AFOSR

BIOENERGY
15 March 2011

Walt Kozumbo
Program Manager
AFOSR/RSL
Air Force Office of Scientific Research
Distribution A: Approved for public release; distribution is unlimited. 88ABW-2011-0770
 2011 AFOSR Spring Review
2308C 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)
2
 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 reactions
Payoffs: - 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 systems
Payoffs: - 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-materials
Payoffs: - ordered enzyme alignments for enhanced power densities in EFC
- enhanced electron transport and power density in biofuel cells
3
- light is harvested and split in artificial photosynthetic solar fuel generator
 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 cognitive
functions, bio-molecular repair and bio-resiliency 4
 Related Research
Funded by Other Agencies
Funding Criteria: Materials
Chemistry
1. Basic research of high quality and relevant to the AF BiologyPhysics
2. Unique or complementary, but non-duplicative—finds a “niche” Engineering Math
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.
5
BioResponse to Photo-electromagnetic Stimulation: Complementary to other funded research.
 Overview of Topic Areas 2308C
Bioenergy: Alternative Energy for Future AF Needs
• Biofuels—Macro-scale Energy • Stable Fuel Supply & Price

• Biosolar Hydrogen • Energy Independence

• Algal Oil for Jet Fuel • Reduced Military Casualties

• Artificial Photosynthesis • Carbon Neutral
• Anti-Climate Change
• Biofuel Cells—Micro-scale Energy
• Reduced Health Effects
• Microbial Fuel Cells • Compact Power Supplies
• Enzymatic Fuel Cells
Aircraft

Photosynthesis Fuel H2
Sun Fuel Cells Vehicles

Robofly
Natural to Artificial
Biofuel Cells MAV

Photo-Electro-Magnetic Stimulation of Bio-responses 6

 FY10 Technology Transfer 2308C
Topic Performer Customer Result Application
Artificial
Nocera, SunCatalytix, An inorganic cobolt-phopshate catalyst To generate hydrogen fuel from water, easily,
Photo-
MIT Cambridge, MA that splits water and self-heals. safely and cheaply.
synthesis
Nano-structured plasmonic meta-
Koder, Phoebus Optoelectronics,
materials for harvesting and splitting To convert solar energy into biofuels.
CCNY LLC, New York, NY light.
A high energy-dense biodegradable
Zhang, Gate Fuels Inc., sugar battery (3 x's > lithium ion To develop a secondary battery charger for
Biofuel Cells
VA Tech. Blacksburg, VA batteries) using cell-free synthetic compact energy sources.
enzyme pathways

An enzyme immobilization technology To improve long-term operational stability

Atanassov, Lynntech Corp,
based on chemical vapor deposition of of enzymes under biofuel cell operational
U of NM College Station, TX silica-encapsulated enzymes. conditions.

An in vitro enzyme cascade system

Minteer, CFD Research Corporation, To enhance anodal energy output via the
that completely oxidizes sucrose
St. Louis University Huntsville, AL complete oxidation of sucrose as fuel.
to CO2 and water.

Multiple Web sites displaying algal genomes on

Merchant and a browser for data visualization and To deduce structures of algal transcripts and
Algal Oil worldwide experimental comparisons patterns of expression for any gene of interest.
Pelligrini, UCLA
institutions http://genomes-merchant.mcdb.ucla.edu

Worldwide A web-based tool that utilizes the To map differentially expressed genes
Merchant and annotated Chlamydomonas genome for to metabolic pathways and convert
Chlamydomonas understanding metabolism protein IDs from one genomic
Pelligrini, UCLA
research community http://pathways.mcdb.ucla.edu/chlamy version to another.

A flow cytometry procedure for single- To evaluate lipid accumulation over time,
Hildebrand, General Atomics Inc.,
cell and subpopulation analyses of lipid advancing the process of lipid analysis on
UCSD San Diego, CA accumulation in microalgae. cultures.

Biosolar Posewitz, ConocoPhillips, Genetic techniques to over-accumulate To obtain glucose as a feedstock

Hydrogen CO School of Mines Bartlesville, OK starch and glucose in Chlamydomonas for a variety of biofuels. 7
 Bioenergy:
A Progressive Research Strategy
Natural to Artificial
Sun Photosynthesis Fuel Biofuel Cells POWER

Generation 1st 2nd 3rd 4th

Natural Optimized Natural Hybrid Artificial

System Biosystems
Type Biosystems Systems Systems

Basic Characterization Metabolic/ Synthetic Chemistry &

Research Mechanisms Protein Biology Materials
Type Models Engineering Science

Disciplinary
Biology
Chemistry
Inputs Math Physics Engineering

8
 Photosynthesis, Systems Biology and Metabolic
Engineering for the Production of Biofuels
Microalgae & Cyanobacteria Make Hydrogen, Lipids & Sugars
Light Reactions PSI and PSII Dark Reactions
Triglyceride (Oil)
light chlorophyll Lipid Synthesis Jet Fuel
_
4e CO2
Sugar/Cellulose
4 H+ Ethanol
water-splitting
Synthesis X
2 H2O
enzyme
H2-generating
carbon-fixing hydrogenase H2
enzyme
Three Key Biocatalysts enzyme

Overview of Research Strategy

AFOSR & DOE (NREL)
Collaboration
mutants

HoxE HoxF ORF? HoxU HoxY HoxH

Nco I (3375) Nco I (6934)
Bam HI (1484)
Cla I (2981) Eco RI (4977)
Nco I (1099) Cla I (7047)

screening genome genes GMOs

diaphorase moiety Ni-Fe hydrogenase moiety
genomic sequence around hox genes in S. platensis
field 7098 bp

9
2011 AFOSR Spring Review:
Bioenergy (2308C)

Biosolar Hydrogen
(MURI and Core Funding)

10
 Bio-Solar Hydrogen Production
Eight Labs Including AFRL & DOE

Objective: Light + 2 H2O  O2 + 2 H2 (H+/e-) Technical Approaches:

• 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
• Bio-prospecting new strains & species
H2 Detectors
• New H2 detection & analytical methods
H2 Rate H2 Yield
• Stress responses and H2 production
• H2ase sequence, maturation & assembly
• Electrode consumes H2
• Extended spectral range
• Increased light source • Systems biology and pathway analyses
intensity 500X with LED
• Genetic engineering of pathways

Accomplishments: DOD Benefit:

•Developed techniques for high throughput Sun 1. Stable fuel supply & price
screening of H2-producing phototrophs
2. Energy independence
•Identified physiological factors for increasing Photosynthesis 3. Reduced military conflicts
rates & yields of cellular H2 production 4. Carbon neutral
5. Anti-climate change
•Engineered metabolic pathways with Fuel
6. Reduced health effects
increased production of H2
POWER
•Engineered transgenic cyanobacteria
containing foreign O2-tolerant NiFe-
hydrogenases 11
 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

purple = dark H2 pathway • Increased starch accumulation (15x

green + blue = light H2 pathways more) without nutrient deprivation.
• Photosynthate is diverted to starch.
#1 • Starch can be oxidized to support H2
production or converted to alcohols
wildtype D66 cells or lipid hydrocarbon fuels.

140

120

% of wildtype rate
Screened 16,000 mutants to
100 yield double mutant (4-5-1)
80

D66 cells over- 60

Water or starch oxidation are the two expressing
40
pathways to algal H2 production. isoamylase gene
20

Conclusions: (1) Over-expression of isoamylase increases 0

D66 -hydA2 -hydA1 16-5 4-5-1 -hydEF
starch accumulation & supports dark production of H2 (and
other biofuels). (2) Disruptions of both endogenous Platform is created to introduce
hydrogenase genes provide “clean” background for future #2 heterologous enzymes that are 12
recombinant techniques to improve enzyme performance. now in hand for first time.
 BioSolar H2 Cyanobacterial Metabolism
Improving Cellular Fuel Production Efficiency
Dismukes (Rutgers)

direct H+ (∆Ψ) + e- (Fdx)

photo-H2 Indirect (dark) H+
H+ H+
H2O O2 ∆Ψ
H+
H+
auto- H H + + H+
photosynthesis
storage fermentation e-
e- e-
-
e- ee-
hydrogenase H2
compounds NADH
e- -
e- e- e- e- e

Targets for Protein Engineering

Channeling reductant Revealed NO3- master Identified the metabolic NADH is reductant for
flux through one of two switch between bottleneck in glycogen phase II H2 and NAD+ is
NADH enzymes glycolysis (GLY) & fermentation feedback inhibitor of
increases photo-H2 oxidative pentose hydrogenase
phosphate (OPP)
+ Flavone
Reductant & “Thauer Limit”

Control
GLY
OPP at GAPDH 13
- NO3 + NO3
 Controlled Hydrogenase Expression
Hydrogenase Maturation and Complex H Cluster Assembly
Peters (MSU)

Understanding the steps to synthesizing active hydrogenase is critical to refining superior

H+ + hydrogenase
biotechnological solutions for biological hydrogen production maturation and
H H+
assembly is intimately linked to metabolism. H+
∆Ψ
H+
+

H+
H

HydF is a scaffold for the HydE modifies a [2Fe- HydG synthesizes The 2Fe subcluster of the
biosynthesis of the 2Fe 2S] cluster with carbon monoxide and 6Fe H cluster is inserted
subcluster of the H cluster nonprotein dithiolate cyanide ligands from the into the HydA protein in
amino acid tyrosine the final step

HydF
activates
hydA
HydA 14
2011 AFOSR Spring Review:
Bioenergy (2308C)

Algal Oil
(Core Funding)

15
 Algal Oil
Ten Labs Including DOE and USAFA

Objective: Gain knowledge of basic algal Technical Approach:

biology needed to engineer and enhance • Partner with DOE’s National Renewable Energy Lab
photosynthetic and lipid biosynthetic pathways
• Bioprospect for new lipid-producing algal strains
AFOSR DOE • Optimize light capture and photosynthetic efficiency
• Optimize environmental factors for lipid biosynthesis
• Use systems biology (“omics”) to map lipid pathways
Industry
• Identify genetic targets and model metabolism
• Build genetic tools for enabling algal bioengineering

Accomplishments: AF Benefit:
• Screened1200 algal strains for oil yield and identified
50 candidate strains for future studies Sun 1. Stable fuel supply & price
2. Oil independence
• High pH raises oil yields further in NO3-stressed cells 3. Reduce military conflicts
Photosynthesis
•Transformed carbonic anhydrase into algal genome, 4. Carbon-neutral
resulting in CO2 availability and enhanced growth rate 5. Anti-climate change
Fuel
6. Reduce health effects
• Cell cycle arrest or silica starvation elevates lipid
production in brown algae (diatoms) POWER

• Identified proteins involved in forming intracellular lipid 16

droplets and in controlling their storage capacity
 Why Not Just Use Known Species/Strains of
Microalgae for Producing Oil?
Domestication of crops
M • No commercial system 7-fold productivity gain
in 70 Years
A uses wild-type organisms
I • All large scale production
Z relies on species that are
genetically modified
E (breeding and engineering)
7,000 Years

VS
Identification
A Annotation
L Cloning
Optimization
G Engineering
A Transformation

E Undiscovered genes Engineering/Genetics Algal production strains

17
Characterize, understand, model and engineer algal lipid production
 Enhanced Photosynthetic Efficiency & Algal Growth
by Optimizing Light Harvesting Antennae Size
Richard Sayre (Danforth Plant Science Center)
1.4
Transgenic algae with FACT: Growth in low light
reduced Chl b have: At full sunlight 75% of the captured 1.2 (50 µmol
1) Reduced antennae energy is wasted as fluorescence or photons m-2s-1 )
1.0

Culture Density (OD 750)

size heat.
2) Reduced steady state 0.8
fluorescence HYPOTHESIS:
Reducing the antennae size 0.6
No Chl
WT Chl Deficient
optimizes energy transfer between
the antennae and reactions centers 0.4
Chl
a/b 2.2 ∞ 4.0 4.9
RESULT: 0.2

Reductions in Chl b levels reduced 0.0

the antennae size resulting in a 30% 1 2 3 4 5 6 7
increase in biomass yield at high Growth in high light
light intensities relative to wild type 1.0
(500 µmol
+30%
photons m-2s-1)
No Chl b 0.8

WT 0.6

Reduced 0.4
CC-424
Chl b
CR-118
0.2 CR-133
cbs3
0.0
18
1 2 3 4 5 6 7
Low Chl fluorescence High Growth (days)
 Regulation of Oil Biosynthesis in Algae
C. 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 Lipases are important Global transcript profiling

a lipid droplet associated factors determining oil and metabolic analysis
protein (MLDP) provides a accumulation. details the metabolic
new diagnostic tool for oil 0.6 shift from +N to –N.
Fatty Acids in Oil/Total
Lipase Mutant
0.5
accumulation. 0.4
Wild type
0.3
A B +N -N 0.2
0.1
0.0
12 24 36 48 72
Time (h) after N deprivation

Plasmid insertion into a lipase gene.

Global transcript profiling using

A, Nile Red staining of lipid droplets following N- Illumina technology also shows
deprivation (-N); B, The MLDP protein is only large number of lipase genes
detected following N- deprivation (arrow) in the differentially regulated following
shown immuno blot using a new MLDP antibody. N-deprivation. Miller et al. 2010. Plant Physiol. 154:1737-52

19
 Moving Bio-oil through the Lipid Bilayer
Chang (Scripps) NEW PROJECT 2010

Objective: Use structure-based methods to design a system for secreting oil. In principle, this
system would “plug-and-play” with a variety of oil-producing organisms for secreting biofuels.
“Leveraging NIH Cancer Research Funding”
1. 3. 4.
1. Concept:
A sustainable
system for
biofuel
production by
3. Structural basis for study:
controlled Representative X-ray structures of
secretion membrane transport proteins
solved by Chang Lab as a starting 4. Techniques: Structural-
point for re-engineering oil based engineering and in vitro
transporters. (He et al., Nature evolution of MsbA (shown)
2. and other oil transporters.
2010)

5. Botryococcus braunii GC/MS of oil extract

5. Culture Conditions: Optimizing growth (doubling time 2-3

2. Background: Based on current days) and oil secretion in Botryococcus braunii and chemically
model of wax transport across cell characterizing secreted oil. 20
membranes (Samuels et al., 2008).
 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

X X
A Transcriptomics Proteomics
T1T1
Metabolomics
P A1A
Benning (MSU)
A1A1 A1A Hildebrand (UCSD)
P
R Merchant (UCLA)
Seibert (NREL)
Sayre (Danforth)
B1B1 B1A
B1A ∆Mi
S1 S P1 P
1 1
O
A Bioinformatics: Computational Biology:
Data collection & Mathematical modeling &
C processing pathway mapping
H Pellegrini (UCLA) Rabinowitz (Princeton)

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
21
(http://pathways.mcdb.ucla.edu/chlamy/)
2011 AFOSR Spring Review:
Bioenergy (2308C)

Artificial Photosynthesis
(Core Funding)

22
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)

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
NEW PROJECT 2010 23
2011 AFOSR Spring Review:
Bioenergy (2308C)

Enzymatic Fuel Cells

(MURI and Core Funding)
Load

Anode V e Cathode
e

H+

24
 Fundamentals and Bioengineering of
Enzymatic Fuel Cells: Seven Labs Including AFRL
Objectives: Technical Approach:
(1) Exploit biochemical reactions for converting chemical
• Provide multi-enzyme
to electrical energy and for generating power from fuels
cascades for full utilization
readily available in the environment.
of complex biofuels
(2) Estimate the specific power and energy limits of
• Protein engineering of
enzyme fuel cells to define
enzymes to improve
potential powering uses
bioelectrocatalysts
(3) Transition technology • Establish mechanisms of electron transfer
towards sub-miniature
sustainable mobile power • Design and fabricate novel electrode architectures for
sources enhanced performance
•
Accomplishments: DoD Benefit: Energy technology platform for
scalable power generation. Particularly useful at
• Developed multi-enzyme cascades for complete
miniature and micro-levels. Enabling
oxidation of biofuels, enhancing energy density
technology for sensors and W
• Modeling identified major obstacles in multi-step MEMS devices 100 mW
enzyme catalysis—electrode surface area and co-factor 10 mW
(NAD) instability mW
• Engineered enzymes to self-assemble into conducting µW
hydro-gels and broadened their specificity to accept
both NAD & NADP
25
• Determined O2 binding site in multi-copper oxidases
 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

NAD+ CH3OH 1
2e- Alcohol 2
NADH dehydrogenase
NAD+ HCHO
2e- Aldehyde
NADH dehydrogenase
HCO2H
NAD+
2e - Formate
dehydrogenase 3 Enzyme
NADH CO2 Hydrogel Mixtures
The hydrogel was applied to a biofuel cell
anode combined with an air breathing cathode
New Catalytic Biomaterial Supports
A Synthetic Pathway
3
Three dehydrogenase enzymes were modified
with helical appendages, enabling them to self-
assemble into functional hydrogels (when mixed)
and to oxidize methanol to CO2

Maximum current density Maximum power density

(mA cm-2) (mW cm-2)
Conclusion: A 3-enzyme catalytic hydrogel was Methanol 26.4±1.76 3.52±0.16
developed as an anode modification in a biofuel cell Formaldehyde 16.5±3.78 2.24±0.57 26
resulting in high current and power densities. Formate 4.82±1.44 1.10±0.21
 Integrated Enzymatic Biofuel Cell
Atanassov (UNM)
Deposition and characterization of poly-(methylene green) catalysts for NADH oxidation
Deposition by cyclic voltammetry Electrochemical characterization
1000 2D glassy carbon 3D reticulated vitreous 1000
carbon
Current density (µA/cm2)

10 cycles
800 800 25 cycles
1st cycle 50 cycles
600 polymerization 200 cycles
600
400 oxidation shoulder PMG

Current (µA)
400
200
10th cycle
GC
0 200

reduction
-200 0
-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0 2 4 6 8 10 12 14
Potential vs. Ag/AgCl (mV) [NADH] (mM)
Integration of poly-(MG) modified Integration with laccase-based Polarization and power curves in 475 ethanol
RVC with NAD+-dependent enzymes bio-cathode in a flow-through E0 cell = 0.618 V, pH = 6.3
immobilized in chitosan/CNTs membrane-less biofuel cell Limiting current = 160 µA
composite scaffold Maximum power density = 27 µW/cm3
30
0.6 Laccase cathode

Power/anode volume (µW/cm3)

vs. Ag/AgCl
0.5

Cell voltage (V)

0.4 20
Anode vs.
3-D Anode cathode
0.3

0.2 10
ADH anode
Cathode
0.1
vs. Ag/AgCl
open to air 0.0
0
0 30 60 90 120 150 0 20 40 60 80 100 120
27
3
Current (µA) Current/anode volume (µA/cm )
 Controlling Direct Electron Transfer (DET)
Between Electrodes and Conductive Materials
Johnson & Pachter (AFRL) & Atanassov (UNM)
Objectives: Devise means to characterize and organize the O2 reduction
interface between redox-active enzymes and nanomaterials

• Background: DET requires an electronic interface for

electrons to “hop” from enzyme to the electrode surface.
Multi-copper containing oxidases (MCO) serve as model PBSE as Enzyme-CNT tether
bioelectrocatalysts for fuel cell cathode, accepting electrons 100
1
from electrode and then catalyzing O2 reduction.

Current (µA cm-2)

0
4
onset
• Approach: Various MCO were linked to carbon nanotubes -100 of O2
reduction
2
(CNT) using a chemical “tethering” reagent (1-pyrene butanoic -200 1 Lac-adsorbed
acid, succinimidyl ester (PBSE)). The method conjugates the Torey paper
-300 3 2 CNT / Lac
enzyme and CNT without changing material conductivity. 3 CNT / PBSE / Lac
4 Electrode (3) in N2
-400
• Results: Electrochemical potential and kinetics of O2 reduction -0.4 -0.2 0 0.2 0.4 0.6 0.8 1
reaction approach theoretical optima (+600 mV vs. Ag/AgCl) Potential (V) 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
28
steps toward application. Cover feature on Chem Comm  Chemical Communications (2010)
46:6045-6047
2011 AFOSR Spring Review:
Bioenergy (2308C)

Microbial Fuel Cells

(MURI and Core Funding)

e - e - e- e- e- e-
e- e-
e-
e - e-
Fumarate Succinate
e - e-
e- Fe 3+ Fe 2+
Proton Exchange Membrane

e -
Acetate e- O2 H 2O
e-
Lactate + CO 2
e - e-
e-
e - e-
H+ e- e-
MtrB?
NADH e- e-
e- e-
e- e-
CymA MtrB e- e- CymA?
e- e- e-
e- e- e- e- e-
Anode electrode H + H+ Cathode electrode
H+ H + + H+
H

29
 Optimizing Microbial Fuel Cells via Genetics,
Modeling and Nanofabrication: Seven Labs
Objective: Technical Approach:
• Identification & regulation of the genes, molecular
To understand the machines and structures used to produce and
mechanism(s) involved in transfer current between microbe and electrode
e- e- e- e- e- e-

microbial current production, Microbial e-

e-
e-
Fuel Cell
• Modeling & WT under anaerobic
e-
e-
e- WT or mutant under aerobic

and to utilize multi-scale conditions e- e- (O2) or anaerobic (fumarate)

e-
bioengineering e-

Proton Exchange Membrane

e- e- conditions

Cathode electrode
e- e-

Anode electrode
modeling to exploit this
e-

MtrC-OmcA
e- O2

e-MtrA/B e-

e-
• Development & Acetate

e-
???
+ CO2 Fumarate

H+
understanding in order to

Reductase
e-
exploitation of

CymA
e-
NADH
e- e- H2O

optimize microbes and microbial consortia Lactate H+ H+

H+
H+
H+ H+

microbial communities for with the ability to utilize a wide range of energy
sources
microbial fuel cells.
Current transfer by nanowires… • Modeling, fabrication & testing of miniaturized MFCs

Accomplishments: …and/or soluble mediators?

• Identified current associated genes in Shewanella

• Developed novel vertical scanning interferometry for
interfacial analysis at electrode surface
DoD Benefit:
• Characterized the bacterial behavior of electrokinesis
This project will enable high performance microbial
• Showed the value Bacterial Biofilm Formation fuel cells as power sources for defense and
of bacterial biofilms biotechnological applications. The ability to use
in current production multiple complex fuels under changing physical and
chemical conditions may permit unlimited persistence30
behind enemy lines.
 Microbial Extracellular Electron Transport
Direct vs Mediated Electron Transfer (DET vs MET)

“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 ----

El-Naggar et al., PNAS USA,107, 18127 (2010) Jiang et al., PNAS USA, 107, 16806 (2010)
Bacterial nanowires conductive enough to Electron transport entirely via soluble
account for all microbes electric output > DET electron shuttles > MET
A B
Before cutting
nanowire
Bacterial
Nanowire

Pt

Current in nano-holes
No cell-electrode contact
C Current After Cut
After cutting
nanowire
Current in large wells
Cell-electrode contact
Current
Before
Cut 31
 Controlled Cultivations to Understand Microbial
Extracellular Electron Transfer at the Nanoscale:
Collaboration between Harvard/NRL/Venter Institute
Microbial Fuel Cells Controlled Cultivation
(NRL) (JCVI)
Objective: Further define
extracellular electron transfer
mechanisms used by S. oneidensis
MR-1 at the single cell level.
Miniature-Microbial Fuel Cell

Nanoelectrode Design and

Fabrication (Harvard University)

Chemostat cultures in batch mode Bacterial

produce maximum power Nanowires

Controlled cultivation with analytical analysis will enable differentiation between electron transfer
pathways including direct (nanowire, membrane) and indirect (mediated) mechanisms 32
 Adaptive Evolution & Survival in MFCs
Finkel (USC)

(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.
Cell density (CFU/ml)
(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.

+Fumarate
Bulk Planktonic Cells
+Lactate

Graphite Felt Cells

Control (“interstitial”)

Day Biofilm Cells

(2) During long-term survival, mutants with (A)
(4) Cells within the graphite felt environment
altered biofilm forming ability and (B) increased
(“red” cells) grow to higher yields than bulk
fitness appear. Cells harvested after three
planktonic cells (“blue” cells), n=21.
rounds of incubation for 10 days (n=9) show
significant increases in relative fitness.

B Avg. = 470-fold
A

33
 2011 AFOSR Spring Review
2308C Portfolio

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)
34
 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).

Early Stimulation
NEW
115.00%

% Change From Baseline

PROJECT 105.00%

2011 95.00%

85.00% Active
SHAM
75.00%

65.00%
0 10 20 30 40 50

Time [Mins]
Blood Flow (Active vs. Sham)
104.00%

% Change From Baseline

102.00%
100.00%
98.00%
96.00% Blood Flow - Sham
94.00% Blood Flow - Active
92.00%
90.00%
Gordon et al., 2009
0 10 20 30 40 50

Time [Mins]
Astrocytes rCBF Moore & Cao,
…? 2008
Anodal Information Vigilance
P(APs) rSO2 CO2 rCBF
Stim. processing Perform.

Potential
Metrics
35
Merzagora et al., Helton et al., 2010 Hellige, 1993 &
2010 Warm et al., 2009
 Coupling Terahertz Radiation to Biomolecules
for Controlling Cell Response: Wilmink (AFRL/RHDR)
Terahertz (THz) Radiation: NEW PROJECT 2011
• Alters lipid membranes and modulates neuronal action potentials.
• Oscillates in the same ps time-scale as breathing modes of DNA & proteins (~40 ps).
Biomolecules display unique spectra in THz region THz energy couples to biomolecules
B Water C Carbohydrates D DNA (nucleotides)
700
250
Glucose THz
1. Lipid membrane
600 200

µa 400
500
150
Galactose
2. Protein
Mannose
) 300 100
200
50 Fructose 3. DNA
100

0 0
0 1 2 3 4 0 1 2 3 4 0 1 2 3 4
Frequency (THz) Frequency (THz) Frequency (THz)

Objectives: Investigate coupling mechanism and exploit the understanding to

activate adaptive responses and modify cellular behaviors
Working Hypothesis: Macromolecule-bound
water
Testing Hypothesis:
THz-coupling is • THz exposure system on a
mediated via microscope
macromolecule-bound • Raman & THz spectroscopy
water on the surface of • Fluorescence & atomic force
membranes and Bulk
microscopy 36
biomolecules water • DNA mutation assays
Questions?

37