•• THERMODYNAMICS OF GROWTH -contTHERMODYNAMICS OF GROWTH -cont’’dd

•• APPLICATIONS of MICROBIAL APPLICATIONS of MICROBIAL CHEMOLITHOTROPHY & ANAEROBIC RESP.CHEMOLITHOTROPHY & ANAEROBIC RESP.

Systems MicrobiologyWeds Sept 20 - Ch 5 & Ch 17 (p 533-555)

Bioenergetics & Metab.Diversity•• BASIC MODES OF ENERGY GENERATIONBASIC MODES OF ENERGY GENERATION

Phototrophs (Use Light as Energy Source )

Photoautotrophs (Use CO2)

Photoheterotrophs (Use Organic Carbon)

Figure by MIT OCW.

Anoxygenic photoautotrophs utilize cyclicphotophosphorylation

Light

Excited electrons (2 e-)

Electron transport chain

Energy for productionof ATP

Electron carrier

Cyclic Photophosphorylation

Chlorophyll

ATP

Figure by MIT OCW.

LOTS OF DIVERSITY IN BACTERIAL ANOXYGENIC PHOTOTROPHS !

+0.5

+0.25

-0.25E0'(V)

-0.5

-0.75

-1.0

-1.25

0

P870

Purple Bacteria Green Sulfur Bacteria Heliobacteria

Fd

P870

NADH

Reverse electron flow

QCytbc1

Light

Cytc2

BChlBPh

Light Light

Fd

P840

Chl a

QQCyt

bc1Cytc553

Cytc553P840

P798

Chl a-OH

FeS FeS

Cytbc1

P798

Figure by MIT OCW.

+1.0

+0.75 H2O

O2 + 2H12

e-

+0.5

+0.25

0.0

-0.25

-0.5

-0.75

-1.0

-1.25

LightPhotosystem I

Photosystem II

E0(V)

'

LightP680

Cyclic electron flow(generates proton motive force)

Noncyclicelectron flow(generates protonmotive force)

FeS

Pc

QB

Q pool

Chl a0

P680*

NAD(P)HNAD(P)+

P700

Cyt bf

P700*

Fd

Fp

QA

Ph

QA

PSII PSI

The Z Scheme

Figure by MIT OCW.

HALOARCHAEAHALOARCHAEALive in Live in hypersaline hypersaline habitatshabitats

Aerial photograph of haloarchaea changing the colors of their saltwater habitats removed due to copyright restrictions.

Figure by MIT OCW.

Asp-85

Arg-82

Asp-96

Glu-204

H+

H+

Glu-194

Retinal

Microbial rhodopsins fall into two main functional classes

Sensory rhodopsinsLight-driven ion pumps

Figure by MIT OCW.

Bacteriorhodpsin

BR

H+

Cl-

HR Htrl

His-kinase

Regulator

Regulator

Regulator

His-kinase

P

P

Methylation helices

SRll HtrllSRl

Flagellar motor

Rhodopsin Functional Diversity

Cytoplasm

Bacteriorhodopsin and proteorhodospinLight driven proton pumps

High H+ concentration

Outside cell

Inside cell

Electrons from NADH or chlorophyll

ADP+ Pi

Low H+ concentration

H+ H+

1

2

3

ATP

} Membrane

ATPsynthase

Electron transport chain (includes proton pumps)

Figure by MIT OCW.

Figure by MIT OCW.

Cell interior

Cell exterior

Asp-85

Arg-82

Asp-96

Glu-204

H+

H+

Glu-194

Retinal

Images and diagrams of various rhodopsins removed due to copyright restrictions.See Science 289 (September 15, 2000). www.sciencemag.org.

Venter et al., Environmental Genome ShotgunSequencing of the Sargasso Sea,

Science 394:66-74 (2004)

Many new bacterialproteorhodopsins discoveredin environmetnal shotgunsequencing

Diagram removed due to copyright restrictions.

Images of a hybrid automobile, hydrocarbons, and electricity removed due to copyright restrictions.

ALL ORGANISMS

chemotrophs phototrophs

chemolithotrophs chemoorganotrophs

Where do organisms get their energy?

Oxidize inorganic compounds Oxidize organic compounds

Derive energy from light

MICROBIALMICROBIALMETABOLIC METABOLIC DIVERSITYDIVERSITY

Relative VoltageRelative VoltageREDUCTANTS (EAT) OXIDANTS (BREATHE)

-10

- 8

- 6

- 4

- 2

0

+ 2

+ 4

+ 6

+ 8

+ 10

+ 12

+ 14

-10

- 8

- 6

- 4

- 2

0

+ 2

+ 4 + 6

+ 8

+ 10 + 12

+ 14

OrganicCarbon

CO2

SO4=

AsO43-

FeOOH

SeO3NO2

-

NO3-

MnO2

NO3-/N2

O2

H2

H2SSo

Fe(II)

NH4+

Mn(II)

A

B

Photoreductants

Microbes can

eat & breathe just

about anything !

Diagrams removed due to copyright restrictions.See Figures 5-22a, 5-20, and 5-23 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.

Chemolithoautotroph (chemo [chemical], litho [rock], auto[self], troph [feeding])Energy source: inorganic substrates (H2, NH3, NO2-, H2S, Fe2+)Carbon source: CO2

e- acceptor: O2(aerobes), or S(some anaerobes), Fe3+, NO3, SO4

Chemolithoautotrophs can be grouped according to the inorganic compounds that they oxidize for energy:Nitrifiers - Oxidize reduced Nitrogen compounds such as NH4+

Sulfur Oxidizers- Oxidize reduced Sulfur compounds such as H2S, S0, and S2O-

Iron Oxidizers- Oxidize reduced Iron-Fe2+ (ferrous iron)

Hydrogen Oxidizers-Oxidize Hydrogen gas-H2

METABOLIC DIVERSITY - Continued ….

Table of energy yields from the oxidation of various inorganic electron donors removed due to copyright restrictions. See Table 17-1 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms. 11th ed.Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.

METABOLIC DIVERSITYCHEMOLITHOAUTOTROPHS - Examples

CHEMOLITHOTROPHIC AMMONIA OXIDATION - AEROBIC

NH4+ + 3/2O2 --> NO2

- + H2O + 2H+

NH2OH

NH2OH+ H2O

+ H2O

+ O2+ 2H+NH3 H2O

O2 + 4H+

O

O-N + 5H+

Oxidation of hydroxylamine

2H+H+

H+

ADP + Pi

ATP

12

Oxidation of ammonia

Reduction of oxygen

HAO

AMO

4e- 2e-Cyt c

Cyt c

Q Cyt aa3

2e-2e-

2e-

Periplasm

Figure by MIT OCW.

Anammox means "anaerobic ammonium oxidation". Anammox is both a new low-cost method ofN-removal in wastewater treatment, and a spectacular microbial way of life - woo - woo !

CHEMOLITHOTROPHIC AMMONIA OXIDATION -

ANAEROBIC

Courtesy of the Department of Microbiology at Radboud University Nijmegen. Used with permission.

ΔEo= Eo (electron acceptor) - Eo (electron donor) = 1233 mV

2NH4+/N2 half reaction (6e-) Eo = -0.277 V

2NO2-/N2 half reaction (6e-) Eo = +0.956 V

ΔGo=-nF ΔEo= -(3) (96.5 kJ/Vmol)(1.233V) = - 357 kJ/mol

Broda predicted, based solely on the thermodynamics, that suchmicroorganisms should exist. (And also the fact that if abioenergetically favorable niche exists, a microbe will evolveto fill it !).

About a decade later, the ’ bugs’ were discovered in bioreactorsstarted from waste water treatment plants.

Diagram removed due to copyright restrictions.

Compared to conventionalnitrification/denitrification, thismethod saves 100% of the carbonsource, & 50% of the requiredoxygen. This leads to a reduction ofoperational costs of 90%, a decreasein CO2 emissions of more than 100%(the process actually consumes CO2),and a decrease in energy demand.

Photograph removed due to copyright restrictions

Anaerobic ammonium oxidation by anammox bacteria in the Black Sea Marcel M. M. Kuypers, A. Olav Sliekers, Gaute Lavik, Markus Schmid, Bo Barker Jørgensen, J. Gijs Kuenen, Jaap S. Sinninghe Damsté, Marc Strous and Mike S. M. Jetten. Nature 422, 608-611 (10 April 2003)

Graphs removed due to copyright restrictions.

ANAEROBIC RESPIRATION =Dumping your electrons on something other than oxygen

Denitrification = Use of NO3- as terminal electron acceptor, that results

in complete conversion to N2 gas.

See Figures 17-35 and 17-37 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms. 11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.

Diagrams removed due to copyright restrictions.

Geobacter growing on iron oxides

CH3COO- Fe3+

e-

Fe2+CO2

Anaerobic respiration

Image of geobacter growing on iron oxides removed due to copyright restrictions.

Complex Organic Matter

Hydrolysis

Fermentable Substrates

H2 Acetate

Microbial Competition for Substrates

Other low molecular weight organic acids}

NO3- reducers

Mn(IV) reducersFe(III) reducersSO4

2- reducersMethanogens

}Organic Matter Degradation In Anaerobic Environments

Fermentation

Figure by MIT OCW.

Diagram removed due to copyright restrictions.

E- acceptor ΔGo’ (using glucose)

Oxygen -3190 kJ/mol

NO3- -3030

Mn (IV) -3090

Fe(III) -1410

Sulfate -380

CO2 -350

From Nealson and Saffarini 1994

Energetic explains order: not all e-acceptors are equal!

25

Complex Assemblage of Organic Matter

Hydrolysis into Constituents

Fermentable Sugars and Amino Acids

Fermentation by a Fermentative Microbial Consortium

Acetate and Minor Products Aromatic CompoundsLong-Chain Fatty Acid

Fe3+ Oxide

Fe2+

Geobacter spp.

CO2

Generalized pathway for the anaerobic oxidation of organic matter to carbon dioxide with Fe3+ oxide serving as an electron acceptor in temperate, freshwater and sedimentary environments. The process is mediated by a consortium of fermentative microorganisms and Geobacter species.

Figure by MIT OCW.

Source

Methanogenic

SO4 reduction2

Fe(III) reduction

Mn(IV) reduction

NO3 (IV) reduction Aerobic

Groundwater flow

-

High O2

Low O2

High organics

Figure by MIT OCW.

The distribution of terminal electron-accepting processes (TEAPs) observed within anaerobic portions of aquiferscontaminated with organic compounds.

Images removed due to copyright restrictions.See Lovley, D. R., E. J. P. Phillips, Y. A. Gorby, and E. R. Landa. "Microbial Reduction of Uranium." Nature 350 (1991): 413-416.

Anaerobic respiration to “clean up” of uranium pollution

Acetate + U (VI) U (IV)s + CO2

CH3C00- + 4 U(VI)U (IV)s + 2HCO3- + 9H+

Carried out by Geobacter

Example of “bioremediation”Lovley DR, Phillips EJP, Gorby YA, Landa ER. Microbial Reduction of Uranium, 1991, Nature.350(6317): 413-6.

Soluble= mobile Insoluble, immobile

Photograph removed due to copyright restrictions.

Diagram removed due to copyright restrictions.

Diagram removed due to copyright restrictions. See Bond, Holmes, Tender, and Lovley. Science 295 (2002): 483-485.

Diagram and photograph of a sediment microbial fuel cell removed due to copyright restrictions.

Correspondence between pilus current and applied voltage demonstrating the linear, ohmic, response characteristic of a true conductor.

Schematic of the electronic connection of the AFM tip in a conducting probe atomic force microscope (CP-AFM). HOPG, highly oriented pyrolytic graphite.

Atomic force microscope stage

V=IR

Extracellular electron transfer via microbial nanowires.Nature. 2005 Jun 23;435(7045):1098-101.

‘Good’ electron acceptors

‘Good’ electron donors

Photograph removed due to copyright restrictions

DNS

SO42-

SO42- Diffusion

Sea FloorSeawater

METHANE FLUX

Sulfa

te Re

ducti

on Z

one

AB

CDNS

Figure by MIT OCW.

AANAEROBIC NAEROBIC MMETHANE ETHANE OOXIDATIONXIDATION

Geochemical Observations:Geochemical Observations:CH4 + SO4

2- HCO3- + HS- + H2O

Microbiologically ???CH4 + 2H2O CO2 + 4H2 “Reverse Methanogenesis”

HSO4- + 4H2 HS- + 4H2O Sulfate reduction ΔGo’ = -156 kJ/mol

ΔGo’ = +131 kJ/mol

CH4 + HSO42- CO2

+ HS- + 2H2O ΔGo’ = -25 kJ/mol

CHCH44

COCO22

SOSO44-2-2

HSHS--HH2 2

World maps showing global methane distribution removed due to copyright restrictions. See D’Hondt et al. Science 295 (2002): 2067.