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Growth and Diversity of H2-Producing BacteriaMary Ann Bruns, Ph.D.
Dept of Crop & Soil SciencesPenn State University
• 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
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
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