Oxygen Evolution Measurement: Light Curve for Algae

Steven J Burgess, Chandra Davies

Published: 2022-08-03 DOI: 10.17504/protocols.io.q26g74no9gwz/v1

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

Before start

Have a checklist - (1) water jacket, (2) stirrer, (3) lid as a reminder to do these steps each time

Steps

Prepare 0.5M NaHCO3 Solution

Prepare 0.5 M NaHCO₃ Solution

1.1.

Dispense 10mL of dH₂O into a 50 mL centrifuge tube

1.2.

Weigh out 0.420g of sodium bicarbonate

1.3.

Add 0.420g of sodium bicarbonate to the dH₂O

1.4.

Close lid and invert until everything is dissolved

Prepare the electrolyte

2.1.

Weigh out 17.5g of anhydrous KCl salt

2.2.

Pour the salt into a 500mL glass bottle

2.3.

Measure 100mL of dH₂O

2.4.

Pour the water into the same bottle as salt

2.5.

Close the lid and swirl the bottle with mild aggression until salt is dissolved

Clean and Prepare the electrode

3.1.

Start the water jacket going at 25 ^oC so it’s ready by the time your electrode is prepared

3.10.

Use the membrane applicator to secure the small O-ring over the membrane and cigarette paper at the base of the electrode

3.11.

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.

3.12.

Tuck any extra edges of membrane into the well using forceps so it doesn't touch the larger O-ring

3.13.

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).

3.14.

Open O2View program on laptop

3.15.

Add port cover blanks to 3 of the open ports and insert the red light to the 4^th open port.

3.16.

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).

3.2.

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

3.3.

Circle the inside of the well with the polish

3.4.

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.

3.5.

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.

3.6.

Use a disposable Pasteur pipette to add a drop of electrolyte to the top of the platinum electrode.

3.7.

Add 3-5 drops of water so that the silver band inside the well is completely covered.

3.8.

Cut a 1.5-2 cm² piece of thin cigarette paper to the top of the electrode.

Note

Recommended brand: Rizla T ultra fine

3.9.

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

Light Calibration

Note

Needed for light curve, and the light curve needs to be done before the bicarbonate curve

4.1.

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.

4.2.

In the O2View program, in the upper menu bar, click Calibrate > calibrate light (automatic)

4.3.

This should take a few minutes but it will do it itself.

Electrode Calibration

Electrode calibration

5.1.

Add 1mL of ASW to the electrode chamber

Note

There should also be one of the small (tiny) magnetic stirrers already be inside

5.2.

Insert plunger (hollow lid-like contraption with glass on one end. Glass end goes into the chamber)

5.3.

Calibrate > Liquid phase calibration (manual)

5.4.

Follow the prompts that pop up on the screen

5.5.

Use their default 25 μmol/mL oxygen concentration setting.

Note

Previously entered 6.75 ppm based on 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. chart and our use of ASW for these samples, still a little unsure about which is the most accurate.

5.6.

Enter stirrer speed of 75

5.7.

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.

5.8.

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

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.

6.1.

In a flow hood, pour 25mL of a healthy culture into a 50 mL centrifuge tube

Note

"Healthy" is entirely subjective but generally suggests a more vibrant yellow-green

6.10.

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.

6.2.

Prepare a balance with another tube of 25mL of dH₂O

6.3.

Centrifuge for 0h 5m 0sat 5000g and 20^oC

6.4.

Discard the supernatant by micropipetting

6.5.

Resuspend in 10mL 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.

6.6.

Balance with dH₂O again in centrifuge for 0h 5m 0s, 5000g, 20 ^oC

6.7.

Discard the supernatant

6.8.

Add 1mL ASW to the pellet

6.9.

Swirl the tube gently

PFD Table Set up for Light Curve

Photon Flux Density Table Set up for Light Cruve

7.1.

Remove ASW from chamber

7.10.

When program is done, turn off stirrer

7.11.

View QY Data tab and save the file

Citation

Graph pictured above shows 400-800umols of light as being the best for O. tauri photosynthesis.

7.2.

Make sure the stirrer is still going (can turn off while removing ASW)

7.3.

In the top menu bar, click Hardware > New PFD table

7.4.

Select auto

7.5.

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.

7.6.

Add 900µL of culture

7.7.

Add 100µL of NaHCO₃ solution

7.8.

Place the plunger into the chamber

7.9.

Click "Go" on PFD window

Clean Up

8.1.

Remove the sample from the electrode chamber using a pasteur pipette

8.10.

Rinse the electrode disc with dH₂O

8.11.

Rinse the magnetic flea with dH₂O

8.12.

Make sure everything is gently and thoroughly dried, use a kimwipe for the electrode disc.

8.13.

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.

8.2.

Make sure everything is turned off (stirrer, light)

8.3.

Take out and put away the optical port blanks

8.4.

Take out the light

8.5.

Unscrew the bottom of the electrode chamber, careful not to let the electrode and magnetic follower fall

8.6.

Pull out the electrode disk, catch the magnetic follower

8.7.

Rinse the inside of the electrode chamber with dH₂O