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.