I daily measure Fv/Fm value in several outdoor Nannochloropsis gaditana cultures. I found that measuring quantum yield after sampling all the cultures (i.e. samples are taken inside the lab) leads to an higher quantum yield than sampling and measuring each culture one by one (i.e. following sample still receives solar light). Moreover I still can not find a precise indication on how much should dark-adaptation time be. I read about 20 min. but I found also 5 min. Thank you.
Hi Milan,
Bringing the samples into the lab and letting them dark adapt for 20 minutes or so, and giving them small amounts of far red light during the last 10 minutes of dark adaptation will give you the highest Fv/Fm value. As the far red will oxidize the quinone pool, giving you a higher Fv value.
If you take the measurement while the cells are in the light, you are essentially measuring PhiII, which is a steady state measurement of quantum yield. I suppose that your methods should be adapted to the specific question you are trying to address...which is what?
Cheers
Ben
Hi Ben
Thanks for your quick answer. I want to measure maximum potential efficiency of PSII in dark adapted sample, that is Fv/Fm, if I'm not wrong. By the way maybe I did not explained myself clearly: when I take the sample and measure directly the QY outside, I still let it 5 min in the dark, otherwise I would have measured light adapted QY.
The difference in my experience is that if take all the samples (at noon) from the different photobioreactors inside the lab I get much higher QY than measuring them outdoor on by one with the same identical method. It seems like, and maybe it makes sense, that in a low light environment such as the lab, PSII recovers efficiency, as in the field Fv/Fm exhibits diurnal depression that are almost symmetric to light intensity. But I have read that usually lowest QY value does not differ more than 15% of the maximum one.
In one case for example I got 0.49 for the indoor measure and 0.14 for the outdoor one. It seems too much difference to me.
Cheers
Claudio
Hi Claudio, 0.49 is still a relatively low QY, in the whole scheme of things. There are so many parameters that can effect this that it is hard to say why your QY is low.
Firstly, are you using steady state cultures, are they in mid log phase, or stationary.
do you put them into fresh media for the measurements?
Do you give them far red light during the dark incubation?
For your outdoor culture, do you get a higher QY if you take them right out of the pond than if you let them dark incubate?
These are a start.....
What instrument do you use for your measurements?
Hi Ben, yes you are right 0.49 is still relatively low. This was obtained at noon (in Almeria, Spain) so I don't know if it can be an acceptable value. The same sample taken in early morning (7:30-8:00) gave 0.64.
To answer your questions:
I'm working with steady states cultures.
I do not put them in fresh medium, I just use directly a sample of the culture
I do not give them far light during dark incubation (I use a Photon Systems Instruments AquaPen-C AP-C 100), I just put the cuvette with the sample inside the instrument, wait 5 min and read.
I did not measure light adapted samples. Tomorrow I will try that and see the difference
Thank you very much for your help
Claudio
I completely agree with all of Bens suggestions above.
I would say that 0.49 is a pretty high Fv/Fm actually ,if the samples were taken at mid day in Spain and dark adapted for 5 minutes only. Taking samples for Fv/Fm at mid-day is probably not a very good idea, there are components of NPQ which can be hard to relax, even if you use far-red light.
I would definitely recommend to take those samples in the morning (at night would be best).
Hi Nina
Thanks for your reply and suggestions. I am taking samples from the morning to mid day every hour to see changes in QY with irradiance increase.
Claudio
You will definitely see changes, even after some dark adaptation. There is quite a bit of literature on this with underway measurements in the ocean. Just search for "diurnal changes in Fv/Fm" and you should find a bunch of papers by Mike Behrenfelds lab. There is a really nice lab study by Allan Milligan et al I think in 2012 or 2013, which explains all the different NPQs really well.
Nina, thank you for your contribution. Yes I guess I will see changes but that's what I want because I am using different cultivation technologies and it will be interesting to see how the culture responds basing on photobioreactor design. I will definitely read the papers you mentioned.
By the way, although all the answers on my question were really useful and I thank you both, maybe we have kind of lost my principal problem that is that I find huge differences, and I can not explain why, between measuring dark-adapted QY outside and measuring QY inside the lab. Here obviously the last sample will "have to wait" in a completely different light environment, probably affecting the measurement.
Should I take the in-situ outdoor value (very low) or the lab value (much higher)?
Thanks again
Claudio
Claudio
I think that Nina was right on about the noon day NPQ being tough to relax, and that your values are certainly going to change throughout the day. Taking measurements every hour starting just before sunrise would be great and you will certainly see a trend throughout the day.
Your photobioreactor....does it have tunable light during the day, or is it a constant square wave light?
Hi Ben
Yes, at the end I found differences in QY during the morning and also in dark-adaptation time. That is, QY value has a positive correlation with it.
I am not sure what do you mean with your question about the photobioreactor. It is an outdoor system so I suppose I do not have a tunable light.
Claudio
Hi Claudio,
I assumed that you were using an indoor PBR. Ours delivers programmable light regimes, and I figured that you could simulate QY of your outdoor culture in your reactor. But I misunderstood your culturing strategy. But no worries. Good luck with your studies.
hello Claudio，for my experience, if you want to detect the photochemical efficiency under solar irradiance, the effective photochemical efficiency is better than optimal photochemical efficiency (dark adaption). because the photochemical efficiency can recover so quickly in several minutes. by the way ,what kind of instrument did you use in your exp? and what do you wanna to find by the photochemical efficiency data?
Can that aqua pen FRRF thing you have measure continuously? With some sort of flow-through system? I think that would be ideal for you.
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