5. kozumbo - bioenergy

BIOENERGY 15 March 2011

Walt KozumboProgram Manager

AFOSR/RSLAir Force Office of Scientific Research

AFOSR

Distribution A: Approved for public release; distribution is unlimited. 88ABW-2011-0770

2

2011 AFOSR Spring Review2308C Portfolio Overview

NAME: Walter J. Kozumbo, Ph.D.

BRIEF DESCRIPTION OF PORTFOLIO:

• Bioenergy is a program that characterizes, models and explains the structural features, metabolic functions and gene regulatory mechanisms utilized by various biological systems to capture, transfer, convert, or store energy for the purpose of producing renewable biofuels and improving the power output of biofuel cells. (~90% of portfolio)

Sub-Areas: (1) BioSolar Hydrogen, (2) Algal Oil for Jet Fuel, (3) Artificial Photosynthesis, and (4) Biofuel Cells (Microbial and Enzymatic)

• Photo-Electro-Magnetic Stimulation of Biological Responses is a beginning program that characterizes, models and explains the stimulatory and inhibitory responses of biological systems to low-level exposures of photo-electro-magnetic stimuli. Potential long-term benefits may include accelerated recovery from mental fatigue and drowsiness, enhanced learning and training, and noninvasive treatment of traumatic brain injuries. (~10% of portfolio)

3

Challenges, Opportunities and Breakthrough Examples

Natural Systems Research:Challenge: Explain gene regulatory mechanisms of metabolic pathways and networks

Payoffs: - economically viable jet biofuels - enhanced energy density of microbial fuel cells (MFC)- enhanced cognition/repair/bio-resiliency via photo-electro-magnetic stimulation

Challenge: Understand mechanisms and kinetics of enzyme-catalyzed reactionsPayoffs: - enhanced energy density of enzymatic fuel cells (EFC)

- sustained oxygen-tolerant hydrogen production by photosynthetic microbes

Artificial Systems Research:Challenge: Discover/fabricate cheap, durable synthetic materials that mimic the

enzymatic or structural functions in natural energy systemsPayoffs: - cheap water-splitting catalysts as platinum replacements in H2-generating devices

- enhanced power and energy densities for EFC

Challenge: Integrate and assemble nano-scale inorganic/organic/bio-materialsPayoffs: - ordered enzyme alignments for enhanced power densities in EFC

- enhanced electron transport and power density in biofuel cells - light is harvested and split in artificial photosynthetic solar fuel generator

4

Visionary Transformational AF Capabilities

Bioenergy: • Jet Biofuel Produced from CO2, H2O and Sunlight:

- Algal systems biology data used to bioengineer lipid biosynthetic pathways in microbes or to create novel synthetic pathways in artificial solar fuel systems

• Portable H2 Fuel Generated from H2O or Cellulose: - Cheap, self-healing inorganic catalysts split water into H2 and O2- Engineered photosynthetic microbes produce H2 fuel - de novo construct of metabolic pathways extract H2 from cellulose.

• Compact Power from Ambient Biomass: - de novo construct of metabolic pathways completely oxidizes biomass - Efficient electron transport coupled with unique electrode architectures enhance power and energy densities of biofuel cells

Photo-electro-magnetic Stimulation of Bio-Responses:• Electromagnetically Enhanced Cognition, Protection and Healing:

- low-level treatment with photo-electro-magnetic stimuli enhance cognitivefunctions, bio-molecular repair and bio-resiliency

5

Related Research Funded by Other Agencies

Funding Criteria:

1. Basic research of high quality and relevant to the AF2. Unique or complementary, but non-duplicative—finds a “niche”3. Leverages research in other agencies 4. Critical mass or team of collaborators with focused, multi-disciplinary research objectives

Algal Oil: DOE and DARPA research application oriented; NSF funds mostly individual grants of smaller size that are not based on a coordinated, multi-disciplinary team approach; USDA interested in farming aquaculture; EPA interested in regulation. AFOSR niche is lipid biosynthesis via systems biology. AFOSR has collaborated with DOE-NREL since 2006 and coordinates research as member of emerging Algal Interagency Working Group.

Biosolar Hydrogen: DOE and NSF fund mostly individual grants of smaller size that are not based on a coordinated, multi-disciplinary team approach. AFOSR niche is systems biology and bioengineering for enhanced H2 production. AFOSR has collaborated with DOE-NREL since 2003.

Biofuel Cells: ONR funds only microbial fuel cell (MFC) research for dissolved nutrients in the marine sediment environment. AFOSR funds enzymatic and MFC research for solid substrates in terrestrial environments and coordinates research via ONR reviews and direct personal contact.

Artificial Photosynthesis: This topic is biologically oriented and part of a 2009 AFOSR Initiative “Catalysts for Solar Fuels” with PMs Berman and Curcic, whose topics are chemically and physically oriented. To our knowledge there are no initiative counterparts at other agencies.

BioResponse to Photo-electromagnetic Stimulation: Complementary to other funded research.

PhysicsChemistry

Engineering Math

BiologyMaterials

6

Bioenergy: Alternative Energy for Future AF Needs• Biofuels—Macro-scale Energy

• Biosolar Hydrogen• Algal Oil for Jet Fuel• Artificial Photosynthesis

• Biofuel Cells—Micro-scale Energy

• Microbial Fuel Cells• Enzymatic Fuel Cells

Sun Photosynthesis Fuel

Aircraft

Fuel Cells

Biofuel Cells

H2 Vehicles

MAV Rob

oflyNatural to Artificial

Overview of Topic Areas 2308C

• Stable Fuel Supply & Price

• Energy Independence

• Reduced Military Casualties

• Carbon Neutral

• Anti-Climate Change

• Reduced Health Effects

• Compact Power Supplies

Photo-Electro-Magnetic Stimulation of Bio-responses

7

Topic Performer Customer Result Application Artificial Photo-

synthesis

Nocera, MIT

SunCatalytix, Cambridge, MA

An inorganic cobolt-phopshate catalyst that splits water and self-heals.

To generate hydrogen fuel from water, easily, safely and cheaply.

Koder, CCNY

Phoebus Optoelectronics, LLC, New York, NY

Nano-structured plasmonic meta-materials for harvesting and splitting

light.To convert solar energy into biofuels.

Biofuel Cells Zhang, VA Tech.

Gate Fuels Inc., Blacksburg, VA

A high energy-dense biodegradable sugar battery (3 x's > lithium ion

batteries) using cell-free synthetic enzyme pathways

To develop a secondary battery charger for compact energy sources.

Atanassov, U of NM

Lynntech Corp, College Station, TX

An enzyme immobilization technology based on chemical vapor deposition of

silica-encapsulated enzymes.

To improve long-term operational stability of enzymes under biofuel cell operational

conditions.

Minteer, St. Louis University

CFD Research Corporation, Huntsville, AL

An in vitro enzyme cascade system that completely oxidizes sucrose

to CO2 and water.

To enhance anodal energy output via the complete oxidation of sucrose as fuel.

Algal Oil Merchant and Pelligrini, UCLA

Multiple worldwide institutions

Web sites displaying algal genomes on a browser for data visualization and

experimental comparisons http://genomes-merchant.mcdb.ucla.edu

To deduce structures of algal transcripts and patterns of expression for any gene of interest.

Merchant and Pelligrini, UCLA

Worldwide Chlamydomonas

research community

A web-based tool that utilizes the annotated Chlamydomonas genome for

understanding metabolism http://pathways.mcdb.ucla.edu/chlamy

To map differentially expressed genesto metabolic pathways and convert

protein IDs from one genomic version to another.

Hildebrand, UCSD

General Atomics Inc., San Diego, CA

A flow cytometry procedure for single-cell and subpopulation analyses of lipid

accumulation in microalgae.

To evaluate lipid accumulation over time, advancing the process of lipid analysis on

cultures.

Biosolar Hydrogen

Posewitz, CO School of Mines

ConocoPhillips, Bartlesville, OK

Genetic techniques to over-accumulate starch and glucose in Chlamydomonas

To obtain glucose as a feedstock for a variety of biofuels.

FY10 Technology Transfer 2308C

8

Bioenergy: A Progressive Research Strategy

Sun Photosynthesis Fuel Biofuel Cells POWERNatural to Artificial

DisciplinaryInputs

BasicResearch

Type

CharacterizationMechanisms

Models

Artificial Systems

Natural Biosystems

Hybrid Systems

SystemType

Optimized Natural Biosystems

Metabolic/Protein

Engineering

SyntheticBiology

Chemistry &Materials Science

BiologyChemistry

Math Physics Engineering

4th3rd2nd1stGeneration

9

Photosynthesis, Systems Biology and Metabolic Engineering for the Production of Biofuels

2 H2Owater-splitting

enzyme

4 e_

4 H+

H2-generating hydrogenase

enzyme

chlorophylllight

Sugar/CelluloseSynthesis

Light Reactions PSI and PSII Dark Reactions

Triglyceride (Oil)Lipid Synthesis

Microalgae & Cyanobacteria Make Hydrogen, Lipids & Sugars

Jet Fuel

H2

Ethanol CO2

carbon-fixingenzymeThree Key Biocatalysts

screening

field genomic sequence around hox genes in S. platensis7098 bp

HoxE HoxF HoxU HoxY HoxHORF?

diaphorase moiety Ni-Fe hydrogenase moiety

BamHI (1484) Eco RI (4977)ClaI (2981) ClaI (7047)Nco I (1099)

Nco I (3375) Nco I (6934)

genome

mutants

genes GMOs

AFOSR & DOE (NREL)Collaboration

Overview of Research Strategy

X

10

2011 AFOSR Spring Review:Bioenergy (2308C)

Biosolar Hydrogen(MURI and Core Funding)

11

Bio-Solar Hydrogen Production Eight Labs Including AFRL & DOE

Objective: Technical Approaches:

Accomplishments:

• Bio-prospecting new strains & species

• New H2 detection & analytical methods

• Stress responses and H2 production

• H2ase sequence, maturation & assembly

• Systems biology and pathway analyses

• Genetic engineering of pathways

Light + 2 H2O O2 + 2 H2 (H+/e-)

• Electrode consumes H2 • Extended spectral range• Increased light source intensity 500X with LED

H2 Detectors

H2 YieldH2 Rate

•Developed techniques for high throughput screening of H2-producing phototrophs•Identified physiological factors for increasing rates & yields of cellular H2 production•Engineered metabolic pathways with increased production of H2

•Engineered transgenic cyanobacteria containing foreign O2-tolerant NiFe-hydrogenases

DOD Benefit:Sun

Photosynthesis

Fuel

POWER

1. Stable fuel supply & price2. Energy independence3. Reduced military conflicts4. Carbon neutral 5. Anti-climate change6. Reduced health effects

• Obtain knowledge of the basic scientific principles governing H2 production in microalgae and cyanobacteria

• Genetically engineer pathways to improve the H2 producing capacity of these phototrophs

12

Building Algal Platforms for Improved Biofuel Yields: Posewitz (Colorado School of Mines)

Objectives: (1) increase starch yields to support H2 production and (2) attain genetic backgrounds to alter hydrogenase expression and enhance metabolic integration

Water or starch oxidation are the two pathways to algal H2 production.

• Increased starch accumulation (15x more) without nutrient deprivation.

• Photosynthate is diverted to starch. • Starch can be oxidized to support H2

production or converted to alcohols or lipid hydrocarbon fuels.

Conclusions: (1) Over-expression of isoamylase increases starch accumulation & supports dark production of H2 (and other biofuels). (2) Disruptions of both endogenous hydrogenase genes provide “clean” background for future recombinant techniques to improve enzyme performance.

wildtype D66 cells

D66 cells over-expressing

isoamylase gene

purple = dark H2 pathway green + blue = light H2 pathways

0

20

40

60

80

100

120

140

D66 -hydA2 -hydA1 16-5 4-5-1 -hydEF%

of

wil

dty

pe r

ate Screened 16,000 mutants to

yield double mutant (4-5-1)

Platform is created to introduce heterologous enzymes that are

now in hand for first time.

#1

#2

13

BioSolar H2 Cyanobacterial MetabolismImproving Cellular Fuel Production Efficiency

Dismukes (Rutgers)

H2storagecompounds

photosynthesis hydrogenasee- e-

e- e-

e-e-e- e-

NADHe-

e- e-

e-

auto-fermentation

Identified the metabolic bottleneck in glycogen fermentation

NADH is reductant for phase II H2 and NAD+ is feedback inhibitor of hydrogenase

Revealed NO3- master

switch between glycolysis (GLY) & oxidative pentose phosphate (OPP)

Channeling reductant flux through one of two

NADH enzymes increases photo-H2

H+ (∆Ψ) + e- (Fdx)

∆ΨH+

H+

H+H+H+

H+

H+

H+H2O O2

direct photo-H2 Indirect (dark)

+ Flavone

Control

Reductant & “Thauer Limit”

GLYOPP

+ NO3- NO3

at GAPDH

Targets for Protein Engineering

14

Controlled Hydrogenase ExpressionHydrogenase Maturation and Complex H Cluster Assembly

Peters (MSU)

HydG synthesizes carbon monoxide and

cyanide ligands from the amino acid tyrosine

The 2Fe subcluster of the 6Fe H cluster is inserted into the HydA protein in

the final step

HydE modifies a [2Fe-2S] cluster with

nonprotein dithiolate

HydF is a scaffold for the biosynthesis of the 2Fe

subcluster of the H cluster

∆ΨH+

H+

H+H+

H+

H+

H+

Understanding the steps to synthesizing active hydrogenase is critical to refining superior biotechnological solutions for biological hydrogen production hydrogenase maturation and assembly is intimately linked to metabolism.

HydF activates HydA hydA

15

Algal Oil(Core Funding)


16

Algal OilTen Labs Including DOE and USAFA

Accomplishments:• Screened1200 algal strains for oil yield and identified 50 candidate strains for future studies

• High pH raises oil yields further in NO3-stressed cells

•Transformed carbonic anhydrase into algal genome, resulting in CO2 availability and enhanced growth rate

• Cell cycle arrest or silica starvation elevates lipid production in brown algae (diatoms)

• Identified proteins involved in forming intracellular lipid droplets and in controlling their storage capacity

Objective: Gain knowledge of basic algal biology needed to engineer and enhance photosynthetic and lipid biosynthetic pathways

Technical Approach: • Partner with DOE’s National Renewable Energy Lab

• Bioprospect for new lipid-producing algal strains

• Optimize light capture and photosynthetic efficiency

• Optimize environmental factors for lipid biosynthesis

• Use systems biology (“omics”) to map lipid pathways

• Identify genetic targets and model metabolism

• Build genetic tools for enabling algal bioengineering

AF Benefit:

Industry

AFOSR DOE

Sun

Photosynthesis

Fuel

POWER

1. Stable fuel supply & price2. Oil independence3. Reduce military conflicts4. Carbon-neutral 5. Anti-climate change6. Reduce health effects

17

Why Not Just Use Known Species/Strains of Microalgae for Producing Oil?

IdentificationAnnotationCloningOptimizationEngineeringTransformation

Undiscovered genes Algal production strainsEngineering/Genetics

Domestication of crops

• No commercial system uses wild-type organisms

• All large scale production relies on species that are genetically modified (breeding and engineering)

7,000 Years

MAIZE

VS

ALGAE

7-fold productivity gainin 70 Years

Characterize, understand, model and engineer algal lipid production

18

Enhanced Photosynthetic Efficiency & Algal Growthby Optimizing Light Harvesting Antennae Size

Richard Sayre (Danforth Plant Science Center)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1 2 3 4 5 6 7

Cul

ture

Den

sity

(OD

750

)

Growth in low light (50 µmol

photons m-2s-1 )

0.0

0.2

0.4

0.6

0.8

1.0

1 2 3 4 5 6 7Growth (days)

CC-424CR-118CR-133cbs3

WT

Reduced Chl b

No Chl b

Growth in high light (500 µmol

photons m-2s-1)

FACT:At full sunlight 75% of the captured energy is wasted as fluorescence or heat.

HYPOTHESIS:Reducing the antennae size optimizes energy transfer between the antennae and reactions centers

RESULT:Reductions in Chl b levels reduced the antennae size resulting in a 30% increase in biomass yield at high light intensities relative to wild type

Low Chl fluorescence High

2.2 ∞ 4.0 4.9Chl a/b

+30%

Transgenic algae with reduced Chl b have:1) Reduced antennae

size 2) Reduced steady state

fluorescence

WTNoChl

ChlDeficient

19

Regulation of Oil Biosynthesis in AlgaeC. Benning (MI State U)

The model green alga Chlamydomonas reinhardtii accumulates oil following nitrogen (N) deprivation. The long-term goal is to identify factors required for oil biosynthesis using genomic and genetic approaches.

A specific antibody against a lipid droplet associated protein (MLDP) provides a new diagnostic tool for oil accumulation.

+N -N

A, Nile Red staining of lipid droplets following N-deprivation (-N); B, The MLDP protein is only detected following N- deprivation (arrow) in the shown immuno blot using a new MLDP antibody.

A B

Lipases are important factors determining oil accumulation.

0.00.10.20.30.40.50.6

Time (h) after N deprivation

Fatty

Aci

ds in

Oil/

Tota

lWild typeLipase Mutant

12 24 36 48 72

Plasmid insertion into a lipase gene.

Global transcript profiling using Illumina technology also shows large number of lipase genes differentially regulated following N-deprivation.

Global transcript profiling and metabolic analysis details the metabolic shift from +N to –N.

Miller et al. 2010. Plant Physiol. 154:1737-52

20

Moving Bio-oil through the Lipid BilayerChang (Scripps)

Objective: Use structure-based methods to design a system for secreting oil. In principle, thissystem would “plug-and-play” with a variety of oil-producing organisms for secreting biofuels.

“Leveraging NIH Cancer Research Funding”

1. Concept: A sustainable system for biofuel production by controlled secretion

3. Structural basis for study:Representative X-ray structures of membrane transport proteins solved by Chang Lab as a starting point for re-engineering oil transporters. (He et al., Nature 2010)

2. Background: Based on current model of wax transport across cell membranes (Samuels et al., 2008).

4. Techniques: Structural-based engineering and in vitro evolution of MsbA (shown) and other oil transporters.

5. Culture Conditions: Optimizing growth (doubling time 2-3 days) and oil secretion in Botryococcus braunii and chemically characterizing secreted oil.

1.

2.

3. 4.

5. GC/MS of oil extractBotryococcus braunii

NEW PROJECT 2010

21

Systems Biology for Algal Lipid Pathway Analyses: A 7 Lab Collaboration

Objectives: Next generation RNA Sequencing technologies are used to compare gene expression profiles in lipid- and non-lipid-producing algae (+/- N) to: 1. Identify sentinel genes for lipid production; 2. Map lipid pathways; 3. Model nutrient allocation and regulation; 4. Reconcile transcriptomics with proteomics and metabolomics data

ProteomicsTranscriptomics

Bioinformatics: Data collection &

processing

Computational Biology: Mathematical modeling &

pathway mapping

A1 A1A

B1 B1A

S1

T1X

P1

∆MiA1 A1A

B1 B1A

S1

T1X

P1

Pellegrini (UCLA)

Merchant (UCLA)Seibert (NREL)Sayre (Danforth)

Benning (MSU)Hildebrand (UCSD)

Rabinowitz (Princeton)

Metabolomics

Recent Findings: • 3 time-course experiments analyzed by RNA-Sequencing: from 0 to 48 h• DGAT1, triglyceride synthesis enzyme, is induced early in the time course• A transcription factor, NRTF1, is co-expressed with DGAT1• Developed a web-based protein function annotation tool for algal genomes

(http://pathways.mcdb.ucla.edu/chlamy/)

APPROACH

22

Artificial Photosynthesis(Core Funding)


23

Research Objective:

Create a hybrid multipurpose platform combining nano-plasmonic metamaterials and de novo designed proteins to power the generation of solar biofuels. (Solid state light harvesting is over 3 orders of magnitude more effective than that found in green plants for 750 nm wavelength light.)

Accomplishment:During first 6 months of project, developed a technique to coat the “protein-metamaterial” combo in the light-harvesting wells with a stabilizing sol-gel matrix that disrupts neither the protein structure nor its function.

Encapsulating the proteins with a porous sol-gel greatly increases the functional lifetime of any protein attached to a metal surface

A Hybrid Solar Fuel-Generating Platform: Enhancing Protein Life Span with a Sol-Gel Thin Film

Ronald Koder & David Crouse (The City College of New York)

NEW PROJECT 2010

24

Enzymatic Fuel Cells(MURI and Core Funding)

Ve

e

Load

CathodeAnode

H+


25

Fundamentals and Bioengineering of Enzymatic Fuel Cells: Seven Labs Including AFRL

Objectives:(1) Exploit biochemical reactions for converting chemical to electrical energy and for generating power from fuels readily available in the environment.

(2) Estimate the specific power and energy limits of enzyme fuel cells to definepotential powering uses

(3) Transition technology towards sub-miniature sustainable mobile powersources

Technical Approach:• Provide multi-enzyme

cascades for full utilizationof complex biofuels

• Protein engineering ofenzymes to improvebioelectrocatalysts

• Establish mechanisms of electron transfer

• Design and fabricate novel electrode architectures for enhanced performance

•Accomplishments: • Developed multi-enzyme cascades for complete oxidation of biofuels, enhancing energy density

• Modeling identified major obstacles in multi-step enzyme catalysis—electrode surface area and co-factor (NAD) instability

• Engineered enzymes to self-assemble into conducting hydro-gels and broadened their specificity to accept both NAD & NADP

• Determined O2 binding site in multi-copper oxidases

DoD Benefit: Energy technology platform for scalable power generation. Particularly useful at miniature and micro-levels. Enablingtechnology for sensors and MEMS devices

µWmW

10 mW100 mW

W

26

Complete Oxidation of Methanol by Catalytic Protein Hydrogels: Banta (Columbia University)

Objective: Engineer bi-functional enzymes that retain their catalytic activities while gaining special material properties to enable their use on electrodes of biofuel cells

New Catalytic Biomaterial SupportsA Synthetic Pathway

Three dehydrogenase enzymes were modified with helical appendages, enabling them to self-assemble into functional hydrogels (when mixed) and to oxidize methanol to CO2

Conclusion: A 3-enzyme catalytic hydrogel was developed as an anode modification in a biofuel cell resulting in high current and power densities.

Alcohol dehydrogenase

Aldehyde dehydrogenase

Formatedehydrogenase

CH3OH

HCHO

HCO2H

CO2

NAD+

NADHNAD+

NADHNAD+

NADH

2e-

2e-

2e-3 Enzyme

Hydrogel Mixtures

Maximum current density(mA cm-2)

Maximum power density(mW cm-2)

Methanol 26.4±1.76 3.52±0.16

Formaldehyde 16.5±3.78 2.24±0.57

Formate 4.82±1.44 1.10±0.21

The hydrogel was applied to a biofuel cell anode combined with an air breathing cathode

12

3

27

Integrated Enzymatic Biofuel Cell Atanassov (UNM)

Integration of poly-(MG) modified RVC with NAD+-dependent enzymes immobilized in chitosan/CNTs composite scaffold

-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

-200

0

200

400

600

800

1000

reduction

10th cycle

Cur

rent

den

sity

(µA/

cm2 )

Potential vs. Ag/AgCl (mV)

1st cycle

oxidationpolymerization

shoulder PMGGC

Deposition and characterization of poly-(methylene green) catalysts for NADH oxidationDeposition by cyclic voltammetry

2D glassy carbon 3D reticulated vitreous carbon

0 2 4 6 8 10 12 14

0

200

400

600

800

1000

10 cycles 25 cycles 50 cycles 200 cycles

Cur

rent

(µA

)

[NADH] (mM)

Electrochemical characterization

0 30 60 90 120 150

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Cel

l vol

tage

(V

)

Current (µA)

ADH anode vs. Ag/AgCl

Laccase cathode vs. Ag/AgCl

Anode vs. cathode

Polarization and power curves in 475 ethanolE0 cell = 0.618 V, pH = 6.3Limiting current = 160 µA

Maximum power density = 27 µW/cm3

Integration with laccase-based bio-cathode in a flow-through membrane-less biofuel cell

3-D Anode

Cathode open to air

0 20 40 60 80 100 1200

10

20

30

Pow

er/anode v

olu

me (µW

/cm

3)

Current/anode volume (µA/cm3)

28

Controlling Direct Electron Transfer (DET) Between Electrodes and Conductive Materials

Johnson & Pachter (AFRL) & Atanassov (UNM)

• Background: DET requires an electronic interface forelectrons to “hop” from enzyme to the electrode surface.

Multi-copper containing oxidases (MCO) serve as model bioelectrocatalysts for fuel cell cathode, accepting electrons from electrode and then catalyzing O2 reduction.

• Approach: Various MCO were linked to carbon nanotubes (CNT) using a chemical “tethering” reagent (1-pyrene butanoic acid, succinimidyl ester (PBSE)). The method conjugates the enzyme and CNT without changing material conductivity.

• Results: Electrochemical potential and kinetics of O2 reduction reaction approach theoretical optima (+600 mV vs. Ag/AgCl)High-potential maintained under increased current density, <100 mV decrease @ 50 mA cm-2

Bioelectrodes provided exceptional DET.

• Conclusion: Materials and processing approach accommodates various biocatalysts and is potentially scalable → significant advance over previous literature reports → key steps toward application. Cover feature on Chem Comm

Objectives: Devise means to characterize and organize the interface between redox-active enzymes and nanomaterials

Chemical Communications (2010) 46:6045-6047

-400

-300

-200

-100

0

100

-0.4 -0.2 0 0.2 0.4 0.6 0.8 1

Cur

rent

(µA

cm-2

)

Potential (V) vs. Ag/AgCl

1

4

2

3

onset of O2

reduction

1 Lac-adsorbed Torey paper

2 CNT / Lac3 CNT / PBSE / Lac4 Electrode (3) in N2

PBSE as Enzyme-CNT tether

O2 reduction

29

Microbial Fuel Cells(MURI and Core Funding)

Proton Exchange Mem

brane

Acetate + CO 2

e-

e-

e-

e-e- e- e-e-

e-

e-

e-e-

e-e-

H+

e-

e-

e- e-

Lactate

NADH

MtrB

e-

Cathode electrodeH+H+

H+

e-e-

e-

e-

e-

Anode electrodeH+

e-

e-

e-

e-

e-

e-CymA e-e-

e- e-

e-

H+

H+MtrB?

CymA?

e-

e- e- e-e-

O 2 H 2OFe 3+ Fe 2+

Fumarate Succinate

Proton Exchange Mem

brane

Acetate + CO 2

e-

e-

e-

e-e- e- e-e-

e-

e-

e-e-

e-e-

H+

e-

e-

e- e-

Lactate

NADH

MtrB

e-


H+

e-e-

e-

e-

e-

Anode electrodeH+

e-

e-

e-

e-

e-

e-CymA e-e-

e- e-

e-

H+

H+MtrB?

CymA?

e-

e- e- e-e-

O 2 H 2OFe 3+ Fe 2+

Fumarate Succinate

Proton Exchange Mem

brane

Acetate + CO 2

e-

e-

e-

e-e- e- e-e-

e-

e-

e-e-

e-e-

H+

e-

e-

e- e-

Lactate

NADH

MtrB

e-


H+

e-e-

e-

e-

e-

Anode electrodeH+

e-

e-

e-

e-

e-

e-CymA e-e-

e- e-

e-

H+

H+MtrB?

CymA?

e-

e- e- e-e-

O 2 H 2OFe 3+ Fe 2+

Fumarate Succinate

Proton Exchange Mem

brane

Acetate + CO 2

e-

e-

e-

e-e- e- e-e-

e-

e-

e-e-

e-e-

H+

e-

e-

e- e-

Lactate

NADH

MtrB

e-


H+

e-e-

e-

e-

e-

Anode electrodeH+

e-

e-

e-

e-

e-

e-CymA e-e-

e- e-

e-

H+

H+MtrB?

CymA?

e-

e- e- e-e-

O 2 H 2OFe 3+ Fe 2+

Fumarate Succinate


30

Optimizing Microbial Fuel Cells via Genetics, Modeling and Nanofabrication: Seven Labs

DoD Benefit:This project will enable high performance microbial fuel cells as power sources for defense and biotechnological applications. The ability to use multiple complex fuels under changing physical and chemical conditions may permit unlimited persistence behind enemy lines.

Technical Approach:• Identification & regulation of the genes, molecular

machines and structures used to produce andtransfer current between microbe and electrode

• Modeling & bioengineering

• Development & exploitation of microbial consortia with the ability to utilize a wide range of energy sources

• Modeling, fabrication & testing of miniaturized MFCs

Accomplishments:• Identified current associated genes in Shewanella

• Developed novel vertical scanning interferometry for interfacial analysis at electrode surface

• Characterized the bacterial behavior of electrokinesis

• Showed the value of bacterial biofilms in current production

Objective:To understand the mechanism(s) involved in microbial current production, and to utilize multi-scale modeling to exploit this understanding in order to optimize microbes and microbial communities for microbial fuel cells.

Current transfer by nanowires…

…and/or soluble mediators?

Lactate

Proton Exchange Membrane

Acetate + CO2

e-

e-e- e- e-e-

e-

e-e-

e- e-

H+H+

e-

e-

NADH

MtrA

/B

Anode electrodeCym

Ae-

e-

e-

H+

e-

O2

H2O

Fumarate

Cathode electrode

Reductase

???

e -

e -e -

H+ H+

e-e-

e-

e-

e-

H+

e-

e-

e-e-

e-

H+

MtrC

-Om

cA

WT under anaerobic conditions

WT or mutant under aerobic (O2) or anaerobic (fumarate)

conditions

Bacterial Biofilm Formation

Microbial Fuel Cell

31

Microbial Extracellular Electron Transport Direct vs Mediated Electron Transfer (DET vs MET)

El-Naggar et al., PNAS USA,107, 18127 (2010) Jiang et al., PNAS USA, 107, 16806 (2010)

Before cutting nanowire

After cutting nanowire

Bacterial Nanowire

C

BA

Current Before

Cut

Current After Cut

Pt

“Two experiments, two takes on electric bacteria. Biologists know that certain kinds of microbes can convert organic waste into useful electric current. They just aren’t yet sure how.”

---- Physics Today, December 10, 2010 ----

Bacterial nanowires conductive enough to account for all microbes electric output > DET

Electron transport entirely via soluble electron shuttles > MET

Current in nano-holesNo cell-electrode contact

Current in large wellsCell-electrode contact

32

Controlled Cultivations to Understand Microbial Extracellular Electron Transfer at the Nanoscale:

Collaboration between Harvard/NRL/Venter Institute

Microbial Fuel Cells (NRL)

Miniature-Microbial Fuel Cell

Chemostat cultures in batch mode produce maximum power

Controlled Cultivation (JCVI)

Nanoelectrode Design and Fabrication (Harvard University)

Objective: Further define extracellular electron transfer

mechanisms used by S. oneidensis MR-1 at the single cell level.

Controlled cultivation with analytical analysis will enable differentiation between electron transfer pathways including direct (nanowire, membrane) and indirect (mediated) mechanisms

Bacterial Nanowires

33

(1) Shewanella can survive long periods of nutrient deprivation, but become limited for electron acceptor. Addition of fumarate maintains cell density. After death, control cells recover, suggesting release of e- acceptors.

(3) The MFC anode has three environments with respect to the bacteria, not two as usually assumed. Cells in the interstitial spaces of the graphite felt are physiologically distinct from cells in the “bulk” medium and the biofilm.

(2) During long-term survival, mutants with (A) altered biofilm forming ability and (B) increased fitness appear. Cells harvested after three rounds of incubation for 10 days (n=9) show significant increases in relative fitness.

(4) Cells within the graphite felt environment (“red” cells) grow to higher yields than bulk planktonic cells (“blue” cells), n=21.

Adaptive Evolution & Survival in MFCsFinkel (USC)

+Fumarate

+Lactate

Control

Cel

l den

sity

(CFU

/ml)

Day

BA

Bulk Planktonic Cells

Graphite Felt Cells(“interstitial”)

Biofilm Cells

Avg. = 470-fold

34

Photo-Electro-Magnetic Stimulation of Biological

Responses(Core Funding)

Photo-Electro-Magnetic Stimulation of Biological Responses is a beginning program that characterizes, models and explains the stimulatory and inhibitory responses of biological systems to low-level exposures of photo-electro-magnetic stimuli. Potential long-term benefits may include accelerated recovery from mental fatigue and drowsiness, enhanced learning and training, and noninvasive treatment of traumatic brain injuries. (~10% of portfolio = 5 AFRL/RH projects)

2011 AFOSR Spring Review2308C Portfolio

35

Electric Stimulation of the Brain, Hemodynamics and Sustained Attention: McKinley (AFRL/RH)

Objective: Quantify effects on human vigilance and hemodynamics due to non-invasive stimulation of the brain by low levels of direct current (1 mA).

Anodal Stim.

rCBFAstrocytes…?

P(APs) rCBFCO2Vigilance Perform.

Information processing rSO2

Potential Metrics

Helton et al., 2010Merzagora et al., 2010

Hellige, 1993 & Warm et al., 2009

Moore & Cao, 2008

Gordon et al., 2009

65.00%

75.00%

85.00%

95.00%

105.00%

115.00%

0 10 20 30 40 50

% C

hang

e Fr

om B

asel

ine

Time [Mins]

Early Stimulation

Active

SHAM

90.00%

92.00%

94.00%

96.00%

98.00%

100.00%

102.00%

104.00%

0 10 20 30 40 50

% C

hang

e Fr

om B

asel

ine

Time [Mins]

Blood Flow (Active vs. Sham)

Blood Flow - Sham

Blood Flow - Active

NEWPROJECT

2011

36

Coupling Terahertz Radiation to Biomoleculesfor Controlling Cell Response: Wilmink (AFRL/RHDR)

2. Protein

1. Lipid membrane

3. DNA

THz

0 1 2 3 4Frequency (THz)


0

100

200

300

400

500

600

700

)

Glucose

Galactose

Mannose

Fructose

Carbohydrates


0

50

100

150

200

250

DNA (nucleotides)

Water B C D

Terahertz (THz) Radiation:• Alters lipid membranes and modulates neuronal action potentials.• Oscillates in the same ps time-scale as breathing modes of DNA & proteins (~40 ps).

Objectives: Investigate coupling mechanism and exploit the understanding to activate adaptive responses and modify cellular behaviors

Biomolecules display unique spectra in THz region THz energy couples to biomolecules

Working Hypothesis: THz-coupling is mediated via macromolecule-bound water on the surface of membranes and biomolecules

Testing Hypothesis: • THz exposure system on a microscope

• Raman & THz spectroscopy• Fluorescence & atomic forcemicroscopy

• DNA mutation assays

Macromolecule-bound water

Bulk water

µa

NEW PROJECT 2011

37

Questions?

5. kozumbo - bioenergy

Documents