biofuel production in microorganisms: from phototrophs to ......nrel is a national laboratory of the...

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


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


RejectedEnergy(62%)

U.S. Energy Flows98

.2 Q

uadr

illio

n Bt

u Fuels61%

Electricity39%

Buildings45%

Industry25%

Transportation30%


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


Energy Challenges are Enormous

Economic Growth

Environmental Impact

Energy Security and Reliability

Natural Disasters


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).

Presenter

Presentation Notes

Data from preliminary AEO 2004 tables (released in December 2003). The data is based on US energy consumption (not production which is significantly different). Overall breakdown is from Table A1 (Total Energy Supply and Disposition). Renewable breakdown is from Table A18 (Renewable Energy, Consumption by Section and Source). Corn starch (3% of total renewable energy) and MSW (6% of total renewable energy) are both considered “Biomass” for this breakdown. Mark Ruth


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


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


Comparing Potential Oil Yields

Crop Oil YieldGallons/acre

Corn 18Cotton 35Soybean 48Mustard seed 61Sunflower 102Rapeseed 127Jatropha 202Oil palm 635Algae “10,000”

Source: Wikipedia.org


…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


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


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

Presenter

Presentation Notes

The process by which hydrogen gas is produced in green algae and cyanobacteria relies on the photosynthetic pathway, the generated electrons and protons from which are utilized by hydrogenase enzymes in these organisms, the FeFe-hydrogenase of green algae, or the NiFe bidirectional hydrogenase found in cyanobacteria, which I will be discussing today in my talk. Two of the major barriers in the use of these organisms in photobiological hydrogen production are the sensitivity of these hydrogenase enzymes to oxygen, a byproduct of oxygenic photosynthesis, and the maintenance of the electron flow to the hydrogenase enzyme, since the electron donors for the enzymes, reduced ferredoxin in the case of the FeFe hydrogenase, and NADPH in the case of the NiFe bidirectional hydrogenase, are also used for other reactions in the cell, notably carbon dioxide fixation via the Calvin Benson Besham cycle. Therefore, a better understanding of how these hydrogenase enzymes function and strategies to maximize electron flow to the hydrogenases are of utmost importance, as well as understanding the role of these enzymes in cell metabolism and physiology.


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


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


Biodiesel

Green gasoline

Jet fuel

Genetic Engineering in cyanobacteria for the Production of Liquid Fuels

Troy Paddock, Damian Carrieri, Ambarish Nag,Jianping Yu


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).


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


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


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


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


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


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


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.


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.


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).


Physiological method to induce anaerobicity

Sulfur deprivation inhibits photosynthetic O2 evolution and induces anaerobicity and H2 production in C. reinhardtii

UC Berkeley


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


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


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.


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.


Design of enzyme‐based or microbial electrodes for fuel cells or reverse fuel cells application

Focus on solar driven hydrogen production


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


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


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


Biohybrids: Hydrogenase linked to carbon nanotubes

Drazenka Svedruzic‐Chang, Paul King, Arizona State U.


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


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


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

Presenter

Presentation Notes

For economic hydrogen production: if molar yield is 4, cost of feedstock has to be less than 3cents/lb for the feedstock cost below $1.20/kg H2


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


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


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.


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


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).

biofuel production in microorganisms: from phototrophs to ......nrel is a national laboratory of the...

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