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