Feb 25, 2023
Measuring photophysiological traits of diatoms from Rapid Light Curves using a Water-PAM
- Phoebe Argyle1,2,
- Jana Hinners3,
- Nathan G. Walworth4,
- Sinead Collins5,
- Naomi M. Levine4,
- Martina A. Doblin1,6
- 1Climate Change Cluster, University of Technology Sydney, Ultimo NSW Australia;
- 2Ministry of Marine Resources, Cook Islands;
- 3Institute of Coastal Ocean Dynamics, Helmholtz-Zentrum Hereon, 21502, Geesthacht, Germany;
- 4Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089-0371;
- 5Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, UK;
- 6Sydney Institute of Marine Science, Mosman, NSW Sydney
Protocol Citation: Phoebe Argyle, Jana Hinners, Nathan G. Walworth, Sinead Collins, Naomi M. Levine, Martina A. Doblin 2023. Measuring photophysiological traits of diatoms from Rapid Light Curves using a Water-PAM . protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq4zmovk5/v1
Manuscript citation:
Argyle, P.A., Hinners, J., Walworth, N.G., Collins, S., Levine, N.M., Doblin, M.A., 2021. A High-Throughput Assay for Quantifying Phenotypic Traits of Microalgae. Frontiers in Microbiology 12(2910).
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: August 25, 2021
Last Modified: February 25, 2023
Protocol Integer ID: 52690
Keywords: diatom, microalgae, photophysiology, fluorometry, traits
Funders Acknowledgement:
Moore Foundation Marine Microbes Initiative
Grant ID: MMI 7397
Abstract
This is a brief protocol for how to measure ETRmax, alpha (α) and Ik for microalgae cultures using the Rapid Light Curve protocol included in the WinControl software used with a Walz Water-PAM instrument.
Based on the protocol developed in:
Ralph, P.J., Gademann, R., 2005. Rapid light curves: a powerful tool to assess photosynthetic activity. Aquat. Bot. 82(3), 222-237.
This method was used in:
Argyle, P. A., Walworth, N. G., Hinners, J., Collins, S., Levine, N. M., & Doblin, M. A. (2021). Multivariate trait analysis reveals diatom plasticity constrained to a reduced set of biological axes. ISME Communications, 1(1), 59.
Argyle, P. A., Hinners, J., Walworth, N. G., Collins, S., Levine, N. M., & Doblin, M. A. (2021). A high-throughput assay for quantifying phenotypic traits of microalgae. Frontiers in microbiology, 12, 706235.
Image Attribution
Michaela Larsson, UTS Sydney
Materials
Equipment
Water-PAM
NAME
PAM fluorometer
TYPE
WALZ
BRAND
WATER-PAM-II
SKU
LINK
Prepare culture
Prepare culture
Take a 2 mL aliquot of microalgae culture into a glass cuvette compatible with the WATER-PAM instrument.
When the overall culture volume is limited (i.e. <2 mL of culture available for the measurement), take 0.5-1 mL and dilute with sterile seawater to a final volume of 2 mL .
Notes:
The optimal volume for the WATER-PAM is 2-3 mL .
Make measurements
Make measurements
Set up the WATER-PAM according to the manufacturer's instructions.
Equipment
Water-PAM
NAME
PAM fluorometer
TYPE
WALZ
BRAND
WATER-PAM-II
SKU
LINK
Initiate a Rapid Light Curve by clicking Program > Light Curve in WinControl.
To achieve a successful RLC, there should be at least 3 points on the rise, multiple points to estimate the ETRmax, and at least one point on the decline at the highest irradiance (see instrument manual).
If the curve does not achieve these criteria, this may require optimization of the instrument settings such as the gain and irradiance steps.
Modify the Light Curve Intensity (LI) to change the first irrandiance step of the curve, keeping in mind there will always be 8 steps to the curve.
Gain settings should be adjusted by the user, if Fo or Fm exceeds the maximum value (>4000) then reduce the gain, or dilute the sample.
NB: Do not adjust the gain settings in between samples as this renders the data incomparable.
The curve width is usually set to 10 seconds.
NB: If comparing different cultures, it is important to conduct all measurements within a similar time window based on the photoperiod. I.e. within a 1-2 hour window 2 hours after the onset of light.