Team:Harvard/Hardware/MFCProcedure

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{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%" =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 Anode Frame Cathode Frame
 * Cut titanium wire into one 8" piece and one 16" piece
 * Using pliers, shape anode and cathode as shown

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

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
 * 2) * Attach 2700 to COM1 port of desktop computer w/ LabVIEW™
 * 3) * Download our LabVIEW™ source code [[Media:MFCs.txt|MFCs.vi]]
 * 4) * Open Program in LabVIEW™, adjust block diagram as necessary

Experiment Preparation
Begin 1 day prior to experiment

Assembling Chambers
Procedure 1) Prepare Electrodes 2) Align Chambers 3) Clamp Chambers
 * Attach Luer Lock injection ports to all chamber screws
 * Poke tip of electrodes through designated ports from the inside
 * 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
 * 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)
 * 2) * 5.844 g/L 100mM NaCl
 * 3) * 15.1185 g/L 50mM PIPES (hydrogen)
 * 4) * 7.0 pH
 * 5) Phosphate buffer (60ml / fuel cell)
 * 6) * 2.918 g/L Monosodium phosphate, monohydrate
 * 7) * 4.095 g/L Disodium phosphate, anhydrous
 * 8) * 5.844 g/L 100mM NaCl
 * 9) * 7.0 pH
 * 10) Resazurin solution
 * 11) * 0.489 g/L 0.8mM Resazurin
 * 12) Lactate Solution
 * 13) * 13.51 g/L Lactic acid
 * 14) * 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


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