxn)
[ ] -D
Concentrations of Products and Reactants (X
Energy Yield of Reaction (
SO
Effect of Sulfate Concentration on H2
42- + 4H2 S2- + 4H2O
4 1
ˆˆ ˜˜¯
˜˜¯
-2[ ] SÊexp
ÊÁÁË
DGT Grxn D o -
[ ]-2SO4
ÁÁË
= ⋅PH2 RT
Increasing Sulfate = Decreasing H2
0.06
0.09
0.12
0.15
0.18
0 120
H2 (
Pa) PH2 4
2-]-
0.255
(R2 = 0.993)
Sulfate (mM)
-32
-29
-26
-23
-20
0 120
D-1
)
Expected DGSR 4 2-
Sulfate (mM)
Impact of Sulfate Concentration Change on DG and H2
H2DG
Deduction: H2
maintain const Dsubstrate limitation?
30 60 90
= 0.25·[SO
30 60 90
G (
kJ·m
ol
-vs- SO
in Sulfate-Reducing CLB Sediments
is drawn down to compensate for increasing sulfate; SRB community G near limit for ‘maintenance’ but max efficiency. An adaptation to
0 5 10 15 20
0
10
20
30
40
50
60
0 0.4
0 5 10 15 20
0
10
20
30
40
50
60
0.0 1.0 2.0
Depth
(cm)
H2 (Pa)H2 (Pa)
NovemberAugust
Sulfate
H 2
H 2
Depth Profiles of H2 in CLB Sediments
0.2 0.6 0.5 1.5
Sulfate (mM) Sulfate (mM)
14.5°C 27°C
Sulfate
Ecosystem
H2 consumer 3
producer 1
producer 3
producer 2
Is Controlled By:
Environmental factors
(temperature, chemistry,
Consumer 1 Controls:
Steady-state H2
Thermodynamics of other
microbial processes
2 Transfer
consumer 1
consumer 2
affecting DG
etc.) Both can be
Addressed
Quantitatively
Inter-Species H
in a Complex Microbial
n 1
rxn T
ox
red H
RT G G
exp X X
P 2
˙ ˙ ˚
˘
Í Í Î
È
˜ ˜ ¯
ˆ Á Á Ë
Ê D -D ⋅ =
o
H2
P
Intracellular
Bioenergetics
Extracellular
Measurement
2Bulk phase (extracellular) H partial pressures are described
quantitatively by intracellular thermodynamics
PH2 measured in bulk fluid > PH2
in HC cell
H2
producer
(HP)
H2
consumer
(HC)
H2 measurement
H2
P
HP bulk fluid
H2
consumer
(HC)
H2 producer
(HP)
Organic
matter
H2
P
bulk fluidHCHP
PH2 measured in bulk fluid = PH2
in HC cell
H2 measurement
Efficient utilization of H2 requires mass transport and high concentration gradient unless
mitigated by spatial arrangements. The fact that quantitative H2
Spatial Constraints
HC
etc measurements reflect
bioenergetic control argues for non-random arrangement of consumers and producers as
illustrated above (see later re AOM)
Depth
(cm
)
D -1)
-40 -30 -20 0-10 +10 0
10
20
30
40
50
60
0
10
20
30
40
50
60
-40 -30 -20 0-10 +10
D -1)
SR
MP
SR
MP
In Situ
August November
G (kJ·molG (kJ·mol
Free Energy Yields in CLB Sediments
T = 27°C T = 14.5°C
Biogeochemical Redox Couples
aerobic respiration
CH2 O + O2 ‡ CO2 + H2O
O2 1 mole glucose 30-32 mole ATP
fermentation 1 mole glucose 2-4 mole ATP
Biosynthesis requires approx. 1mole ATP per 4g of cell carbon
Biogeochemical Redox Couples
oxygenic photosynthesis
CO2 + H2O ‡ CH2 O + O2
http://www.bris.ac.uk/Depts/Chemistry/MOTM/atp/atp1.htmMolecule of the Month
Adenosine Triphosphate - ATPPaul May - Bristol University
The 1997 Nobel prize for Chemistry has been awarded to 3 biochemists for the study
of the important biological molecule, adenosine triphosphate . This makes it a fitting
molecule with which to begin the 1998 collection of Molecule's of the Month. Other
versions of this page are: a Chime version and a Chemsymphony version.
ATP - Nature's Energy Store
All living things, plants and animals, require a continual supply of energy in order to function.
The energy is used for all the processes which keep the organism alive. Some of these
processes occur continually, such as the metabolism of foods, the synthesis of large,
biologically important molecules, e.g. proteins and DNA, and the transport of molecules and ions
throughout the organism. Other processes occur only at certain times, such as muscle
contraction and other cellular movements. Animals obtain their energy by oxidation of foods,
plants do so by trapping the sunlight using chlorophyll. However, before the energy can be
used, it is first transformed into a form which the organism can handle easily. This special carrier
of energy is the molecule adenosine triphosphate, or ATP
Its Structure
The ATP molecule is composed of three components. At the centre is a sugar molecule, ribose (the
same sugar that forms the basis of DNA). Attached to one side of this is a base (a
group consisting of linked rings of carbon and nitrogen atoms); in this case the
base is adenine. The other side of the sugar is attached to a string of phosphate
groups. These phosphates are the key to the activity of ATP.
ATP consists of a base, in
this case adenine (red), a
ribose (magenta) and a
phosphate chain (blue).
AMP ADP ATPHow it works
ATP works by losing the endmost phosphate group when instructed to do so by
an enzyme. This reaction releases a lot of energy, which the organism can then
use to build proteins, contact muscles, etc. The reaction product is adenosine
diphosphate (ADP), and the phosphate group either ends up as orthophosphate
(HPO4) or attached to another molecule (e.g. an alcohol). Even more energy can
be extracted by removing a second phosphate group to produce adenosine
monophosphate (AMP).
When the organism is resting and energy is not immediately needed, the reverse
reaction takes place and the phosphate group is reattached to the molecule
using energy obtained from food or sunlight. Thus the ATP molecule acts as a
chemical 'battery', storing energy when it is not needed, but able to release it
instantly when the organism requires i
The 1997 Nobel Prize for Chemistry
The Nobel prize for Chemistry in 1997 has been shared by:
Dr John Walker of the Medical Research Council's Laboratory of Molecular Biology (LMB)
at Cambridge (an institution which has been responsible for 10 Nobel prizes since 1958!)
Dr Paul Boyer of the University of California at Los Angeles
and Dr Jens Skou of Aarhus University in Denmark.
The prize was for the determination of the detailed mechanism by which ATP shuttles
energy. The enzyme which makes ATP is called ATP synthase, or ATPase, and sits on the
mitochondria in animal cells or chloroplasts in plant cells. Walker first determined the amino
acid sequence of this enzyme, and then elaborated its 3 dimensional structure. Boyer
showed that contrary to the previously accepted belief, the energy requiring step in making
ATP is not the synthesis from ADP and phosphate, but the initial binding of the ADP and the
phosphate to the enzyme. Skou was the first to show that this enzyme promoted ion
transport through membranes, giving an explanation for nerve cell ion transport as well as
fundamental properties of all living cells. He later showed that the phosphate group that is
ripped from ATP binds to the enzyme directly. This enzyme is capable of transporting
sodium ions when phosphorylated like this, but potassium ions when it is not. More details
on the chemistry of ATPase can be found here, and you can download the 2 Mbyte pdb file
for Bovine ATPase from here.
References: Chemistry in Britain, November 1997, and much more information on the
history of ATP and ATPase can be found at the Swedish Academy of Sciences and at
Oxford University.