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: 575582. 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

Microbiology Ecology Biogeochemistry

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 Couples What is the energy currency of metabolic reactions in cells ??

How do cells make it ? What powers those reactions?

How do we measure the energy outputs or requirements of metabolism?

How can we use this kind of information in an ecological and biogeochemical sense?

Biogeochemical Redox Couples

CO2 + H2O CH2 O + O2 oxygenic photosynthesis

CH2 O + O2 CO2 + H2O (+)

CH4 + 2O2 CO2 + 2H2O(+) oxidative methanotrophy

CO2 + HS-+ H2O biomass + SO42-

C6H12 O6 2CO2 + 2C2H6O (+ ) fermentation

4H2+SO42- S2-+ 4H2O (+) sulfate reduction

CO2 +2H2 CH4 + 2H2O (+) methanogenesis

Interdependency?

aerobic respiration

anoxygenic photosynthesis

Redox Potentials& Energy Yields The electron tower……..

Strongest reductants, or e donors,on top LHS

Electrons ‘fall’ until they are‘caught’ by available acceptors

The further they fall before beingcaught, the greater the differencein reduction potential and energyreleased by the coupled reactions

(Last Common Ances

Redox Potentials& Energy Yields

The energetically most favored

The energetically most favoredreaction proceeds first ie

CH2O first degraded with O2 CH2O degraded with NO3 nexCH2O degraded with Mn4+ next

followed by SO42-,

and CO2 last (methanogenesis)

Energy Calculations

aA +bB ‡ cC + cD

G = Gf°’ (aA + bB) – Gf°’ (cC + dD)

Where Gfo’ is the free energy of formation of 1 mole under ‘standard’ conditions (pH 7, 25C)

G = G° (T) + RT·ln K .

K=CcDd/AaBb R= 1.98cal.mol-1.°K-1

G = G° (T) +RT·ln

[C]c[D]d [A]a[B]b

How do microbes garner energy and carbon?

Organic compound

Electron flow

Carbon flow

Carbon flow

Carbon flow

Electron flow

Electron flow

Inorganic compound H2 H2S NH2 Fe2+

Biosynthesislithotrophy

Other organic compound

anaerobic respiration

Organic compound

respiration

Mechanisms and Balance SheetsElectron Donor

Electron “Carrier”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 G = 688kcal Therefore aerobic respiration ca. 39% efficient

In contrast, glucose fermentation lactate = 29 kcal/mol ca. 50% efficient

Reactions of the TCA Cycle

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

Pyruvate

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 G = 688kcal Therefore, in this case, aerobic

respiration ca. 39% efficient

In contrast, glucose fermentation lactate = 29 kcal/mol ca. 50%efficient

Multi-Step Organic Matter Remineralization in Anoxic Systems

Biopolymers

(CH2O)n

Monomers

Small Organics

CO2

NO3- NH4+

Mn4+ Mn2+

Fe3+ Fe2+

So42- H2S

CO2 CH4

oxidation reduction

Requires numerous extracellular electron transfers

A nearly ubiquitous means of

extracellular electron transport in

microbial redox chemistry

H2 2H+ + 2e-

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 + G

• CH3COOH + O2 2H2

+ 2CO2 + G

Opposite biochemistry when methanogen present

Anaerobic oxidation of methane is energetically marginal unless????

• 2CH4 +SO4

2- S2-+ 2CO2 +4H2

H2 has a High Relative Stoichiometry in Many Anaerobi

c Remineralization Processes

Production

CH3CH2COOH + 2H2O CH3COOH + CO2 + 3H2

Consumption

CO2 +4H2 CH4 +2H2O

Free Energy Yield Depends Exponentially onStoichiometry in Reaction

CO2 +4H2 CH4 +2H2O

G= G°(T)+RT In‧ PCH4

PCO2(PH2)4

Gmp is much more sensitive to PH2 than to PCH4 or PCO2

Thermodynamics of Inter-Species H2 Transfer

producer

consumer

Both Organisms Depend Highly on H2 Partial Pressure:

Too High Alters Production Pathway Shifts, Inhibition, Reversa

Too Low Inhibits Consumption

producer

consumer

H2 in the Environment

PH2 controlled by the balancebetween production andconsumption

For constant or decreasing H2

production rate (e.g. sediments), PH2

in practice reflects control by H2

consumptionConsumption very efficientlycoupled to production; PH2 held atvery low steady-state levels;residence times short (seconds orless)

Free Energy Regulation in Methanogenesis

4H2 + CO2 CH4 + 2H2O

CH3COOH CH4 + CO2

Data for methanogenic sediments from Cape Lookout Bight at 22°C;Responsiveness [X] and Dt required to change free energy yield by 10kJ/mole

Inter-Species H2 Transferin a Complex Microbial Ecosystem

producer1producer2

producer3

comsumer 1

comsumer 2

comsumer 3H2

producer

consumer

Controls on Hin Anoxic Sediments

PH2 in sediments is controlled

by H2 consumers

Steady-state PH2 reflects efficiencyof consumption; constrained byphysiologic limitations of H2 consumers

Ultimate physiologic limitation:requirement to extract sufficient freeenergy from H2 consumption to permit

continued metabolism

Steady State H2 Concentrations Sensitive To:

Concentrations of Products and Reactants (Xox and Xred)

Specific Redox Couple (e.g. CO2/CH4 -vs- SO42-/S2-)

Temperature

Energy Yield of Reaction (Grxn)

Effect of Sulfate Concentration on H2

SO42- + 4H2 S2- + 4H2O

Increasing Sulfate = Decreasing H2

Impact of Sulfate Concentration Change on DG and H2

in Sulfate-Reducing CLB Sediments

Deduction: H2 is drawn down to compensate for increasing sulfate; SRB communityMaintainconst G near limit for ‘maintenance’ but max efficiency. An adaptation tosubstrate limitation?

Sulfate (mM)

G H2

Sulfate (mM)

Expected GSR-vs-SO42-

Depth Profiles of H2 in CLB Sediments

Sulfate (mM)

Sulfate (mM)

Depth(cm)

August 27oC

November 14.5oC

Sulfate Sulfate

H2 (Pa) H2 (Pa)

Inter-Species H2 Transferin a Complex Microbial

comsumer 1

affecting G

etc.)Both can beAddressQuantitatively

comsumer 2

Bulk phase (extracellular) H2 partial pressures are described quantitatively by intracellular thermodynamics

ExtracellularMeasurement

IntracellularBioenergetics

PH2

Spatial Constraints

consumer consumer

producer producer H2

(HP) (HP)

(HC) (HC)

H2 H2

H2 measurementH2 measurement

HP bulk fluid HC HP HC bulk fluid

PH2 measured in bulk fluid > PH2 in HC cell PH2 measured in bulk fluid = PH2 in HC cell

Efficient utilization of H2 requires mass transport and high concentration gradient unless

mitigated by spatial arrangements. The fact that quantitative H2 etc measurements reflect

bioenergetic control argues for non-random arrangement of consumers and producers asillustrated above (see later re AOM)

Organic matter

In Situ Free Energy Yields in CLB Sediments

G(KJ·mol-1) G(KJ·mol-1)

August T=27oC

November T=14.5oC

Dep

th (

cm) MP MP

Biogeochemical Redox Couples

aerobic respiration

CH2 O + O2 CO2 + H2O

1 mole glucose 30-32 mole ATP

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

Molecule 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

http://www.bris.ac.uk/Depts/Chemistry/MOTM/atp/atp1.htm

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 ATP How 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)

ATP + H2O ADP + HPO4When 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.