NREL is a national laboratory of the U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, operated by the Alliance for Sustainable Energy, LLC.
Biofuel production in microorganisms: From phototrophs to obligate anaerobes
Carrie Eckert, Ph.D.
NREL Photobiology Group
Biosciences Center
6/23/10
Innovation for Our Energy Future
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U.S. Energy Consumption by source ‐ 1850‐2000
0
20
40
60
80
100
120
1850 1870 1890 1910 1930 1950 1970 1990
Qua
drill
ion
BTU
s
Source: 1850-1949, Energy Perspectives: A Presentation of Major Energy and Energy-Related Data, U.S. Department of the Interior, 1975; 1950-2000, Annual Energy Review 2000, Table 1.3.
Coal
Crude Oil
Natural Gas
Nuclear
Hydro
Non-hydro Renewables
Wood
Innovation for Our Energy Future
RejectedEnergy(62%)
U.S. Energy Flows98
.2 Q
uadr
illio
n Bt
u Fuels61%
Electricity39%
Buildings45%
Industry25%
Transportation30%
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Dependence on Foreign OilU.S. Use of Petroleum
1970 1980 1990 2000 2010 2020
Today
Domestic Petroleum Usage
Imports
Actual: Annual Energy Review 2000 Tbls 1.2, 5.1 and 5.12Forecast: Annual Energy Outlook 2002 Tbls 7 and 11Split between Autos and Lt Truck: Transportation Energy Data Book Edition 21 Tbl 2.6Updated October 2002
0
5
10
15
20
25
30
Non-transportation
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Energy Challenges are Enormous
Economic Growth
Environmental Impact
Energy Security and Reliability
Natural Disasters
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The Role of Renewables in U.S. Energy Consumption- 2003
Coal 23%
Petroleum 39%
Renewable 6%
Natural Gas 24%
Nuclear 8%
Wind 2%
Biomass 46%
Hydroelectric 46%
Geothermal 5%
Solar <1%
Source: AEO 2004 tables (released in December 2003) based on US energy consumption. Overall breakdown Table A1 (Total Energy Supply and Disposition), and Renewable breakdown Table A18 (Renewable Energy, Consumption by Section and Source).
Innovation for Our Energy Future
Technology-based Solutions:There is no single nor simple answer
Energy efficiencyRenewable energyNon-polluting transportation fuels Separation and capture of CO2 from fossil fuelsNext generation of nuclear fission and fusion technologyTransition to smart, resilient, distributed energy systems coupled with pollution-free energy carriers, e.g. hydrogen and electricity
Innovation for Our Energy Future
Current Biology-Based Strategies for Production of Carbon-Neutral Fuels
Ethanol from corn (non R&D)Ethanol from cellulosic material
R&D: cellulolytic enzymes, molecular engineering of crops for decreased lignin content
Biodiesel from plantsBiodiesel from algaeBiodiesel/fuels from other microorganisms
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Comparing Potential Oil Yields
Crop Oil YieldGallons/acre
Corn 18Cotton 35Soybean 48Mustard seed 61Sunflower 102Rapeseed 127Jatropha 202Oil palm 635Algae “10,000”
Source: Wikipedia.org
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…Using Waste CO2 from Coal-fired Power Plants
Carbon dioxide rich streams from combustion of fossil fuels or other industrial processes ideal for algae production
Double benefit: provide food for algae or other microorganisms that can utilize CO2, and remediate waste stream (recycling of fossil CO2)
Carbon credits may become economic driver
Innovation for Our Energy Future
H2 Diesel
Biodiesel from Hydrogen and Carbon Dioxide
Biological catalyst
RenewableSource
(biological/wind/solar)
CO2+
Jianping Yu, PinChing Maness,Carrie Eckert,OPX, JM
NREL is a national laboratory of the U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, operated by the Alliance for Sustainable Energy, LLC.
Ralstonia eutropha H16
‐soil microbe that can utilize H2 as an energy source and fix
CO2 to produce sugars for growth
‐Wide distribution in ecosystem
‐Versatile metabolic modes
‐Robust growth
‐High productivity: commercial bioplastic producer
Project involves engineering to produce
Fatty acid methyl esters (FAMEs) as a
source of biodiesel
With OPX Biotechnologies (Boulder) and Johnson-Matthey (U.S. & U.K.)
Innovation for Our Energy FutureNational Renewable Energy Laboratory
Photoproduction of fuels from water
Charge separatingdevice
Fuel producing catalyst
Sun
electron (‐)
FUELS(H2, lipids, alcohols, carbohydrates)
2H2O4H+ + O2
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Photosynthesis and Biofuel Production
14
2H2O O2 + 4H+
NiFe Hydrogenase
NADPH
NADP+
CO2 fixation
Carbohydrate
FeFe Hydrogenase
H2
2H+
e‐
H+
H+
ATPase
ATP
ADP + Pi
PSII PQ pool PSIe‐
Chloroph
yll
Anten
na
Chloroph
yll
Anten
na
e‐ FD e‐ FNR
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Microalgal Cultivation
Open raceway pond systems stirred with paddle wheelsSuch systems can be 50x more productive per acre than traditional cropsRequire CO2 injection for maximal productivity
More intensive cultivation systems becoming available
Potential for ocean culture
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High-lipid accumulating species identifiedGenetic “toolbox” developedWork in diatoms initiated to push carbon flow toward TAGs at expense of carbohydrates (chrysolaminarin)Identification of key enzymes in each pathway (Cyclotella cryptica)
– Acetyl CoA Carboxylase (ACCase) enzyme for lipid pathway
– UDP-glucose pyrophosphorylase(UGPase) enzyme for carbohydrate pathway
NREL Aquatic Species Program: Genetic Engineering
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Biodiesel
Green gasoline
Jet fuel
Genetic Engineering in cyanobacteria for the Production of Liquid Fuels
Troy Paddock, Damian Carrieri, Ambarish Nag,Jianping Yu
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Potential of Photobiological Hydrogen Production
• Maximum conversion efficiency of about 10-13% of incident sunlight;
• Land area of about 100 x 100 square kilometers (or 4,500 square miles) is required to provide enough energy to fully supply the U.S. transportation needs (263 million vehicles, 60 mi/gge); this equals about 0.12% of the U.S. surface area.
• Estimated cost of photobiologically-produced H2could be as low as $3/kg (or gge).
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Major Challenges for Photobiological H2 Production
How to achieve H2 production in the presence of co-evolved O2
How to achieve high electron transport rates if the proton gradient is not consumed by ATP production
How to maintain high quantum yield of incident light at sunlight intensity
What photobioreactor material to use to ensure that it is transparent, impermeable to hydrogen, durable and low cost
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Biological H2 Production1. Photobiological (photosynthetic microorganisms) 2H2O → H2 + O2
(requires light and a catalyst, a NiFe or FeFe-hydrogenase)
2. Fermentative (anaerobic bacteria)1C6H12O6 + 6H20 → 2 C3H6O3+ 2 or 4 H2 (requires a NiFe or FeFe-hydrogenase catalyst)
3. Integrated
4. Biomimetic
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H2 Production: Photobiological
Analyze the structure and function of a bidirectional [NiFe]-hydrogenase in cyanobacteria
Look at metabolic changes in algae under conditions where the hydrogenase is active vs. inactive
Engineer an algal [FeFe]-hydrogenase that is resistant to O2 inactivation, since O2 is an obligatory by-product of photosynthesis;
Introduce the gene encoding a [NiFe]-hydrogenase with increased O2resistance into a water-splitting, photosynthetic cyanobacterial system;
Use a sulfur-switch to induce culture anaerobiosis and subsequent H2-production activity in algae.
Raimund Fromme, ASU
Klaus Schulten, Univ. Illinois
Craig Venter Institute
Russian Academy of SciencesA. Melis, UC Berkeley
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Synechocystis sp. PCC 6803 Bidirectional Hydrogenase
Carrie Eckert, Jianping Yu,Pin‐Ching ManessArizona State University
Genetic dissection of the endogenous hydrogenase to further understand its structure and function
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2D DIGE analysis of C. reinhardtii grown under oxic vs anoxic conditions
Cy2 Cy3 Cy5
Oxic + Anoxic Oxic Anoxic
SYPRO Ruby stained gel
Venkat Subramanian, Alexandra Dubini,Mike Seibert
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Molecular Engineering Oxygen Tolerance into the Algal Hydrogenase
We focused our engineering efforts in the area the high energy barrier. We substituted local amino acid residues for larger ones, sterically hindering the access of O2 to the catalytic site.
Christine English, Paul King, Klaus Schulten, Univ. Illinois
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Introducing NiFe hydrogenases in cyanobacteria
The NiFe-hydrogenase from the bacterium Rubrivivax gelatinosumhas high tolerance to O2inactivation;We have cloned genes involved in its expression and introduced them in the cyanobacterium;Synechocystis 6803On-going work focuses on finding the minimum number of genes require for expression of an active hydrogenase in Synechocystis.
Karen Wawrousek, Jianping Yu,Pin‐Ching Maness, J. Craig Venter Inst.
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O2-Tolerant Hydrogenase System(conceptual)
(from fertilizers)
Chemostat II: 2 days of initial growth, followed by indefinite H2 photoproduction (by limiting CO2 bubbling); nutrients supplemented at 1/4th of amount required for growth during the H2 production phase.
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General Assumptions: O2-tolerant hydrogenase algae
Reactor types: 20 covered racetracks of 40 ft vs 1090 ft; 10 cm depth, covered by 6cm polyethylene film (0.016% H2 lost from the system, as per Dan Blake’s analysis; 90% of incident light transmitted through), paddlewheels for stirring and mixing; nutrients from commercial grade fertilizer and bubbled CO2 supplied through an inlet valve;
National Renewable Energy Laboratory
biomass removed from slip stream by rotary drum filters (used in aquaculture industry); no heat exchangers; gas produced will be a flammable mixture of H2 and O2; gases will be compressed and separated by pressure swing adsorption (PSA).
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Physiological method to induce anaerobicity
Sulfur deprivation inhibits photosynthetic O2 evolution and induces anaerobicity and H2 production in C. reinhardtii
UC Berkeley
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Cell suspensions Immobilized onto glass fibers
Immobilized in alginate films
Low cell‐density, short duration
Higher cell density, longer duration, non‐degradable matrix
High cell density,long duration, controllable thickness,degradable matrix
Immobilization of Sulfur-deprived Chlamydomonas Cells
Days0 10 20 30 40 50 60 70 80 90
H2,
ml/d
02468
1012141618
H2,
ml
0
100
200
300
400
500Rate Volume
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Cell Immobilization using Polymeric Matrices
Immobilization in alginateStick tapetape
Screen
Alginate with algal cells 50 mM CaCl2
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 50 100 150 200
Time -S -P, hH
ydro
gen,
mol
/ m
2
0.125 g0.25 g0.5 g1 g2 g
Hydrogen production after argon treatment
H2 production occurs at lower rates in the presence of atmospheric O2!
NREL and Anatoly Tsygankov and Sergey Kosourov, Russian Academy of Sciences,
Pushchino, Russia
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Sulfur-deprived, immobilized algae (actual)
Dual‐bed reactor system: immobilized algae are grown in one reactor for 2 days and then transferred to a second reactor for 3 days of H2‐production; alternating cycles of growth and H2 production in the second reactor last for a total of 180 days.
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General Assumptions: Sulfur-deprived, immobilized algae
Reactor types: 90 covered racetracks of 40 ft vs 1060 ft; 10 cm depth for H2production and 2 racetracks for growth. One pre-growth subassembly. Racetracks covered by 6cm polyethylene film (0.16% H2 lost from the system, as per Dan Blake’s analysis; 90% of incident light transmitted through), paddlewheels for stirring and mixing; nutrients from commercial grade fertilizer and bubbled CO2 supplied through
National Renewable Energy Laboratory
an inlet valve; no heat exchangers; gas produced will be H2 and CO2, which will be compressed and separated by PSA.
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Design of enzyme‐based or microbial electrodes for fuel cells or reverse fuel cells application
Focus on solar driven hydrogen production
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Photobiomimetic Research at NREL
Biological Photosynthesis
hν
h+
e‐
2H+2e‐
H2
CO2
2e‐
bicarbonate
Nanoparticles/hydrogenases Dye‐sensitized TiO2/hydrogenases Photosytem II/hydrogenases
Inorganic charge separation/biological catalyst Biological charge separation/catalyst
Kate Brown, Paul King,Drazenka Svedruzic‐Chang,Arizona State University
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Photobiological and Photobiohybrid H2-Production Processes
Biological
Biomimetic
H2Fuelcell
Electricity
H2O
O2
Sunlight
Algae
Expectedmaximum light conversion efficiency to H2 of about 10% with biological systems
Paul King,Arizona State University
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H2
Xox
Xred
hν‐
+
2H+ + 2e‐
0
50
100
150
200
250
300
nmol
e H
2
nc-CdTe:H2ase ratio
Illuminated
dark
Biohybrids: Hydrogenase linked to nanoparticles
Kate Brown, Paul King
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Biohybrids: Hydrogenase linked to carbon nanotubes
Drazenka Svedruzic‐Chang, Paul King, Arizona State U.
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H2 Production: Fermentative
H2 molar yield: 2.8260 ml H2/hr/reactor
Steam explosion
Hemicellulose Lignocellulose
Clostridium thermocellum
Sewagesludge
H2 molar yield: 2.245 ml H2/hr/reactorSharon Smolinski,
Lauren Magnusson,Shiv Thammanagowda, JiHye Jo,Pin‐Ching Maness
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Lignocellulosic Biomass
US cultivates > 80 million acres of corn yielding a billion tons of corn stover annually
Billion Ton Vision Study*: to displace 30% of the petroleum consumption
* A US Dept. of Agriculture and US DOE joint study, 2005
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Fermentation in Clostridium thermocellum
GLUCOSE
2 Pyruvate2 Lactate
2 Acetyl-CoA
2 H2
2 H2
Ethanol2 Acetate
Solvent ProductionH2 and
Acid Production
Glycolysis
Adapted from Demain et al., Microbiol. Mol. Rev. 2005, p. 124-154
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Integrated Biological System for H2production (with U.C. Berkeley)
Use stacked reactors containing green algae and photosynthetic bacteria (that utilize different regions of the solar spectrum) to photoproduce H2; use a consortium of H2‐producing fermentative bacteria that is capable of metabolizing spent algal and bacterial biomass; add the organic acid generated from the fermentor as a substrate to photosynthetic bacteria and as an estimulator of photosynthetic green algal H2 production.
Green algaePhotosynthetic bacteria
H2
Fermentativebacteria
CO2 acetateand other organic acids
algal biomass
H2
H2O
CO2
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H2 Production: Integrated
• Combined Yield: 8.52 mol H2/mol hexose
– The NREL‐PSU integrated system exceeds DOE 2013 target of “4 mol H2/mol hexose.”
– $6.61/Kg H2 based on DTI Economic analysis
– Improvement in MEC necessary to lower the cost
• Fermentation: 1.64 mol H2/mol hexose
– Fermentation is fast and easily scalable, using recalcitrant cellulosic substrates.
• MEC: 6.88 mol/mol (based on actual cellobiose effluent)
– First demonstration of H2 from fermentation effluent via MEC.
Penn State University
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H2 Production: Integrated
• We demonstrated that a fermentative consortium is able to digest different components of algal biomass: starch, lipids and proteins;
• On‐going research aims at demonstrating metabolization of alginate as well.
• The fermentation of potato waste to H2 was accomplished by a bacterial consortium, yielding a mixture of C2, C3 and C4 organic acids;• Dilute organic acids were successfully metabolized by photosynthetic bacteria;• The combined yield of the two processes of about 5 H2/hexose.
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Summary of Technoeconomic Analysis
System Estimated Light Conversion Efficiency (% incident)
Hydrogen Cost ($/kg)
Photobiological• O2‐tolerant H2ase• Sulfur‐deprived
10%3%
$2.99$6.02
Fermentative (lignocelluloses feedstock)• with by‐product (acetate) sales• w/o by‐product sales
H2/hexose: 3.6$2.09$4.33
Integrated• Photo + Fermentative• Fermentative + MFC
N/AH2/hexose: 10.8
$3.21$6.61
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Members of the Team and Collaborators
Mike Seibert, Paul King, Pin-Ching Maness, Alexandra Dubini, Jianping Yu, Sharon Smolinski, Christine English, Drazenka Svedruzic-Chang, Kate Brown, Damian Carrieri, Carrie Eckert, Shiv Thammanagowda, Venkat Subramanian, Lauren Magnusson, Murthy Narayantha, Seth Noone, Troy Paddock, Damian Carrieri, Ji-Hye Jo, Erin Peden, Grant Pennington, Kath Ratcliff (NREL)
Kwiseon Kim, Chris Chang, Hai Long, Ambarish Nag (NREL)
Matthew Posewitz (Colorado School of Mines), Matthew Wecker (GeneBiologics), Mace Golden (Golden Bioenergy), Sergey Kosourovand Anatoly Tsygankov (Russian Academy of Sciences), Klaus Schulten and Jordi Cohen (Beckman Institute), Raimund Fromme, Tom Moore, Ana Moore, and Devens Gust (Arizona State University), Anastasios Melis (University of California-Berkeley), Bruce Logan (Penn State University).