Aug 02, 2022
Oxygen Evolution Measurement: Light Curve for Algae
- 1University of Illinois at Urbana-Champaign;
- 2PRECS;
- 3Contra Costa College
Protocol Citation: Steven J Burgess, Chandra Davies 2022. Oxygen Evolution Measurement: Light Curve for Algae . protocols.io https://dx.doi.org/10.17504/protocols.io.q26g74no9gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 21, 2022
Last Modified: July 11, 2023
Protocol Integer ID: 65055
Keywords: Oxygen electrode, light curve, Ostreococcus tauri, Hansatech, Oxygraph+, Photosynthesis
Abstract
Thanks to a lovely combination of trial and error I present to you an optimized* protocol for using an oxygen electrode to create a light curve for Ostrecoccus tauri. The light curve can be used to determine what light intensity is ideal for photosynthesis in O. tauri, which can then inform the light conditions for a subsequent bicarbonate curve. An O. tauri bicarb curve could potentially indicate (when compared to other algal bicarb curves) whether or not this species has a carbon-concentrating mechanism.
*This protocol could be further optimized by making a growth curve for O. tauri first.
*The protocol is written for O. tauri and can be adapted to other algal species. When adapting it will be necessary to reassess optimal cell density for measurements, and consider the choice of buffer for washing and incubating cells. ASW is used in this situation but will not be suitable for fresh water species
Materials
- Hansatech Oxygen Electrode Kit, which should include:
- OXYG1+ - Oxygraph+ electrode control unit
- DW1 - Oxygen electrode chamber
- S1 - Oxygen electrode disc
- S1/SMB - SMB-SMB Electrode cable
- A2 - Electrode disc membrane applicator tool
- A3 - Top plate key & alignment jig
- S2/P - Pack of 5 magnetic followers
- S4 - 30m reel of PTFE membrane
- S7A - Spare O-rings set for DW1/AD
- S6B Assorted Spare ‘O’ rings for DW2/2
- S16 - Electrode cleaning kit
- Rapid electrode disc polish
- USB2.0 A to B - 2m USB cable with A-plug to B-plug
- DW1MAN - User Manual
- 980266T - 12V Power Supply
- 980257 - DW Accessory Kit
- Auxiliary red light
- Port cover blanks
- Ultra-fine cigarette paper (Rizla T. Thin Blue)
- Cotton buds (small enough to fit in the well of the disc)
- Sharp scissors
- Forceps
- Gloves
- Paintbrush (small, the kind you'd use for painting precise lines)
- dH2O
- Kimwipes
- 1 mL Pasteur pipettes
- Laptop with Oxygraph+ program O2View installed
- Electrolyte solution (prepare from KCl anhydrous salt and de-ionized water)
- Water Jacket
- Circulating water bath
Before start
Have a checklist - (1) water jacket, (2) stirrer, (3) lid as a reminder to do these steps each time
Prepare 0.5M NaHCO3 Solution
Prepare 0.5M NaHCO3 Solution
Prepare 0.5 M NaHCO3 Solution
Dispense 10 mL of dH2O into a 50 mL centrifuge tube
Weigh out 0.420 g of sodium bicarbonate
Add 0.420 g of sodium bicarbonate to the dH2O
Close lid and invert until everything is dissolved
Prepare the electrolyte
Prepare the electrolyte
Prepare the electrolyte
Weigh out 17.5 g of anhydrous KCl salt
Pour the salt into a 500 mL glass bottle
Measure 100 mL of dH2O
Pour the water into the same bottle as salt
Close the lid and swirl the bottle with mild aggression until salt is dissolved
Clean and Prepare the electrode
Clean and Prepare the electrode
Clean and Prepare the electrode
Start the water jacket going at 25 oC so it’s ready by the time your electrode is prepared
Dampen a cotton bud with dH2O and use it to pick up a thin layer of Hansatech Rapid Electrode Disc Polish to clean the electrode
Circle the inside of the well with the polish
Polish the platinum electrode as well.
Note
Try not to polish the epoxy resin surrounding the electrode as it can alter the shape of the dome, thus affecting accuracy.
Use a clean, dH2O-dampened cotton bud to remove the residual polish. A kimwipe can be used to dry the electrode after this step, too.
Use a disposable Pasteur pipette to add a drop of electrolyte to the top of the platinum electrode.
Add 3-5 drops of water so that the silver band inside the well is completely covered.
Cut a 1.5-2 cm2 piece of thin cigarette paper to the top of the electrode.
Note
Recommended brand: Rizla T ultra fine
Cut a slightly larger square of the provided PTFE membrane tape and place it on top of the paper.
Note
If the membrane tape is a little dusty, run it between your thumb and index fingers gently to try and wipe off any debris
What electrode looks like after this step
Use the membrane applicator to secure the small O-ring over the membrane and cigarette paper at the base of the electrode
Membrane applicator with o-ring
Place the large O-ring in the indentation outside the well
Note
Make sure this is one of the thicker large o-rings, otherwise your sample will leak out of the electrode chamber and your curve will be less curvey and more zig-zaggy.
Tuck any extra edges of membrane into the well using forceps so it doesn't touch the larger O-ring
Fully set up electrode disc
Attach the cord that connects the control unit to the electrode, then fit the disc into the bottom of the electrode chamber (pictures in steps 4.1-4.2).
Open O2View program on laptop
Add port cover blanks to 3 of the open ports and insert the red light to the 4th open port.
Insert the light sensor to the top opening of the electrode chamber, ensuring the circular patch is facing the light source (pictures in steps 4.1-4.2).
Light Calibration
Light Calibration
Light Calibration
Note
Needed for light curve, and the light curve needs to be done before the bicarbonate curve
Ensure both the light and light sensor are inserted to their appropriate ports, and that the light sensor is actually facing the light
Electrode chamber before light sensor and electrode disc are in place
Electrode chamber with electrode disc fastened in place and light sensor being inserted to the chamber.
In the O2View program, in the upper menu bar, click Calibrate > calibrate light (automatic)
This should take a few minutes but it will do it itself.
Electrode Calibration
Electrode Calibration
Electrode calibration
Add 1 mL of ASW to the electrode chamber
Note
There should also be one of the small (tiny) magnetic stirrers already be inside
Insert plunger (hollow lid-like contraption with glass on one end. Glass end goes into the chamber)
Calibrate > Liquid phase calibration (manual)
Follow the prompts that pop up on the screen
Use their default 25 μmol/mL oxygen concentration setting.
Note
Previously entered 6.75 ppm based on this chart and our use of ASW for these samples, still a little unsure about which is the most accurate.
Enter stirrer speed of 75
Continue to follow prompts:
Note
Don't click "OK" on prompt 4 of 5 until you have your zeroing chemical ready. Nitrogen gas was the preferred method of zeroing the oxygen due to its availability and lessened risk of affecting any subsequent measurements (unlike Sodium Dithionite, which can also be used). The nitrogen tank is intimidating and can take a few tries to get used to.
Nitrogen gas was introduced to the chamber from a large tank at the end of the bench through a tube with a 1000 μL pipette tip attached. The pipette tip goes directly into the ASW in the electrode chamber, preferably without jamming it into the electrode disc. Leave it in until the signal has plateaued and the calibration is saved.
Prepare Light Curve Culture Samples
Prepare Light Curve Culture Samples
Preparing Light Curve Culture Samples
Note
It's suggested to prepare each sample right before performing a light curve on it, rather than preparing them all together and having some sit out for longer.
In a flow hood, pour 25 mL of a healthy culture into a 50 mL centrifuge tube
Note
"Healthy" is entirely subjective but generally suggests a more vibrant yellow-green
Prepare a balance with another tube of 25 mL of dH2O
Centrifuge for 00:05:00 at 5000 g and 20oC
5m
Discard the supernatant by micropipetting
Resuspend in 10 mL of ASW by gently disturbing the pellet with a small paintbrush
Note
Too large of a paintbrush will absorb all your cells and media. It can be done, but it is alarming.
If you only have a large paintbrush and it does eat your cells, you can try adding another 1.5mL directly onto the brush to sort of wash the cells off. It might not look convincing, but it should allow you to have enough left for a test.
Balance with dH2O again in centrifuge for 00:05:00 , 5000 g , 20 oC
5m
Discard the supernatant
Add 1 mL ASW to the pellet
Swirl the tube gently
Use the paintbrush again to resuspend. Press the paintbrush against the inside of the tube when pulling it out to try and save as much of the sample as possible.
PFD Table Set up for Light Curve
PFD Table Set up for Light Curve
Photon Flux Density Table Set up for Light Cruve
Remove ASW from chamber
Make sure the stirrer is still going (can turn off while removing ASW)
In the top menu bar, click Hardware > New PFD table
Select auto
Adjust so the following intensities of light (umoles) are selected for 1 minute each: 0, 5, 20, 40, 80, 120, 200, 400, 800, 1600. Click save if you need to close the window, otherwise leave the window up until you clock "go."
Note
This screenshot is from before time was adjusted to 1 min. 2 mins tends to result in a flatline because it allows too much oxygen to build up in the chamber.
Add 900 µL of culture
Add 100 µL of NaHCO3 solution
Place the plunger into the chamber
Click "Go" on PFD window
When program is done, turn off stirrer
View QY Data tab and save the file
Expected result
Graph pictured above shows 400-800umols of light as being the best for O. tauri photosynthesis.
Clean Up
Clean Up
Clean Up
Remove the sample from the electrode chamber using a pasteur pipette
Make sure everything is turned off (stirrer, light)
Take out and put away the optical port blanks
Take out the light
Unscrew the bottom of the electrode chamber, careful not to let the electrode and magnetic follower fall
Pull out the electrode disk, catch the magnetic follower
Rinse the inside of the electrode chamber with dH2O
Take the O-rings off the electrode and rinse them with dH2O
Take off the membrane and paper from the electrode and dispose of them in the bio-waste bin
Rinse the electrode disc with dH2O
Rinse the magnetic flea with dH2O
Make sure everything is gently and thoroughly dried, use a kimwipe for the electrode disc.
Repeat the same polish process as done at the beginning on the electrode/electrode disc. It's important to clean the electrode disc thoroughly right after you're done using it to prevent any tarnish buildup, which can happen after one day of use. The electrode disc should also be stored with a dehydration packet.