A staggering number of organism-organism and organism- environment interactions underlie global biogeochemistry These can be studied at vastly

Transcription

1 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 O 2 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: Hoehler TM, et al., Comparative ecology of H2 cycling in sedimentary and phototrophic ecosystems Antonie von Leeuwenhoek 81: Hoehler et al., Apparent minimum free energy requirements for methanogenic Archaea and Sulfate reducing bacteria in an anoxic marine sediment. FEMS Microbial Ecol. 38;

2 A staggering number of organism-organism and organism- environment interactions underlie global biogeochemistry These can be studied at vastly different spatial and time scales

3 PRESS RELEASE Date Released: Thursday, February 21, 2002 Texas A&M University Rock-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.

4 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?

5 Biogeochemical Redox Couples D CO 2 + H 2 O CH 2 O + O 2 CH 2 O + O 2 CO 2 + H 2 O (+D) oxygenic photosynthesis Interdependency? aerobic respiration CH 4 + 2O 2 CO 2 + 2H 2 O(+D) D CO 2 + HS - + H 2 O biomass + SO 2-4 C 6 H 12 O 6 2CO 2 + 2C 2 H 6 O (+D) oxidative methanotrophy anoxygenic photosynthesis fermentation 4H 2 + SO 4 2- S H 2 O (+D) CO 2 +2H 2 CH 4 + 2H 2 O (+D) sulfate reduction methanogenesis

6 P680* P680+ CH 2 O CO 2 10 pe(w) 10 H H H NH 4 + CO 2 H 2 N 2 NH 4 + N 2 CH 2 O E o(v) DG kj/mol e Redox Potentials & Energy Yields The electron tower.. CH 4 CO 2 CO 2 CH 4 H 2 S S S H 2 S 100 H 2 S 2 SO 4 2 SO 4 H 2 S Strongest reductants, or e donors, on top LHS Fe 2+ Fe(OH) Fe(OH) 3 Fe 2+ 0 Electrons fall until they are caught by available acceptors OXIDATION REDUCTION The further they fall before being NO 3 NH 4 caught, the greater the difference NH NO in reduction potential and energy 2 NO 2 NO 3 NO 3 NO released by the coupled reactions Mn 2+ MnO 2 MnO 2 Mn2+ CO CO 2 CO 2 CO N NO 3 NO 3 N 2 H 2 O O 2 O 2 H 2 O 0 (Last Common Ances P680+ P Fe 3+ Fe V

7 P680* P680+ pe(w) DG E (V) o CO 2 CH 2 O kj/mol -0.5 CH Redox Potentials e- 2 O CO & Energy Yields H 2 H H H2 + + NH 4 N 2 NH 4 N 2 CH 4 CO 2 CO 2 CH 4 Reaction must be exergonic (-ve DG) H 2 S S S H 2 S H SO SO4 2 2 S 4 H 2 S The energetically most favored reaction proceeds first ie Fe 2+ Fe(OH) 0 0 Fe 2+ 3 Fe(OH) 3 0 CH 2 O first degraded with O 2 OXIDATION REDUCTION CH - 2 O degraded with NO 3 next CH 2 O degraded with Mn 4+ next NH 4 NO 3 NO 3 NH 4 followed by SO4 2-, 2 NO 2 NO 3 NO 3 NO 2 Mn 2+ MnO Mn 2+ 2 MnO and CO 2 last (methanogenesis) CO CO 2 CO 2 CO N 2 NO 3 NO 3 N 2 O 2 0 H 2 O O 2 H 2 O (Last Common Ancestor) P680+ P Fe 3+ Fe V

8 Energy Calculations aa +bb cc + cd DG = G f (aa + bb) G f (cc + dd) Where G fo is the free energy of formation of 1 mole under standard conditions (ph 7, 25C) DG = DG +RT ln K (T). K=C c D d /A a B b R= 1.98cal.mol -1. K -1 DG = DG (T) +RT ln [C] c[ D] d [A] a [B] b

9 How do microbes garner energy and carbon? Organic compound Carbon flow CO 2 O 2 Electron flow respiration Organic compound Carbon flow CO 2 Electron flow anaerobic respiration NO - SO Fe 3+ Other organic compound Inorganic compound H 2 H 2 S NH 3 Fe 2+ CO 2 Carbon flow O 2 Electron flow Biosynthesis lithotrophy

10 Electron Donor Mechanisms and Balance Sheets Electron Carrier Organic compound Electron flow Carbon flow CO 2 NAD + H2 NADH (catab) or NO - SO Fe 3+ Other organic compound Terminal Electron Acceptor NADP + H2 NADPH (anab) 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 O 2 DG = 688kcal Therefore aerobic respiration ca. 39% efficient In contrast, glucose fermentation lactate = 29 kcal/mol ca. 50% efficient

11 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 FADH 2 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 FADH 2 leads to 2 moles of ATP. Therefore, for each mole of pyruvate which enters the TCA cycle, 12 moles of ATP can be generated

12 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 O 2 DG = 688kcal Therefore, in this case, aerobic respiration ca. 39% efficient In contrast, glucose fermentation lactate = 29 kcal/mol ca. 50% efficient

13 Multi-Step Organic Matter Remineralization in Anoxic Systems Biopolymers (CH 2 O) n Monomers e NO 3 Æ NH 4 Mn 4+ Æ Mn 2+ Fe 3+ Æ Fe 2+ Small Organics SO 4 2- Æ H 2 S CO 2 CO 2 Æ CH 4 oxidation reduction Requires numerous extracellular electron transfers

14 H 2 2H + + 2e - A nearly ubiquitous means of extracellular electron transport in microbial redox chemistry

15 Hydrogen Anaerobic metabolism strongly sensitive to ph 2 Fermentation frequently characterized by obligate (1-2 C s) or facultative (>3 C s) H 2 production Reaction only energetically feasibly with H 2 sink Obligate H 2 producers don t grow in pure culture Readily grown in co-culture H 2 consuming reactions affected oppositely

16 Hydrogen H 2 consuming reactions affected oppositely e.g. with mm SO 2-4 SRB can maintain very low ph 2. In presence of active SRB, H 2 too low for methane production to be energetically feasible Often see zonation between SR and MP under thermodynamic control

17 Hydrogen 2H 2 + 2CO 2 CH 3 COOH + O 2 + DG CH 3 COOH + O 2 2H 2 + 2CO 2 + DG Opposite biochemistry when methanogen present Anaerobic oxidation of methane is energetically marginal unless???? 2CH 4 +SO 4 2- S CO 2 +4H 2

18 H 2 has a High Relative Stoichiometry in Many Anaerobic Remineralization Processes Production CH 3 CH 2 COOH + 2H 2 O Æ CH 3 COOH + CO 2 + 3H 2 Consumption CO 2 +4H 2 Æ CH 4 +2H 2 O

19 Free Energy Yield Depends Exponentially on Stoichiometry in Reaction CO 2 + 4H 2 Æ CH 4 + 2H 2 O DG = DG (T) +RT ln P CH4. (P H ) 4 P CO2 2 DG mp is much more sensitive to P H2 than to P CH4 or P CO2

20 Thermodynamics of Inter-Species H 2 Transfer producer Both Organisms Depend Highly on H 2 Partial Pressure: H 2 Too High Alters Production Æ Pathway Shifts, Inhibition, Reversal consumer Too Low Inhibits Consumption

21 H 2 in the Environment producer H 2 consumer P H2 controlled by the balance between production and consumption For constant or decreasing H 2 production rate (e.g. sediments), P H2 in practice reflects control by H 2 consumption Consumption very efficiently coupled to production; P H2 held at very low steady-state levels; residence times short (seconds or less)

22 Free Energy Regulation in Methanogenesis 4H 2 + CO 2 CH 4 + 2H 2 O Conc.( M)t res (s)[x] m CH 3 COOH CH 4 + CO 2 Æ (s)h 2 DD D x3.5 DD Data for methanogenic sediments from Cape Lookout Bight at 22 C; Responsiveness D [X] and Dt required to change free energy yield by 10kJ/mole

23 Inter-Species H2 Transfer in a Complex Microbial Ecosystem producer 1 producer 2 H consumer 1 2 consumer 3 consumer 2 producer 3

24 Controls on H in Anoxic Sediments 2 producer H 2 consumer P H2 in sediments is controlled by H 2 consumers Steady-state P H2 reflects efficiency of consumption; constrained by physiologic limitations of H2 consumers Ultimate physiologic limitation: requirement to extract sufficient free energy from H2 consumption to permit continued metabolism

25 P H 2 = Ê Á Á Ë [ ] X D o red G - T [ ] exp X ox Ê Á Ë RT D G rxn ˆ ˆ 1 n Steady State H 2 Concentrations Sensitive To: Concentrations of Products and Reactants (Xox and X red ) Specific Redox Couple (e.g. CO 2 /CH 4 -vs- SO 2-4 /S 2- ) Temperature Energy Yield of Reaction ( DG rxn )

26 Effect of Sulfate Concentration on H 2 SO H 2 S 2- +4H 2 O P H2 [] 2 - S Ê =Á Á[ 2 ] Ë - SO 4 Ê exp Á Ë D o -D G T RT G rxn 1 ˆ 4 ˆ Increasing Sulfate = Decreasing H 2

27 Impact of Sulfate Concentration Change on DG and H 2 in Sulfate-Reducing CLB Sediments DG H D G (kj mol -1 ) H 2 (Pa) P H2 = 0.25 [SO4 2- ] (R 2 = 0.993) -32 Expected DG SR -vs- SO Sulfate (mm) Sulfate (mm) Deduction: H 2 is drawn down to compensate for increasing sulfate; SRB community maintain const DG near limit for maintenance but max efficiency. An adaptation to substrate limitation?

28 Depth Profiles of H 2 in CLB Sediments 0 10 Sulfate (mm) Sulfate Sulfate (mm) Sulfate Depth (cm) H H 2 50 August 27 C H 2 (Pa) November 14.5 C H 2 (Pa)

29 consumer 1 Inter-Species H2 Transfer in a Complex Microbial Ecosystem Consumer 1 Controls: Steady-state H 2 Thermodynamics of other microbial processes Is Controlled By: Environmental factors affecting DG (temperature, chemistry, etc.) Both can be Addressed Quantitatively producer 1 consumer 2 producer 2 H 2 consumer 3 producer 3

30 2 Bulk phase (extracellular) H partial pressures are described quantitatively by intracellular thermodynamics P H 2 P H 2 = È X Í Î Í X red ox Ê G exp Á D Ë o T - D G RT rxn ˆ 1 n Extracellular Measurement Intracellular Bioenergetics

31 Spatial Constraints H 2 measurement H 2 consumer (HC) H 2 producer (HP) Organic matter H 2 consumer (HC) H 2 measurement H 2 producer (HP) P H 2 P H 2 HP bulk fluid HC HP HC bulk fluid P H2 measured in bulk fluid > P H2 in HC cell P H2 measured in bulk fluid = P H2 in HC cell Efficient utilization of H 2 requires mass transport and high concentration gradient unless mitigated by spatial arrangements. The fact that quantitative H 2 etc measurements reflect bioenergetic control argues for non-random arrangement of consumers and producers as illustrated above (see later re AOM)

32 In Situ Free Energy Yields in CLB Sediments DG (kj mol-1 ) SR 10 DG (kj mol-1 ) SR Depth (cm) MP MP August T = 27 C November T = 14.5 C

33 Biogeochemical Redox Couples aerobic respiration CH 2 O + O 2 CO 2 + H 2 O O 2 1 mole glucose mole ATP fermentation 1 mole glucose 2-4 mole ATP Biosynthesis requires approx. 1mole ATP per 4g of cell carbon

34 Biogeochemical Redox Couples oxygenic photosynthesis CO 2 + H 2 O CH 2 O + O 2

35 Molecule of the Month Adenosine Triphosphate - ATP Paul 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

36 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).

37 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 (HPO 4 ) 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

38 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.