Running an MFC Experiment

This page is intended as a comprehensive guide to completing a microbial fuel cell setup and running an experiment from start to finish.

Creating a Testing Environment

Begin 1-2 weeks prior to experiment

Constructing Fuel Cell Components

Materials (per fuel cell)

• 4" Polycarbonate Square Tube, 2" Outer Diameter
• 6" x 6" Polycarbonate Sheet, 1/4" Thick
• 4 Steel Fully Threaded Stud, 1/4"-20 Thread, 6" Length
• 8 Zinc Alloy Wing Flange Nut, 1/4"-20 Screw Size, 1" Wing Spread
• 1" x 1" Nafion® membrane, 0.180mm thick
• 1" x 1" Carbon felt, 0.25" thick
• 1.5" x 1.5" E-TEK ELAT™ GDE (platinum on carbon)
• 2' Titanium Grade 2 Wire .046" Diameter
• Teflon Tape, 1/4" Width
• 5" x 2.5" Silicone Sheet
• Silicone Glue
• Spiral Point Tap 1/4"-28
• 8 Plastic Luer Lock Coupling Nylon, Female to Male Thread, 1/4"-28
• 8 Luer Lock Injection Ports

Procedure

1) Mill Polycarbonate

• Cut polycarbonate sheet into 4 equal 3" x 3" pieces
• Drill four 3/8" holes through each piece, 1 per corner, indented 5mm from both sides
• Drill a 1/2" hole in the center of each piece

• Cut polycarbonate tube into two equal 2" halves
• Drill four 1/4" holes through each half in configuration shown

• Tap each hole with 1/4" -28 spiral tap

2) Glue Chambers (repeat for each half)

• Center tube on endplate by marking plate with 'X' from corner to corner
• Squirt 2mm thick line of silicone on edge of tube (edge furthest from holes)
• Press tube firmly against marked location on endplate
• Quickly spread excess silicone along edge
• Let stand 24h to harden

3) Construct Gaskets

• Cut silicone sheet into two equal 2.25" x 2.25" pieces
• Cut out centered inner squares in each piece, 1.75" x 1.75"
• Using inner squares, cut two 'O' rings, inner diameter 1/4", outer diameter 1/2"

4) Construct Electrodes

• Cut titanium wire into one 8" piece and one 16" piece
• Using pliers, shape anode and cathode as shown

Anode Frame

Cathode Frame

• Spear carbon felt with tip of anode titanium wire and wedge into frame
• Weave platinum carbon cloth through cathode titanium wire

Anode

Cathode

5) Seal Injection Ports

• Wrap threads of all eight Luer Lock screws with 1' of teflon tape in opposite direction of screwing
• Screw Luer Locks into all tapped holes in both chambers

Setup of Measurement Device

Materials

• Keithley 2700 Digital Multimeter
• Keithley 7700 Multiplexer
• Small Breadboard
• Supply of insulted thin copper wire
• 470 Ohm resistors (one/fuel cell)

Procedure 1) Wire Multiplexer

• Open multiplexer, note channels
• Cut two wire 18" wire leads per fuel cell
• Strip ends, place one wire in each screw terminal, screw tight
• Tape paired wires (two are attached to each channel) near non-attached ends and label
• Clamp wire bundles near back of device with provided plastic latch clamps
• Close Multiplexer and slide into 2700 DMM

2) Create Resistor Array

• Connect resistors across middle of breadboard (one per fuel cell)
• Connect leads from multiplexer across resistors (one pair across each resistor)

Controlling the DMM with LabVIEW™

1. Initialize Multimeter
   • Attach 2700 to COM1 port of desktop computer w/ LabVIEW™
   • Download our LabVIEW™ source code MFCs.vi
   • Open Program in LabVIEW™, adjust block diagram as necessary

Experiment Preparation

Begin 1 day prior to experiment

Assembling Chambers

Procedure
1) Prepare Electrodes

• Attach Luer Lock injection ports to all chamber screws
• Poke tip of electrodes through designated ports from the inside

2) Align Chambers

• Lay one chamber on a flat surface
• Place silicone square ring on top edge of tube
• Place polycarbonate square on silicone
• Place silicone 'O' ring around central pore
• Place Nafion membrane on top of 'O' ring
• Sandwich membrane between second 'O' ring
• Align second polycarbonate square on top of 'O' ring
• Center second silicone square ring on polycarbonate square
• Set second chamber on top of silicone, ensuring ports facing same direction as first chamber

3) Clamp Chambers

• Move assembly into vice or clamp
• Insert rods through holes in end plates and screw on wing nuts
• Tighten evenly

Solutions Prep

1. Chamber media (150ml / fuel cell)
   • 5.844 g/L 100mM NaCl
   • 15.1185 g/L 50mM PIPES (hydrogen)
   • 7.0 pH
2. Phosphate buffer (60ml / fuel cell)
   • 2.918 g/L Monosodium phosphate, monohydrate
   • 4.095 g/L Disodium phosphate, anhydrous
   • 5.844 g/L 100mM NaCl
   • 7.0 pH
3. Resazurin solution
   • 0.489 g/L 0.8mM Resazurin
4. Lactate Solution
   • 13.51 g/L Lactic acid
   • neutralize to pH 7 with NaOH

Gas Tubing Assembly

Materials

• 25' Silicone Soft Rubber Tubing, 3/32" ID, 7/32" OD, 1/16" Wall
• Tank of Compressed Nitrogen
• Gas Regulator
• Lab Supply of Air
• 4 Plastic Luer Lock Coupling Nylon, Male X Barb, for 3/32" Tube
• 4 Plastic Luer Lock Coupling Nylon, Female X Barb, for 3/32" Tube
• Plastic Luer Lock Coupling Nylon, T junctions, for 3/32" Tube
• Syringe needles - 27 gauge
• 2 Aspirator Flasks
• 2 Rubber Stoppers

Procedure 1) Make Flow Regulators

• Insert nozzle of female Luer Lock into rubber stoppers (poke hole if necessary)
• Cap aspirator flasks with rubber stoppers
• Attach tubing from gas sources to each glass nozzle of aspirator flask

2) Make Manifolds

• Attach T-junction Luer Lock pieces into manifold (1 junction/ fuel cell ; 2 manifods total)
• Turn last juction such that off is facing end of manifold
• Attach tubing from stopper of flow regulators to beginning of each mainfold

Growing Strains

Materials

• 150mL LB / strain
• 1000mL airating flask / strain
• Antibiotics (if desired strain must be selected for)
• Plate or Glycerol Stock with desired strain

Procedure

1. Fill flasks with LB
2. Add correct concentration of selection antibiotic
3. Pick single colony from plate, add to flask
4. Incubate overnight in shaker at 30C

Runtime

Begin 2 hours prior to experiment

Bacteria

Procedure

1. Pipet the cells out of the flask and into 250mL centrifuge containers
2. Make sure the containers are close in weight (within 0.5g of each other)
3. For a culture more than 250mL split it into two containers
4. Set the centrifuge temperature to 22-23C, spin speed to 5000RPM, and time to 15 min
5. After first spin, drain each container of the LB, making sure to leave the bacteria pellet intact
6. Resuspend pellet in 50mL of potassium buffer
7. After bacteria are fully resuspended (no pellets at all), spin down again, 22-23C, 5000RPM, 15min.
8. Pour potassium buffer out of container slowly.
9. Resuspend pellet in 50mL of potassium buffer
10. Spin down again, 22-23C, 5000RPM, 15min.
11. Pour potassium buffer out of container slowly.
12. Resuspend pellet in 4 mL of sodium pipes
13. Check OD (100microliters in 15mL or 1:150 dilution)
14. If OD is in linear range, calculate dilution for desired quantity of bacteria in 1mL (typically 10^8 cells /mL of chamber media)
15. Repeat dilution if not in linear range (using different ratio)
16. Inject 1mL of bacteria into each chamber (see below)

Fuel Cells

Procedure

1. Pipet 75mL of NaPiPES solution into each side of fuel cells
2. Inject 1mL of Resazurin solution into each side of chamber
3. Using Luer Lock nozzles, connect tubing from top ports to beaker w/ distilled water
4. Cut tubing to span distance from manifolds to each fuel cell (1 from nitrogen, 1 from air)
5. Attach syringe needles to tubing via Luer Lock nozzles
6. Using Luer Lock nozzles, connect tubing from manifolds
7. Start gas flow
8. Poke needles through bottom ports on fuel cells

Measurements

Procedure

1. Turn on computer and digital multimeter
2. Open LabVIEW program
3. Click Run arrow to take resistance readings
4. Connect fuel cells to resistor array via alligator clips
5. Click "Begin Current Readings" Icon on instrument display

Injections/Variables

Procedure

1. Once current readings reach equilibrium, inject bacteria into fuels cells using syringes
2. Allow bacteria to consume any carbon sources left in their media (approximately 12 hours)
3. Once current levels reach stable baseline, inject 1ml lactate solution
4. Inject additional variables as desired

Clean Up

Procedure

1. Drain chambers and soak in 70% ethanol
2. Remove carbon felt from anodes and discard (save titanium)
3. Scrub all parts in ethanol and distilled water successively
4. Use pipe cleaners on ports and tubes