Towards biological hydrogen production. Ed van Niel

Transcription

1 Towards biological hydrogen production Ed van Niel The biofuels of today & tomorrow 24 August 2011

2 Hydrogen is the fuel of the future.. and it always will be! Production Transport 20% 33% 4% Natural gas Storage Heavy oils & Naphtha 43% Coal Electrolysis

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4 Why hydrogen as energy carrier? o Contains 3x energy of gasoline by weight o No diffuse CO 2 production (only central) o Clean waste product: H 2 O o Fuel cell: high operational efficiency

5 Renewable H 2 can be produced in many ways!

6 For private use

7 Story Choices Organism Processes Impact Metabolism Kinetics

8 Biological hydrogen production: the choices H 2 O O r g a n i c c o m p o u n d s Biophotolysis Photofermentation Dark fermentation H Y D R O G E N Hallenbeck (2011) Biofuels 2: Hallenbeck & Gosh (2009) Trends Biotechnol 27:

9 Photosynthetic H 2 principle Cyanobacteria Algae H 2 Advantages o Abundant substrate (H 2 O) o Simple products (H 2 & O 2 ) o Non-terrestrial o CO 2 fixation Disadvantages o Low light conversion efficiency Algae 3% (plants 1%) o O 2 -sensitive H 2 ase or expensive N 2 ase o Expensive reactors (area/material!)

10 Photobioreactors Needs costly fertilizer Evaporation in open pond Material: light penetrable H 2 impenetrable Pumping/mixing (energy!) How will this be cost-efficient? Water evaporation

11 Fermentation processes: Biomass as energy source Organic wastes (agriculture, domestic, industry) Energy crops Forest Algae & waterplants (but: fertilization!)

12 Fermentations of wet biomass Fermentation product hexose based G in product kj/mol glucose 3 methane acetone+butanol+ethanol (ABE) 4 H methane ethanol H (average) for comparison: G glucose -2700

13 Biomass fermentation to H 2 C 6 H 12 O H 2 O 6 CO H 2 G 0 = 3 kj/mol glucose

14 Organic acids Two-stage processes Photofermentation Hyvolution H 2 H 2 Energy crops Organic waste Dark fermentation Electrohydrogenesis H 2 Methanogenesis CH 4

15 Photofermentation 2 CH 3 COOH + 4 H 2 O 4 CO H 2 2 H e ATP H ADP Hallenbeck & Gosh (2009) Trends Biotechnol 27:

16 100 L prototype modular Flat Plate Photobioreactor (RWTH) Biggest challenge: Photobioreactor - Penetration of light - Higher photochemical efficiency - Internal cooling (heat from the sun) - Mixing -Area 100 L prototype nearly horizontal tubular reactor (RWTH/TECHNOGROW) H 2 produced for 3 months with R.capsulatus in fed-batch mode

17 Microbial Electrolysis Cell Anode Ion exchange membrane

18 Challenges to realization of bioh 2 Microbial process Microbial electrolysis Biophotolysis Photofermentation Dark fermentation (mesophilic) Technical barrier Cathode material Increase current densities Lower voltage for high H 2 yields O 2 -sensitive hydrogenase Low light-conversion efficiencies Low cost photobioreactors Excess energy demand by nitrogenase Low light-conversion efficiencies Low cost photobioreactors Metabolic constrictions low yield Production of undesirable side products PC Hallenbeck (2011) Biofuels 2:

19 Dark fermentation step C 6 H 12 O H 2 O 2 CO CH 3 COOH + 4 H 2 Thermophilic versus mesophilic H 2 producers: Higher efficiency (~ 3-4 H 2 /glu vs ~ 2 H 2 /glu) Less undesired byproducts Less chance of contamination (e.g. methanogens) However, low volumetric productivities (> 10 x)

20 Where are we? Bottom up Schnitzhofer et al 2007 Zeidan et al 2007 De Vrije et al 2007 Van Niel et al 2002 Thong et al 2008 UASB Granules 60 C Thermoanaerobacterium sp. Productivity (mol H 2 m -3 h-1 ) 3-4 H 2 /glucose 1-2 H 2 /glucose Goal Top down Trickling bed - preliminary Defined co-culture - preliminary Continuous cultures

21 Story Choices Organism Processes Impact Status Metabolism A case study: Hyvolution Two stage fermentation process Kinetics

22 Comparison of energy efficiencies With other H 2 & CH 4 production systems Miltner et al 2009 Chem Eng Trans 18: Hyvolution process

23 Allocation of the environmental impact to in/outputs Substrate: Potato Steam Peels PSP1: basic case Ochs et al 2010 J Clean Prod 18:S88-S94 Option PSP2: recirculation of sewage

24 Environmental impact of dark/photofermentation combination Substrate: Potato Steam Peels Ochs et al 2010 J Clean Prod 18:S88-S94

25 Story Choices Organism Processes Impact Metabolism Kinetics

26 Dark fermentation process Our selection: Caldicellulosiruptor species G-positive, obligate anaerobes Class of Clostridia Extreme thermophiles (T opt 70 C) Polysaccharolytic Acetate is the main end product C. saccharolyticus:. physiological studies. first genome sequence (van de Werken et al AEM 2008;74:6720-9) Grand Prismatic, New Zealand C. saccharolyticus cells (Courtesy of M. Verhaart) Organism first described: Rainey et al 1994 FEMS Microbiol Lett 120:263-6

27 Interactions between microorganism and environment Understanding fermentation process: Enviromics approach Lidén (2002) Bioproc Biosyst Eng 24:273

28 Physiological study approach P H 2 π Sugar type NADH/NAD ATP/ADP Y SH 2 [Sugar] Nutrients (PPi) q H 2

29 Van de Werken et al (2008) AEM 74:6920-9

30 Anabolism PP i : connection catabolism - anabolism ATP production flux ATP consumption flux Catabolism PP i FBP / ATP-PFK Anabolism ATP AMP+PP i PP i -PK/ ATP-PK AMP+PP i ATP Catabolism PPi Macromolecule ATP (mmol/g) Protein (60%) 36.4 RNA 15.3 DNA 18.8 Proton pump ATP Transport H + -PP i :ase ATP demanding Bielen et al 2010 FEMS Microbiol Lett 307:48-54

31 Central Carbon metabolism: electron flow 2 H 2 4 e - glucose Lactate production: - High ph 2 (high NADH) but also: - Transition phase 4 e - 2 lactate 2 pyruvate LDH 4 e - 2 ethanol 4 e - 2 acetate - LDH is constitutively expressed - How does LDH remain inactive? 2 H 2

32 Lactate formation at the end of growth Lactate Lactate Willquist & van Niel 2010 Met Eng 277:1843

33 PPi-depending regulation of LDH Activity (U/mL) Concentration (mm) Affinity for NADH ATP ATP NADH ATP ATP NADH ATP 1 PPi NADH (mm) A) 13-fold change in PPi/ATP ratio 7 6 PPi 5 PPi PPi PPi PPi PPi PPi PPi ATP exponential phase ATP PPi stationary phase Willquist & van Niel 2010 Met Eng 277:1843

34 NADH/NAD Simulation PPi-depending regulation of LDH Ln(OD) [PPi] High Low [PPi] Low High Time (h) -4 Lactate formation B) NADH/NAD Low High Low High Willquist & van Niel 2010 Met Eng 277:1843

35 Mindmapping overall outcome T, ph π, ph 2 [sugar] Growth boundaries Genetic protocol Scale up Hydrolysates 600 L 30L 1 L Transcriptomics Proteomics Metabolomics Omics C. saccharolyticus Genomewide model Biomass composition Medium composition Improve annotation C. kristjansonii C.owensensis Stimulates Kinetics & regulation Enzyme kinetics metabolite concentrations Hyvolution: Wageningen Un, Un of Szeged, Lund Un, Profactor, A&F

36 Remember: the technology is already there!

37 Acknowledgements Dr. Pieternel Claassen project coordinator A&F, Netherlands Dr Karin Willquist former PhD-student Lund Un Dr Ahmad Zeidan former PhD-student Lund Un Sudhanshu Pawar PhD student Lund Un Dr. Wolfgang Schnitzhofer Profactor, Austria Prof Inci Eroglu Ankara University, Turkey Funded by EU (FP ENERGY ) & Energimyndigheten

38 Conclusions overall H 2 comes and goes, but... It ll be back!

39 Concentration (mm) Concentration (mm) C. saccharolyticus C. kristjanssonii 150 ml inoculum of C. kristjansonii: Effect of supernatant of C. saccharolyticus culture OD GLC Time (h) 3 OD OD 620 OD 620 Test: + 50 ml supernatant 100 ml inoculum: C. kristjanssonii alone Control o Lag phase = 53 h + 50 ml supernatant o Lag phase = 5 h Time (h) 0

40 Low D C. saccharolyticus C. kristjanssonii D = 0.06 h -1 co-culture microbial interaction (altruism) max 2 0 max 2 ' ( max 2 0 max 2 ) E E C High D D = 0.15 h -1 Y Y ) 0 ' sx Ysx ( Y 2 2 sx 2 0 sx 2 E E C Zeidan et al 2010 Microb Cell Fact 9:102

41 Overconcentration Overconcentrations of H 2 in liquid phase Pauss et al (1990) AEM 56: Equation for overconcentration: 12 [gas] l Q V = + 1 [gas] * l K H R T k l a 9 6 Q V = vol. gas production rate 3 K H = Henry s law constant R = ideal gas constant T = temperature (K) k l a = mass transfer coefficient k l a (h -1 ) ( ) = H 2 and ( ) = CO 2 at 35 C ( ) = H 2 at 70 C