Growth and Diversity of H2-Producing BacteriaMary Ann Bruns, Ph.D.

Dept of Crop & Soil SciencesPenn State University

EarlyArchaeanlandscape

3.5 billionyears ago

• Abundance and diversity of H2-producing microorganisms

• Known biochemical pathways for biohydrogen production

• Microbial insights which might help us develop a human-scale hydrogen economy

Carl Woese at Univ of Illinois,

Champaign-Urbana

With DNA sequencing

films revealinga universal tree

of life

Five Kingdoms

Animals

Fungi

Monera

Protists

Plants

Five Kingdoms

Animals

Fungi

Prokaryotes

Monera

Protists

Plants

Eukaryotes

Eukaryotes represent four of “Five Kingdoms”

but just one Domain

Archaea

Prokaryotes make up two Domains in the Universal Tree of Life

Eukarya

Bacteria

3052Marine

22-215560Terrestrial

ProkaryotesPlants & algae

Global estimates of biomass carbon1 in Petagrams(1015 g) includes surface and subsurface soils

& ocean sediments

1Biomass C includes live protoplasm, cell walls, and structural materials

Evolutionary lineages with species known to produce H2

Protists

Some anaerobic fungi & protistscontain “hydrogenosomes”,H2-producing organelles evolved from bacterialsymbionts

Akhmanova et al. Nature 1998

Hydrogenases are a diverse family of enzymes thatcatalyze the reversible reaction 2H+ + 2e- ↔ H2

Theoretical & Computational Biophysics GroupUniv of Illinois-Champaign Urbana

H+ and e- passage through the Cpl hydrogenase of Clostridium pasteurianum

Prokaryotic H2-generation

Oxygenic photosynthesisCyanobacteria (formerly called “blue-green algae”)Genera include Anabaena, Nostoc, SynechocystisMechanisms:

Direct biophotolysisIndirect H2 generation from reduced carbon

Key Advantage:Availability of solar radiation

Key Drawback:O2 sensitivity of hydrogenaseLow efficiency of solar energy capture

DIRECT (O2-SENSITIVE)

INDIRECT

Prokaryotic H2-generation

Anaerobic photosynthesisPhotosynthetic bacteriaGenera in betaproteobacteria include Rhodobacter, RhodospirillumMechanisms:

Direct biophotolysis (electrons from H2O)Indirect H2 from fermentation (electrons from reduced carbon)H2 production by nitrogenase during N2 fixation

Key advantage:Availability of solar radiationNo O2 to inhibit hydrogenase

Key drawback:Low efficiency of solar energy captureHigh cost of photobioreactors

Prokaryotic H2-generation

Dark-fermentationAnaerobes and facultative anaerobesSporeforming genera (Clostridium)Non-sporeforming genera (Enterobacter, Citrobacter)Mechanisms:

Electrons derived from reduced carbon to generate reductantReductant is oxidized and electrons transferred to H+

Key advantage:Use renewable wastes as electron donorsLower cost bioreactors

Key drawback:Low molar H2 yields from organic wastes compared to methane generationPure culture of Clostridium

isolate from PSU soil

Cyanobacteria

Anaerobic photosynthetic bacteria

Anaerobic bacteria

H2O

H2 + ½ O2

Energy of formationG = 242 kJ/mol

Activation energy

Activation energy

C6H12O6 12 H2 + 6CO2

G = -3 kJ/mol

4H2 + 2 AcetateG = -46 kJ/mol

Prokaryotic H2 generation

Early experiments to create “hybrid” H2-producing systems from chloroplasts and hydrogenase enzymes from

fermenting Clostridium bacteria

Only about 1% of extant prokaryotic species have been studied.

Need for continued discovery of new species, enzymes, pathways for energy production.

H2 metabolism of Shewanella oneidensis MR-1

Other mechanisms for prokaryotic H2 generation

Pyruvate conversion during stationary phase

Meshulam-Simon et al. 2007. Appl Environ Microbiol

Other mechanisms for prokaryotic H2-generation

N2 fixationFree-living N2 fixers (cyanobacteria, photosynthetic bacteria,

Azotobacter spp.)Symbiotic N2 fixers (e.g., Rhizobium, Frankia spp.)Mechanism:

H2 is byproduct of nitrogenase enzyme

Key advantage:Use organic wastes as electron donorsCreate artificial conditions in low-cost bioreactors

Key drawback:Need for induction of nitrogenaseNitrogenase’s O2 sensitivityRelatively low H2 yields N2-fixing nodules

on legume roots

Bacteroidsin nodules

If H2 production by prokaryotes is so prevalent, why doesn’t H2 build up in the atmosphere?

Prokaryotes’ ability to take up and oxidize H2 is probably even more widespread than the ability to

produce H2

H2-oxidizing bacteria are diverse and widespread“syntrophs” consume H2 as fast as it’s produced

H2 is readily consumed by microbial biomass when injected into soil ( 33 nmol H2 cm-3 hr-1 )

Dong et al. 2001

Hypothesis: Legumes “fertilize” soils not only with fixed N but also with H2.

( Dong et al., 2003)

( Invention disclosure for patent application: http://www.wipo.int )

Some rhizobia lack “uptake hydrogenases” and cannot recycle H2 from nitrogenase

H2 leaking from these nodules is consumed within 5 cm of the nodule surface

d = 4.5 cm( 30-254 nmol H2 cm-3 hr-1 )

Favre et al 1983

Popelier et al., 1985

Relationship between the rate of H2-uptake by soil and microbial biomass content

If we prevent microbes from consuming H2, how much do we produce in experimental systems?

Photochemical production mmol H2/g-hr

Oxygenic photosynthetic bacteria 0.4-1.3

Anoxygenic photosynthetic bacteria 3-10

Dark fermentation production

Spore-forming anaerobic bacteria 7-25

Nonspore-forming, facultative anaerobic bacteria 10-17

From Tanisho, BioHydrogen, 1998

How feasible is “Industrial Revolution-Style” H2production?

Large-scale production and storageUtility-based dissemination

What would a “Biological Revolution” energy system look like?

Prokaryotic Life Exists Predominantly in Biofilms

Source: USDOE Genomes to Life

Tight coupling between energy production and energy consumption

Can humans learn how to be “syntrophs” tomicrobial energy partners?

Biologically based energy systems:• Broad distribution of energy production sites• Less distance between points of energy

production and use • Energy production tightly linked to consumption

Report from the American Academy of MicrobiologyNovember 2006

Questions?