Renewable Hydrogen Production from Biological Systems
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Renewable Hydrogen Production from Biological Systems Matthew Posewitz Colorado School of Mines DOE Biological Hydrogen Production Workshop September 24th, 2013 Photosynthetic H2 pathways Peters JW et al. Science 1998 Nicolet Y et al. Structure and Folding Des. 1999 H2 production PSII/PSI pathway PSI/nonphotochemical PQ Dark fermentation H2 uptake oxyhydrogen reaction photoreduction 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 CO CN CN Fe Cys2 S S Cys4 Ni Cys3 S S Cys1 • [NiFe]-Hydrogenases • [FeFe]-Hydrogenases – Reversible O2 inhibition – Algal enzymes are simplest – Group 1- Uptake Enzymes to date Membrane bound – Often ferredoxin linked – Group 2- Cyanobacterial uptake – Hnd multimeric enzymes and H Sensing Enzymes 2 are NAD(P)(H) linked • Latter are O tolerant 2 – Differences in O tolerance – Group 3- Bidirectional 2 reported but typically • NAD(P)(H) linked irreversible inhibition – Group 4- H Production Enzymes 2 – Capable of very high • Ferredoxin linked 3 turnover >10 /s Nitrogenases + - Nitrogenase MoFe Protein 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 Nitrogenase Fe Protein Mutants with increased H2 production reported P Clusters FeMo-cofactor Prolonged H2 Production in Algae Induced by Nutrient Stress 140 120 1 - hr 100 1 - 80 P mg Chl 60 R 2 40 400 20 moles O moles 300 m 0 0 20 40 60 80 100 200 Incubation time -S, h 100 Melis et al., Plant Phys. 2000 ml collected, gas of Volume Kosourov et al., 2002 0 40 60 80 100 120 140 160 180 Time, h Photosynthetic antennae mutant with enhanced H2 production Kosourov, Ghirardi and Seibert 2011 tla1 mutant Polle and Melis (UCB) Nitrogenase Catalyzed H2 Production in Cyanothece: Sustained Biophotolysis • Rates of H2 production up to ~ 400 mmole/hr . mg chl • O2/H2 coproduction • Predominately light driven Melnicki et al., 2012, Beliaev laboratory PNNL Reddy et al., 1993 Photosynthesis and Starch- Interplay for H2 Production Initial Hydrogen Production Rates -1 160 330 hr 140 sta6 -1 120 sta7 100 sta7(J4)::STA7 80 mg Chl 2 60 40 330 sta6(J5) 20 moles H 0 0.5 1.5 4.0 7.0 o/n 0 0.5 1.5 4.0 7.0 o/n m 0 0 0.5 1.5 4 7 o/n Time (h) 250 WT 200 sta7-10 g/ml 150 m 100 Glucose, Glucose, 50 0 0 20 40 60 80 100 120 140 Libessart et al., The Plant Cell 1995 Time -S, h A 140 120 Starch Hyperaccumulation 100 80 cells 6 60 40 20 0 µg starch / 10 / starch µg c5 c5 10 10 - - c19 c19 sta6 sta6 CC124 CC124 Starch sheath surronding pyrenoid sta7 sta7 TAP + N TAP - N 0 hrs 96 hrs TAP+N TAP-N B 500 450 400 350 300 D66 250 200 (wildtype) 150 100 50 0 c5 c5 10 10 µg starch / ml culture ml / µgstarch - - c19 c19 sta6 sta6 CC124 CC124 sta7 sta7 TAP + N TAP - N 2.2.23 D66 0 hrs 96 hrs Hydrogenase Maturation Heterologous Expression and Screening in Alternate hosts Insertion site in TAA HydEF-1 mutant TAA HydG HydEF 1 kb H2 Production Rates 1 - 50 h -1 40 30 mg protein mg 20 2 10 mole H mole m 0 A1 A1 + EF A1 + G A1 + EF + G Hyd transformants Hydrogenase Maturation Heterologous Expression and Screening in Alternate hosts Screening mutant libraries for enhanced O2 tolerance Stapleton and Swartz 2010 Stanford Expression of [FeFe]-hydrogenase in Anabaena heterocysts. Gartner et al. 2012, Hegg lab MSU Reverse Genetics via PCR-Based Mutant Library Screening PSAD PRO APHVIII Int CYC6 Single colonies in 96-well microtiter plate Pool 96 colonies and isolate genomic DNA Combine DNA from 10 pools into “superpool” Screen for specific gene disruptions by PCR (gene-specific and APHVIII-specific (RB2) primers RB2 PSAD PRO APHVIII Int CYC6 F1 F2 F3 F4 Target Gene R1 R2 R3 R4 Arthur Grossman, Claudia Catalanotti, Wenqiang Yang, Venkat Subramanian, Alexandra Dubini, Mike Seibert Chlamydomonas reinhardtii [FeFe]-hydrogenases and KO lines • 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. HindIII SalI XhoI MfeI NotI Avr IIAfl II ClaI Kpn I Bgl II H-cluster 5’ UTR 3’ UTR Meuser et al., 2012 Genomic HYDA1 Exon Genomic HYDA1 Intron Chloroplast Transit peptide Multiple Cloning Site C. r. HYDA1 cDNA HYDA paralogs are reciprocally monophyletic hydrogenase oxygen sensitivity Meuser et al., 2011 Planta Algae Algae 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 Inferred HydA Structural Variation Conserved Accessory Domains: [a] [b] 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 Eric Boyd, John Peters Montana State University Updated from Meyer, 2007 Area Required for US Transportation Needs 10% Solar- to-Fuel Conversion Efficiency 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 . 2 ~ 8 kg H2/yr m Current efficiencies are considerably less than 1% and short term. Among most productive systems are heterocyst based nitrogenase systems in cyanobacteria and nutrient deprived Dismukes et al., 2008 eukaryotic algae Recent Advances Accelerating Progress • Enzyme structure/function/maturation • Genetic/metabolic tools • Synthetic biology • Metabolic engineering • Generation of key mutants Turner, Science 1999 10,000 square miles (100 x 100 miles) .