Justin C. Biffinger1, Bradley R. Ringeisen1, Emily R. Petersen2, Lisa A. Fitzgerald1

1) US Naval Research Lab, Chemistry Division, Washington, DC. 2) NOVA Research Inc., 1900 Elkin St., Suite 230, Alexandria, VA 22308, The only autonomous submersible water column platform capable of accepting biological electrochemical systems as sole source of power

Biffinger, J.C., Ringeisen, B.R., Wu, P.K., “Enlisting Bacteria to power autonomous water column sensors”, Sea Technology, 2011,52(10), 29-32. Advantages of Working in the Water Column: Surface & Submerge • Diverse (and more complex) carbon content an Autonomous Sensor for USW • Higher dissolved O2 content (cathode) • Better nutrient Applications or • Warmer Environmental • Accessible RF/UHF communication (surface) Applications

< 0.5 L Approach • Microbial power in the water column (not sediment) • Microbial gas generation for ultra-low power ballast control

Sediment Distributed Sensor Systems • Acoustic or Wired COMMS Sediment-Based Microbial Power Source

1 meter Stone Aerospace- DEPTHX CHINARE, Sami-pH sensor Sami-pH aquatic sensor (US Davis)

Robotic Fish –M.I.T. U of Montana, Sami-pHSUAV – US Navy sensor in Bahamas Power requirements: mW – kW (mission dependent)

700

600

500

400

Patents 300 Total Number Total Publications

200

100

Multi figure image from: Logan, B., et al.,

0 Environ. Sci. Technol.,

1962 2008 1984 1986 1988 1995 1997 2000 2002 2004 2006 2010 2012 1981 40 (17), 5181 -5192, 2006

Year

Links and references provided on request Anode Cathode Load - e- e e- e- - - - e- e e e

M+ H+

M+ O 2 • Regenerated Cellulose and Nafion are Osmotic Membranes H+ • Nylon, Cellulose Nitrate, and Polycarbonate are all passive diffusion M+ H+ • All membranes have pore sizes less that O 0.2 µm M+ 2

Nanoporous Membrane Biffinger, J.C., et al. Environ. Sci. Technol. 41, 1444 (2007) Power up to 50% greater than Nafion!

• Similar polarization 0.6 0.5 curves indicate 0.025 0.4 0.3 similar resistance of E (V) 0.2 membranes 0.1 0.02 0 0 0.1 0.2 0.3 0.4 0.5 I (mA) 0.015 Regenerated Cellulose Nafion P (mW) P 0.01 Cellulose Nitrate

Polycarbonate 0.005 Nylon

0 0 0.1 0.2 0.3 0.4 0.5 I (mA)

Biffinger, J.C., et al. Environ. Sci. Technol. 41, 1444 (2007) MFC Stack Cathode E Anode OC I (mA) (Connection type) Type (V) sc Volume (mL) “Barnacle” MFC Uncoated 4 “Barnacle” MFCs Carbon 2.4 0.11 15 (Series) (ORR) Cathode Uncoated 4 “Barnacle” MFCs Carbon 0.58 0.58 15 (Parallel) (ORR) Anode

4 “Cyprid” MFC Bare carbon 2.6 0.042 2 (Series) (ORR) Electric- bacteria

4 “Cyprid” MFC Bare Carbon 0.61 0.13 2 (Parallel) (ORR)

3 mini-MFC Fe(CN) 2.2 1.3 3.6 (Series) 6 3 mini-MFC Fe(CN) 0.75 3.4 3.6 (Parallel) 6

Table Footnote: Fe(CN)6: 50 mM potassium ferricyanide in 100 mM phosphate buffer (pH 7); Isc: Short Circuit ; EOC: Open circuit voltage • 611 cm2 of graphite felt in 1.2 cm3 chamber - 509 cm2 GF/cm3 • Typical Void Volume Graphite Felt (95%) • 1.14 cm3 void volume in chamber Membrane So, 95% of the chamber volume is filled with a culture (avg: 108 CFU/mL) but the void is within the electrode (3D electrode) : 2x105 Shewanella/cm2 GF

Once chamber is filled with culture…interaction of bacteria (-) with electrode = rate of diffusion in water (rate of diffusion in water inversely proportional to distance)

Electrode acting more like a filter than typical electrode in MFCs

System by definition a microbial fuel cell but engineering more like in-line sensor

Ringeisen, BR., et al., Environ. Sci. Technol., 2006, 40(8) 2629-2634. J. Biffinger, R. Ray, B. Little (NRL Stennis Space Center) • Isolated from Ice in • One of 28 sequenced Shewanella

Big differences between S. frigidmarina and S. oneidensis MR-1: • No typical redox mediators observed • Power output in salt water > 10x

Fitzgerald, L.A., et al., Biosensors and Bioelectronics, 2012 (Special Issue for Biosensors 2012) doi:10.1016/j.bios.2012.06.039., LB ½ MB

Acetate MM Modified Marine Broth Modified Luria Bertani

Modified MB

Mg2+ and Ca2+

Just Mg2+ salts Bulk conductivity unchanged 17 (LB) to 24 (for modified) mS/cm Bulk conductivity unchanged 24 +/- 2 mS/cm Polarization Curves for Evaluation of Ca2+ on S. oneidensis MR-1

350 0.8 0.7 0.6

300 0.5 0.4 E (V) E 0.3 250 0.2 0.1 0

200 0 500 1000 1500 I (µA)

P (µW) P 150 0 µM CaCl2 300 µM CaCl2 100 680 µM CaCl2 50 1400 µM CaCl2 2080 µM CaCl2 0 0 500 1000 1500 I (µA)

Fitzgerald, LA.; Petersen ER.; Gross BJ.; Soto CM.; Ringeisen BR.; El-Naggar MY.; Biffinger JC. Biosensors & Bioelectronics 2012, 31 (1), 492-8. Current output from cultures grown or spiked with 0 or

1400 µM CaCl2 *

0 µM CaCl2

Delayed CaCl2 Addition

1400 µM CaCl2 Comparison of Lactate consumption by S. oneidensis MR-1 in

selected of CaCl2

30

25

20

0 µM CaCl2 15 300 µM CaCl2 680 µM CaCl2 1400 µM CaCl2

10 2080 µM CaCl2 Lactate (mM) concentration Lactate

5

0 0 5 10 15 20 25 30 Time (hrs)

Food Food Food CO2

CO2

CO2

e- Med Med ox Red e- Med Med Direct Electron Transfer Direct Electron Transfer Ox Red Medox MedRed (Membrane) (Nanowires)

Mediated Electron Transfer

Geobacter uses direct electron transfer pathways Shewanella uses both direct electron transfer and mediated pathways

Adapted from Shröder, U., Phys.Chem.Chem.Phys., 2007, 9, 2619-2629 Coursolle, D., Gralnick, J., Mol. Microbiol., 2010, 995

Lall, R., Mitchell, J., Bioinformatics, 2007. 23, 2754. El-Naggar, M.Y., et al., Proc Natl Acad Sci., 2010, 107(42), 18127; Gorby, Y.A., et al., Proc Natl Acad Sci, 2006, 103(30) 11358–11363

Fredrickson, JK., et al., Nat. Reviews: Microbiology, 2008, 592. Msh-type pilin

Flagellum

“nanowire”

Pil(IV)-type pilin 3D model of PilX

Helaine, S., et al., PNAS,2007, 104(40), 15888-15893 Selected Main Role Cell envelope Primary Annotation Summary 25 Total Selected Sub Role Surface structures Total genes: 5066 100.00% Type IV pilus biogensis protein 12 Protein coding genes: 4938 97.47% Pilin (includes type IV) 4 Genes assigned a role category: 2915 59.03% Curli production assembly/transport component 2 Flagellar biosynthesis protein 1 Conserved hypothetical genes: 864 17.49% Fimbrial protein 1 Hypothetical genes: 1159 23.47% Other 5

Selected Main Cellular processes Role 27 Total Selected Pathogenesis Selected Main Role Cellular processes 107 Total Sub Role Selected Sub Role Chemotaxis and motility Curli Subunit/ 4 Fimbrial biogenesis and twitching motility 1 Production Assembly Flagellin/flagellar associated proteins 43 MSHA biogenesis protein 12 Twitching motility protein 2 MSHA pilin protein 4 Other 61 Agglutination protein 1 Other 6 Information from http://cmr.jcvi.org Strain Description

MR-1 Wild-type; Lake Oneida isolate MR-1 ΔpilMNOPQ; type IV pili biogenesis ΔpilM-Q mutant MR-1 ΔmshHIJKLMNEGBACDOPQ; Msh pili ΔmshH-Q biogenesis mutant MR-1 mutant that lacks type IV and Msh pili ΔpilM-Q/ΔmshH-Q biogenesis genes Δflg MR-1 mutant that lacks the flagellin gene MR-1 mutant that lacks the decaheme ΔmtrC/ΔomcA cytochrome c complex, involved in metal oxide reduction

R.A. Bouhenni., et al., Electroanalysis, 2010, 22(7) 856-864 1. Biffinger, J. C.; Ray, R.; Little, B. J.; Fitzgerald, L. A.; Ribbens, M.; Finkel, S. E.; Ringeisen, B. R., Biotechnol. Bioengineering 2009, 103 (3), 524-531 2. Biffinger, J.; Ribbens, M.; Ringeisen, B.; Pietron, J.; Finkel, S.; Nealson, K., Biotechnol. Bioengineering 2009, 102 (2), Benefits: 436-444. Flexibility 3. Biffinger JC., Ribbens, M., Nealson, KH.; Designed for Long Duration Ringeisen, BR., 2010, “High-throughput Scalable to 96 wells (largest device 18 wells) Biological Screening Assay Using Voltage Gradients”, US 20100176005, Filed: January 2010.

Anodes

( - ) ( - ) ( - )

Marine Carbon Paper Epoxy PEM Cathode (Reference) ( + )

On average msh mutants generated less current than wild type and so possible candidate of “nanowire”. In the VBSA, biofilm formation clearly linked with msh pilin and would be attributable to lower current.

R.A. Bouhenni., et al., Electroanalysis, 2010, 22(7) 856-864 Strain Description

MR-1 Wild-type; Lake Oneida isolate MR-1 ΔmshH-Q ΔmshHIJKLMNEGBACDOPQ; Msh pili biogenesis mutant MR-1 ΔmshA-D; Msh pili ΔmshA-D structural mutant

Fitzgerald, L. A.; Petersen, E. R.; Ray, R. I.; Little, B. J.; Cooper, C. J.; Howard, E. C.; Ringeisen, B. R.; Biffinger, J. C. Process Biochemistry, 2012, 47 (1), 170-174. 1.4

1.2

1

0.8

WT OD 0.6 ΔmshH-Q ΔmshA-D

0.4

0.2

0 0 20 40 60 80 100 Time (hr)

Similar OD and growth characteristics observed across all Shewanella tested Msh A (major pilin subunit)

Msh B, Msh C, Msh D, Msh O (minor pilin subunit)

Msh Msh Outer Membrane Q Q Proteins Msh J Ms Ms Msh J h L h L

Msh K Msh K

Msh P Msh P Periplasm

Inner Msh I Msh I Membrane Msh Proteins N Msh N

Msh G Msh E Msh E Msh G Cytoplasm

Msh H Msh Msh Msh H M M

Demonstrate 3-6 month fully functional optical and water column sensor powered using only energy harvesting power sources at NRL Potomac River Dock starting June 2015.

FY11-12 FY09-11 FY13-15

Deliverables and Milestones Milestones - Bacterial ballast - Powering optical sensor with Deliverables : manipulation biological power source - Bacterial ballast control - Onboard electrical - Improved long term cathode - Powered and unpowered measurements materials for biological power source prototypes - First deployed hybrid - 10 fold increase in duration and MFC/EFC system sustainable power output from floating system Duration: 1 week Duration: 1-3 weeks Duration: 9-18 weeks Autonomous Sensor Platform for Coastal Security

Biologically-driven system that will support versatile, sustainable detection of:

Motion & acoustic signatures

Chemical & biological agents

Nuclear & radiological threats

• Zero power • Signature free • Unattended

Glenn R. Johnson Justin C. Biffinger Plamen Atanassov Successful Field Demonstration Trapped gas Thailand 2009-2010 Float/Payload (RF COMM, GPS, etc.) Gas vent hole

Weight

Fluid Vent Hole Tube connecting bacterial chamber with ballast chamber

Sealed container with H Gas Autonomous 2 headspace Surfacing & Re- Submerging Solid-Phase Bacterial Culture Gas-generating bacteria w/ sustainable, time- released nutrients

Tom Boyd, Joe Smith Tether (NRL, Navy Reservists) *Provisional U.S. Patent Filed, February 2009 • Zero power consumed to surface and submerge autonomous system • Operation of system left entirely to rate of gas production from bacteria Fundamentals for ballast control Emerald Raptor - 2011 Crimson Viper (2009-2010) Initial test of floating platform

Deployment of EFC/MFC in water column

P.K. Wu, L.A. Fitzgerald, J.C. Biffinger, B.J. Spargo, B.H. Houston, J. Bucaro, B.R. Ringeisen, “Zero-Power Autonomous System Controlled by Microbial Gas Production”, Review of Scientific Instruments, 2011, 82, 055108; doi:10.1063/1.3587623.; J.C. Biffinger, P.K. Wu, L.A. Fitzgerald, S.E. Lizewski, B.R. Ringeisen, “Advancements Toward a Zero-Power Autonomous Aquatic Littoral Sensor Framework”, 44th Power Sources Conference Proceedings, 2010, P- 9.; B.R. Ringeisen, P.K. Wu, J.C. Biffinger, B.J. Spargo, L.A. Fitzgerald. “Autonomous buoyancy control system” NC#99,745, US Patent Application# 20100199907, August 12 2010.; B. Spargo, J.C. Biffinger, B.R. Ringeisen, E.C. Howard, L.A. Fitzgerald, P.K. Wu, M. Molito, “Self refilling gas tanks for fuels Manipulating rate of surfacing using and ballast”, provisional submitted 5/10/2010 NC# 100,525. media viscosity 100% = 1.5% agar in water *NRL Memo Report, 2009 Unrestricted gas release Re-pressurization (Restricted)

Variable Agar Concentrations 1.5% Agar Gas Liberating Gel

Greatest generated: 24 hrs, 30C, 56 psi

Biffinger,JC., et al., Applied Microbiology and Biotechnology, 2012, doi: 10.1007/s00253-012-4296-5.

Time (min) 0 20 40 0.0 30 0.5 29

1.0 0.37% Agar

C) ° 1.5 28 2.0 1.50% Agar 2.5 27 Depth (m) Depth 3.0

26 ( Temperature 3.5 4.0 Temperature 25 4.5 100% = 1.5% agar in water 5.0 24

Biffinger,JC., et al., Applied Microbiology and Biotechnology, 2012, doi: 10.1007/s00253-012-4296-5. Booster circuit: HOBO data logger MFC Vout/2 0 - 0.7 V Capable of boosting the MFC voltage to Vout Input resistance isolated from MFC > 10 V. Loading circuit (see below) keep Resolution = 1.2 mV/step output voltage from 2.8 to 3.0 V

Synapse Wireless Engine Follower: Input resistance isolated from MFC Increase the internal resistance to > 13 MΩ Resolution = 5 mV/step Conditioning: Control load Divide Booster output voltage by 2. To load applied when V > match the resolution for the HOBO and out 3.0 V Synapse engine. load removed when V > Load: Load Control out 2.8 V Load (a resistor) applied when Vout is within the operation voltage range: External Battery (3.0 V) 2.8 < V < 3.0 V out Two-way wireless link

Beach based computer External Battery (4.5 V)

Deployed Stacked Hybrid Enzyme/Microbe Fuel Cells on floating platform Charge/Discharge of capacitors Wireless communication with system for data download . Autonomous long duration sensors will require the highest energy efficiency standards in order to integrate MFCs . MFCs should be engineered toward a specific application . Fabricated DC/DC Booster circuited designed for MFCs . Demonstration of completely autonomous sensor platform with minimal electronic and acoustic signature In Press Publications 1. Fitzgerald, L.A., Petersen, E.R., Leary, D.H., Nadeau, L.J., Soto, C.M., Ray, R.I., Little, B.J., Ringeisen, B.R., Johnson, G.R., Vora, G.J., Biffinger, J.C., “Shewanella frigidimarina microbial fuel cells and the influence of divalent cations on current output,” Biosensors and Bioelectronics, 2012 (Special Issue for Biosensors 2012) . doi:10.1016/j.bios.2012.06.039., 2. Fitzgerald, LA.; Petersen ER.; Gross BJ.; Soto CM.; Ringeisen BR.; El-Naggar MY.; Biffinger JC. Aggrandizing power output from Shewanella oneidensis MR-1 microbial fuel cells using calcium chloride. Biosensors & Bioelectronics 2012, 31 (1), 492-8. 3. Fitzgerald, L. A.; Petersen, E. R.; Ray, R. I.; Little, B. J.; Cooper, C. J.; Howard, E. C.; Ringeisen, B. R.; Biffinger, J. C. “Shewanella oneidensis MR-1 Msh pilin proteins are involved in extracellular electron transfer in microbial fuel cells”. Process Biochemistry, 2012, 47 (1), 170-174.

Patent Biffinger JC., Ribbens, M., Nealson, KH.; Ringeisen, BR., 2010, “High-throughput Biological Screening Assay Using Voltage Gradients”, US 20100176005, Filed: January 2010.

Presentations 1. [KEYNOTE PRESENTATION] Biffinger, JC., Johnson, GR., Atanassov, PB., Luckarift, HR., Strack, G., Sizemore, SR , Nichols, R., Farrington, KE., Lau, C., Wu, PK., Ringeisen, BR., Petersen, ER., Fitzgerald, LA., “Emerald Raptor: Autonomy Derived From Microbiology”., Biosensors 2012, Cancun, Mexico., May 15-18, 2012. 2. Luckarift, HR, Strack, G, Sizemore, SR, Nichols, R, Farrington, K., Lau, C, Wu, PK, Atanassov, P, Biffinger, JC, Johnson, GR, Power generation of a hybrid microbial/enzymatic biological fuel cell operating in seawater, 221st National Meeting (2012), May 6-12, 2012 ECS-1464. 3. Petersen, E.R., Fitzgerald, L.A., Ringeisen, B.R., Biffinger, J.C., “Influence of Calcium Chloride with Shewanella oneidensis MR-1 Microbial Fuel Cells,” 111th General Meeting of the American Society for Microbiology, New Orleans, LA, 21-24 May 2011. • Prof. Charles Lieber • Xiaocheng Jiang • Funding provided by Air • Jinsong Hu Office of Scientific • Prof. Steven Finkel Research and the Office of Naval Research through NRL 6.2 BLK •GRJ (AFRL) for funding expenses and travel for Emerald Raptor I and II exercise