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Renewable Hydrogen Production from Biological Systems
Matthew Posewitz
Colorado School of Mines
DOE Biological Hydrogen Production Workshop
September 24th, 2013
H2 production
PSII/PSI pathway
PSI/nonphotochemical PQ
Dark fermentation
H2 uptake
oxyhydrogen reaction
photoreduction
Photosynthetic H2 pathways
Peters JW et al. Science 1998 Nicolet Y et al. Structure and Folding Des. 1999
Phototroph Hydrogenases • Cyanobacteria
– Only [NiFe]-hydrogenases identified to date.
– Typically dark H2 production.
– Linked to NAD(P)H.
• Eukaryotic Algae – Only [FeFe]-hydrogenases
identified to date.
– Typically two hydrogenases with only the H-cluster domain.
– Linked to ferredoxin and photosynthetic electron transport.
Hydrogenases
• [NiFe]-Hydrogenases
– Reversible O2 inhibition
– Group 1- Uptake Enzymes Membrane bound
– Group 2- Cyanobacterial uptake and H2 Sensing Enzymes
• Latter are O2 tolerant
– Group 3- Bidirectional
• NAD(P)(H) linked
– Group 4- H2 Production Enzymes
• Ferredoxin linked
• [FeFe]-Hydrogenases
– Algal enzymes are simplest to date
– Often ferredoxin linked
– Hnd multimeric enzymes are NAD(P)(H) linked
– Differences in O2 tolerance reported but typically irreversible inhibition
– Capable of very high turnover >103/s
S SCys2
Ni
Fe
COCN CN
Cys4
SCys3
Cys1
S
S SCys2
Ni
Fe
COCN CN
Cys4
SCys3
Cys1
S
S SCys2
Ni
Fe
COCN CN
Cys4
SCys3
Cys1
S
Cys2
Ni
Fe
COCN CN
Cys4
Fe
COCN CN
Cys4
SCys3
Cys1
S
Nitrogenase MoFe Protein
P Clusters FeMo-cofactor
Nitrogenase Fe Protein
Nitrogenases
Mo N2 + 8H+ + 9e- = 2NH3 + H2
Fe N2 + 21 H+ + 12e- = 2NH3 + 7.5H2
V N2 + 12H+ + 12e- = 2NH3 + 3H2
~ 2 ATP/e-
Strong thermodynamic driving force
essentially irreversible H2 producti on
Oxygen sensitive
Heterocyst differentiation or temporal separation
Mutants with increased H2 production reported
Prolonged H2 Production in Algae Induced by Nutrient
Stress
P R
0
20
40
60
80
100
120
140
0 20 40 60 80 100
Incubation time -S, h
mm
ole
s O
2 m
g C
hl-1
hr-
1
Melis et al., Plant Phys. 2000 Kosourov et al., 2002
Time, h
40 60 80 100 120 140 160 180
Volu
me o
f gas c
olle
cte
d, m
l
0
100
200
300
400
Photosynthetic antennae mutant with enhanced H2 production
tla1 mutant Polle and Melis (UCB) Kosourov, Ghirardi and Seibert 2011
Nitrogenase Catalyzed H2 Production in Cyanothece: Sustained Biophotolysis
Reddy et al., 1993
• Rates of H2
production up to ~
400 mmole/hr . mg chl
• O2/H2 coproduction
• Predominately light
driven
Melnicki et al., 2012, Beliaev laboratory PNNL
Photosynthesis and Starch- Interplay for H2 Production
Time -S, h
0 20 40 60 80 100 120 140
Glu
cose
, m
g/m
l
0
50
100
150
200
250
WT
sta7-10
Libessart et al., The Plant Cell 1995
Initial Hydrogen Production Rates
0
20
40
60
80
100
120
140
160
0 0.5 1.5 4 7 o/n
Time (h)
mm
ole
s H
2 m
g C
hl-1
hr-1 330
sta6
sta7
sta7(J4)::STA7
330 sta6(J5) 0 0.5 1.5 4.0 7.0 o/n 0 0.5 1.5 4.0 7.0 o/n
Starch Hyperaccumulation
D66 2
.2.2
3
D66
(wild
typ
e)
TAP+N TAP-N
Starch sheath surronding pyrenoid
050
100150200250300350400450500
CC
12
4
sta6
sta7
-10 c5
c19
CC
124
sta6
sta7
-10 c5
c19
µg
sta
rch
/ m
l cu
ltu
re
0 hrs 96 hrs
TAP + N TAP - N
0
20
40
60
80
100
120
140
CC
12
4
sta
6
sta7
-10 c5
c19
CC
12
4
sta
6
sta7
-10 c5
c19µg
star
ch /
10
6ce
lls
0 hrs 96 hrs
TAP + N TAP - N
A
B
Hydrogenase Maturation Heterologous Expression and Screening in Alternate hosts
HydEFHydG
TAA TAA
1 kbHydEFHydG
TAA TAA
1 kb
Insertion site in
HydEF-1 mutant
H2 Production Rates
0
10
20
30
40
50
A1 A1 + EF A1 + G A1 + EF + G
Hyd transformants
mm
ole
H2 m
g p
rote
in-1
h-1
Hydrogenase Maturation Heterologous Expression and Screening in Alternate hosts
Expression of [FeFe]-hydrogenase
in Anabaena heterocysts. Gartner
et al. 2012, Hegg lab MSU
Screening mutant libraries for enhanced O2 tolerance
Stapleton and Swartz 2010 Stanford
Single colonies in 96-well microtiter plate
R4R3R2R1
Combine DNA from 10 pools into “superpool”
Screen for specific gene disruptions by PCR (gene-specific and APHVIII-specific (RB2) primers
Pool 96 colonies and isolate genomic DNA
RB2
Target Gene
F1 F2 F3 F4
PSAD PRO Int CYC6APHVIII
PSAD PRO Int CYC6APHVIII
Arthur Grossman, Claudia Catalanotti, Wenqiang Yang, Venkat Subramanian, Alexandra
Dubini, Mike Seibert
Reverse Genetics via PCR-Based Mutant Library Screening
Chlamydomonas reinhardtii [FeFe]-hydrogenases and KO lines
H-cluster
Afl II ClaI Kpn I
HindIII SalI XhoI MfeI NotI
Bgl II
5’ UTR 3’ UTR
Genomic HYDA1 Exon
Chloroplast Transit peptide
Genomic HYDA1 Intron
Multiple Cloning Site
C. r. HYDA1 cDNA
Avr II
• HYDA1 is the dominant photolinked enzyme
• The two H2ases are not uniquely dedicated to photo/ferm hydrogen
production
• Both enzymes can participate in photo/ferm H2 production.
• Increases in ferm H2 production can be achieved by increasing
HYDA1 levels
• Sufficient H2ase is present in WT cells to handle all PS electron flux.
Meuser et al., 2012
HYDA paralogs are reciprocally monophyletic
Alg
ae Meuser et al., 2011 Planta
hydrogenase oxygen sensitivity
Docking Hydrogenase into Cellular Metabolism
Terashima et al. 2011
Fermentation to H2: PFL1 and ADH1 Deletions in Chlamydomonas
Hypotheses:
1) PFL competes with
PFR andH2 production
2) ADH1 competes with
H2ase for reductant at
the level of NADH
Elimination of either or
both should stimulate H2
production
Reality: neither the
single mutants nor the
double mutant increase
fermentative H2.
Hydrogen From Starch Using in vitro Pentose Phosphate Pathway or Acetate Microbial Fuel Cells
Zhang et al., 2007 PLoSOne
Prospecting for New Enzymes and Organisms
H2 production in GSL halophilic Algae
H2 production in high salt
Hydrogenase Diversity in Naure: hydA along
Great Salt Lake Vertical Gradient
Eric Boyd, John Peters Montana State University
Jon Meuser, CSM
[a] [b] Conserved Accessory Domains:
ACS: acetyl-CoA synthase
AAA: ATPase associated with a
with a wide variety of cellular
activity
PAS: Sensory domain
Rubredoxin: Iron containing
protein, FeS4 domain at the active
site
Rubrerythrin: Iron containing
protein, FeS4 diiron domain at the
active site
Inferred HydA Structural Variation
Eric Boyd, John Peters
Montana State University
Updated from Meyer, 2007
Turner, Science 1999
Area Required for US Transportation Needs 10% Solar-to-Fuel Conversion Efficiency
10,000 square miles (100 x 100 miles)
1000 mmoles PAR m-2s-1
12 hours light/day
15768 moles of PS photons/yr
7884 moles of reductant
3942 moles H2/yr
1 kg H2 = 495 moles of H2 = 1 gallon of gasoline
~12100 L H2 gas
~ 8 kg H2/yr .m2
Current efficiencies are considerably less than
1% and short term. Among most productive
systems are heterocyst based nitrogenase
systems in cyanobacteria and nutrient deprived
eukaryotic algae Dismukes et al., 2008
Recent Advances Accelerating Progress
• Enzyme structure/function/maturation
• Genetic/metabolic tools
• Synthetic biology
• Metabolic engineering
• Generation of key mutants