A Spectrophotometer view of Photosynthetic Organisms in our Tanks

[taricha]

Is it possible to roughly translate pigment composition percentages to action spectra? When you look at the pigment concentrations in this study. It would appear that the action spectra would be very different between the acro and brain corals.

That's an awesome paper for this discussion. Thank you!
Also, yes. I think since it gives us pigment mass concentrations, we can build an absorption spectrum for the zoox. And as the last few posts discussed - it looks a lot like an action spectrum (give or take a few quirks).
I'll give it a shot anyway. See if the result is sensible.

P.s. and a tidbit for later...
What photosynthetic organism in my tank has this insane pink as its pigments? [emoji848]
(All that was done to it was freeze, smash, thaw, pour through paper towel)

 

[Dana Riddle]

That's an awesome paper for this discussion. Thank you!
Also, yes. I think since it gives us pigment mass concentrations, we can build an absorption spectrum for the zoox. And as the last few posts discussed - it looks a lot like an action spectrum (give or take a few quirks).
I'll give it a shot anyway. See if the result is sensible.

P.s. and a tidbit for later...
What photosynthetic organism in my tank has this insane pink as its pigments? [emoji848]
(All that was done to it was freeze, smash, thaw, pour through paper towel)

There are a number of pink/red 'pigments' (technically not correct in some cases, but I use it anyway.) Is this pink one water-soluble? Fluorescent or no?

 

[taricha]

There are a number of pink/red 'pigments' (technically not correct in some cases, but I use it anyway.) Is this pink one water-soluble? Fluorescent or no?

Water soluble.
Pretty dang fluorescent.

 

[Dana Riddle]

Water soluble.
Pretty dang fluorescent.

I'll hedge my bet... LOL. 'P' stands for protein (or pigment if you will) and the number refers to the emission maximum. Most are fluorescent proteins and in a case or two, phycoerythrin.
P-574 Zoanthus
P-575 Dendronephthya
P-575 Dendronephthya
P-575 Montastraea cavernosa
P-575 Montipora (digitata/angulata)
P-575 Phycoerythrin within symbiotic cyanobacteria in coral
P-575 Scolymia sp.
P-576 Montipora efflorescens
P-576 Ricordea florida
P-576 Zoanthus - Mature form of P-522
P-578 Scolymia cubensis
P-579 Mycedium elephantotus
P-580 Lobophyllia hemprichii (red)
P-580 Montastraea cavernosa (mcavRFP)
P-580 Plesiastrea verispora (blue morph)
P-580 Plesiastrea verispora (green morph)
P-581 Lobophyllia hemprichii (red)
P-582 Catalaphyllia jardinei
P-582 Echinophyllia echinata
P-582 Goniopora tenuidens
P-582 Montastraea cavernosa (mc1)
P-582 Montastraea cavernosa (R1_2)
P-582 Montastraea cavernosa (G1_1)
P-582 Montastrea cavernosa
P-582 Trachyphyllia geoffroyi
P-583 Discosoma sp.
P-587 Madracis pharensis @ 40m (brown tissue)
P-587-590 Ricordea florida (mouth)
P-590 Acropora digitifera
P-590 Porites compressa
P-593 Acropora millepora
P-593 Discosoma sp. 2
P-593 Discosoma sp. 3
P-593 Favia favus
P-595 Anemonia sculata
P-597 Acropora millepora
P-610 Montipora monasteriata
P-611 Entacmaea quadricolor
P-620 Montipora sp.
P-620 Plesiastrea verispora (blue morph)
P-620 Plesiastrea verispora (green morph)
P-620 Porites astreoides
P-625 Acropora horrida
P-625 Pocillopora damicornis
P-625 Porites murrayensis
P-630 Acropora aspera
P-685 Chlorophyll - common to healthy hermatypic corals

 

[taricha]

Is it possible to roughly translate pigment composition percentages to action spectra? When you look at the pigment concentrations in this study. It would appear that the action spectra would be very different between the acro and brain corals.

I think since it gives us pigment mass concentrations, we can build an absorption spectrum for the zoox.

Ok. here's my attempt. I did 2 branchy types, 2 brain types, and one lobe-type kind of in-between.
These are absorption spectra for the coral zoox based on the masses of pigments provided in the paper.
See here for a picture of the corals.

[process notes: I had to sub some carotenoids for others, but I tried to find the closest match carotenoid, and scaled the strength of the absorption based on known absorption/extinction coefficients.
The neoxanthin family I replaced into dinoxanthin. P457 I replaced into fucoxanthin. Chl B showed up in O. annularis, but that's got to be contamination from green algae in the skeleton, so I removed it, and an equal amount of chl A that likely came with it.]

Overall, I think @SDchris idea that the brains and the sticks had notably different pigment profiles was right. Acro had the highest Chl A relative to Peridinin (and other carotenoid) levels. The brains had the reverse: highest peridinin/carotenoids relative to Chl A.

 

[Dana Riddle]

Is it possible to roughly translate pigment composition percentages to action spectra? When you look at the pigment concentrations in this study. It would appear that the action spectra would be very different between the acro and brain corals.

I need some time to digest the results listed in the referenced paper. It appears corals were sampled from similar biotopes, but the clades listed seem to be from a number of sites.

 

[taricha]

It appears corals were sampled from similar biotopes, but the clades listed seem to be from a number of sites.

Yeah, they couldn't actually do the clade identification so they provided a lit rundown of which clades were in those corals in similar areas.

"As we were unable to perform genetic identification of the different symbiont clades found in the studied species, we base our Principal Components Analysis, Hierarchical Cluster Analysis, and further discussion on the most common symbiont clades found in the Caribbean (including Puerto Rico) within colonies living under similar environmental conditions as the ones we sampled here..."

 

[Dana Riddle]

Their

Yeah, they couldn't actually do the clade identification so they provided a lit rundown of which clades were in those corals in similar areas.

"As we were unable to perform genetic identification of the different symbiont clades found in the studied species, we base our Principal Components Analysis, Hierarchical Cluster Analysis, and further discussion on the most common symbiont clades found in the Caribbean (including Puerto Rico) within colonies living under similar environmental conditions as the ones we sampled here..."

Their research is not without credence, but requires further research. I'm almost completely tied up with doctor's appointments and other until Friday. I'll carry my laptop and try to keep abreast!

 

[Dana Riddle]

I carried my laptop to the offices today. I need some more time to look into the referenced paper (clade v. photopigment.) I don't like to question published results but do have some questions that weren't addressed in the paper.

 

[Dana Riddle]

If I were asked to peer-review this paper (Torres-Perez), I would make these comments:

Expand the Symbiodinium clades to these:

Acropora cervicornis: A, A3, C, C1, C12

Colpophyllia natans: B, B1, B6, B9

Orbicella (Montastraea) annularis: A, A13=A1.1, B, B1, B10, C, C3, C7, C31, D1a

Porites astreoides: A, A3, A4, A4a, B, B1, C, C1, C1a

Porites furcata: A, A4, B, B1, C, C4

Porites strigosa: B1, C1

Siderastrea siderea: A, A3, B5a, C, C1, C3, D, D1a

Chlorophyll content and its relationship to depth is poorly related. Analyses of chlorophyll content across fine scales in Hawaiian corals found microenvironments (folds, creases, self-shading, etc.) that can profoundly affect photopigment concentrations. The researchers seem to admit this with this quote from their paper:

Nonetheless, keeping in mind that all samples were collected at the same depth (1m), the

range in symbiont concentration among species may reflect the influence of different skeletal

arrangements and the consequent differences in light regime reaching the endosymbiotic

dinoflagellates within the coral tissue.

This is a very interesting research paper and I applaud the time and effort involved.
If I were asked to peer-review this paper (Torres-Perez), I would make these comments:

Expand the Symbiodinium clades to these:

Acropora cervicornis: A, A3, C, C1, C12

Colpophyllia natans: B, B1, B6, B9

Orbicella (Montastraea) annularis: A, A13=A1.1, B, B1, B10, C, C3, C7, C31, D1a

Porites astreoides: A, A3, A4, A4a, B, B1, C, C1, C1a

Porites furcata: A, A4, B, B1, C, C4

Porites strigosa: B1, C1

Siderastrea siderea: A, A3, B5a, C, C1, C3, D, D1a

Chlorophyll content and its relationship to depth is poorly related. Analyses of chlorophyll content across fine scales in Hawaiian corals found microenvironments (folds, creases, self-shading, etc.) that can profoundly affect photopigment concentrations. The researchers seem to admit this with this quote from their paper:

Nonetheless, keeping in mind that all samples were collected at the same depth (1m), the

range in symbiont concentration among species may reflect the influence of different skeletal

arrangements and the consequent differences in light regime reaching the endosymbiotic

dinoflagellates within the coral tissue.

This is a very interesting research paper and I applaud the time and effort involved.

 

[taricha]

Expand the Symbiodinium clades to these:
[...alphabet soup of clades...]

Do you expect all these clades would show up in these corals in the Caribbean, or is that a worldwide association of clades and corals?

Chlorophyll content and its relationship to depth is poorly related. Analyses of chlorophyll content across fine scales in Hawaiian corals found microenvironments (folds, creases, self-shading, etc.) that can profoundly affect photopigment concentrations. The researchers seem to admit this with this quote from their paper:

"Nonetheless, keeping in mind that all samples were collected at the same depth (1m), the
range in symbiont concentration among species may reflect the influence of different skeletal
arrangements and the consequent differences in light regime reaching the endosymbiotic
dinoflagellates within the coral tissue."

I need to catch up on my reading here regarding how much pigment amounts & ratios change when symbiodinium acclimatize to a different light spectrum, but it sounds like you're saying that the pigment variation within a single zoox strain (even within one coral) due to available light may be larger and more important than the pigment differences between different clades of zoox?

After I catch up on reading, I'll think about how to change lights for a coral or two of mine,

one last comment. The profile of the brain (massive) corals - high in Peridinin, low in Chl A versus the profile in the acro/branching corals - low Peri, high Chl A seems advantageous when you consider how quickly the short wavelength blue light would be extinguished upon reaching the coral tissue.
Thin branching geometry means higher surface area to volume ratio so more zoox are closer to surface so more blue on average is available hence Chl A. In the massive coral geometry, more zoox will on average be farther from the surface and so the shortest wavelengths will be less present at some depth in the coral tissue and therefore having absorption at longer wavelengths is more helpful, hence Peridinin.

If we want to get really wierd with it, we can ask the chicken or egg question. If pigments are shaped by light available due to geometry, can light available force a change in geometry? Some corals can change to be branchier or thicker - and maybe the light provided can influence that one way or another. Maybe more extreme short wavelength blue that wouldn't penetrate coral tissue well would encourage more surface area: branchier structure and thinner branches. hmmm.....

 

[Dana Riddle]

Yes, the zoox clades I added to the list are all found in the Caribbean.

There was a paper written some time back that found corals' photopigments' concentrations varied within single colonies along the north/south/east/west axes. It has been years since I read the paper but self-shading played a part.

And I agree that light availability can at least in some cases define morphology (columns flattening to plates in deeper water.) This was apparent in a Hawaiian Montipora species.

What I don't recall is the number of peridinin per chlorophyll a - if my poor memory serves me, this ratio varied among dinoflagellate genera. Does it vary among Symbiodinium species/clades?

Do you expect all these clades would show up in these corals in the Caribbean, or is that a worldwide association of clades and corals?



I need to catch up on my reading here regarding how much pigment amounts & ratios change when symbiodinium acclimatize to a different light spectrum, but it sounds like you're saying that the pigment variation within a single zoox strain (even within one coral) due to available light may be larger and more important than the pigment differences between different clades of zoox?

After I catch up on reading, I'll think about how to change lights for a coral or two of mine,

one last comment. The profile of the brain (massive) corals - high in Peridinin, low in Chl A versus the profile in the acro/branching corals - low Peri, high Chl A seems advantageous when you consider how quickly the short wavelength blue light would be extinguished upon reaching the coral tissue.
Thin branching geometry means higher surface area to volume ratio so more zoox are closer to surface so more blue on average is available hence Chl A. In the massive coral geometry, more zoox will on average be farther from the surface and so the shortest wavelengths will be less present at some depth in the coral tissue and therefore having absorption at longer wavelengths is more helpful, hence Peridinin.

If we want to get really wierd with it, we can ask the chicken or egg question. If pigments are shaped by light available due to geometry, can light available force a change in geometry? Some corals can change to be branchier or thicker - and maybe the light provided can influence that one way or another. Maybe more extreme short wavelength blue that wouldn't penetrate coral tissue well would encourage more surface area: branchier structure and thinner branches. hmmm.....

 

[taricha]

I’m going to circle back to symbiodinium later, but first let’s talk about this crazy pink sample.

P-575 Phycoerythrin within symbiotic cyanobacteria

It comes from a purple photosynthetic sponge.
It gets sold sometimes as “collospongia auris” or just blue/purple photosynthetic sponge but was ID’d last year as actually Lendenfeldia chondrodes (paper).

It has a few interesting features. First, it can grow like a weed for some people and not grow at all for others. The photosynthetic symbiont is a cyanobacteria and different specimens can have slight color variations on purple from green to blue to red, which is believed to be due to the exact cyano living inside it.

[top: sponge in tank, and under room LEDs showing purple color. bottom: 90% acetone extracts Chl A and carotenoids into green liquid, and leaves behind the pink from the phycobilins.]

The other thing I find interesting about it is the challenge to get the weird pink/purple pigments out. When I first started playing with it, all I could manage to extract from the purple sponge was just green, and what was left behind was bright pink sponge. And later I want to look at coralline algae, so needed to figure out the pigments.

Here’s the end result after getting all pigments extracted…it's a pink phycoerythrin party.

[Black line is actual measured absorbance and grey dotted line is reconstruction from adding pigments together to match.]

Chl A and Carotenoids appear, but the pink Phycoerythrin from the cyanobacteria is king here - fluoresces strongly at 575nm, with a small contribution from phycocyanin. This may explain why it grows like crazy in my tank, but some people I've given it to have said it never grew much at all for them. My tank gets some morning sun so plenty of green light, and if others lighting didn't include much green, it may be a struggle to grow this.

 

[Dana Riddle]

I’m going to circle back to symbiodinium later, but first let’s talk about this crazy pink sample.

It comes from a purple photosynthetic sponge.
It gets sold sometimes as “collospongia auris” or just blue/purple photosynthetic sponge but was ID’d last year as actually Lendenfeldia chondrodes (paper).

It has a few interesting features. First, it can grow like a weed for some people and not grow at all for others. The photosynthetic symbiont is a cyanobacteria and different specimens can have slight color variations on purple from green to blue to red, which is believed to be due to the exact cyano living inside it.

[top: sponge in tank, and under room LEDs showing purple color. bottom: 90% acetone extracts Chl A and carotenoids into green liquid, and leaves behind the pink from the phycobilins.]

The other thing I find interesting about it is the challenge to get the weird pink/purple pigments out. When I first started playing with it, all I could manage to extract from the purple sponge was just green, and what was left behind was bright pink sponge. And later I want to look at coralline algae, so needed to figure out the pigments.

Here’s the end result after getting all pigments extracted…it's a pink phycoerythrin party.

[Black line is actual measured absorbance and grey dotted line is reconstruction from adding pigments together to match.]

Chl A and Carotenoids appear, but the pink Phycoerythrin from the cyanobacteria is king here - fluoresces strongly at 575nm, with a small contribution from phycocyanin. This may explain why it grows like crazy in my tank, but some people I've given it to have said it never grew much at all for them. My tank gets some morning sun so plenty of green light, and if others lighting didn't include much green, it may be a struggle to grow this.

I think it was Charlie Mazel who identified P-575 in cyanobacteria/corals. We saw that in some of the Montipora spp. we cultured at Aquatic Wildlife back in the 90's.

 

[Rybren]

I have this sponge in my tank and it is an incredibly fast grower. Tank is lit by two 165W Black Boxes, each with two green LEDs. It also grew really well in an older tank under T5s (2xATI Blue Plus and 2x ATI Aquablue Special)

 

[taricha]

Next organism will be coralline algae.

Hmmm... maybe some phycoerythrin in there too?

 

[Dana Riddle]

Next organism will be coralline algae.

Hmmm... maybe some phycoerythrin in there too?

Yes, I think so, but not in all coralline species. Some fluoresce an orange color very nicely when the proper excitation source is used.

 

[AltitudeAquarium]

Recently I got my hands on a spectrophotometer. I wanted to take a peek at absorption spectra of organisms that grow in our tanks and see if I could identify pigments other than Chlorophyll A - the accessory pigments - to start to drill down on possible differences between the ways they use light.
I eventually figured out how to extract pigments, and found some published methods that I could replicate to see if my results made sense before I went too far down the road.

My first three organisms are Green Hair Algae (Derbesia) clumps out of my tank, Chaetomorpha out of my fuge, and Isochrysis (T-Iso golden brown phyto) from live culture.
Below is cleaned up data from pigments extracted with some combo of crushing and freeze/thaw in 90% acetone. (I overshot on the acetone % and it shifted a couple of the peaks by 1-2nm so the textbook 664nm actually is at 662...)
anyway...

Chlorophyll A dominates all 3 spectra as expected with a blue peak at ~430nm and a red peak at ~664nm. These are the locations of the peaks in 90% acetone. Below, I'll compare with the more natural peaks of the un-extracted pigments.

I was able to find signs of 3 pigments other than Chl A: Chl B, C, and Fucoxanthin.
The green algaes Derbesia and Chaetomorpha have Chl A & B. The T-Iso (prymnesiophyte) has pigment spectra a lot like dinoflagellates in our coral. It has Chl A, C and Fucoxanthin (whoo! a non-chlorophyll!)

Additionally, there are simple equations for calculating the amount of individual Chlorophylls in a sample by looking at the sizes of the peaks on the red end of the spectrum (indicated by arrows at 630, 647, and 664nm). Seems to work - I calculated Chlorophyll ratios in the ballpark of data I could find published elsewhere.
Derbesia had ~1.6x the ChlA as B
Chaeto had ~2.1x the ChlA as B
T-Iso had ~3.3x the ChlA as C
(expressed as decimal ratios of Chl A on the above graph).
Looking at the spectra, I found it a little surprising I calculated as much secondary Chlorophyll as I did, considering how Chl A totally dominates the shape of the spectrum.
[I couldn't find comparable eqn for calculating relative amounts of fucoxanthin, but maybe I'll run across something.]

So solvents like acetone are great for getting clean curves, with reliable peak heights that let you calculate ratios of pigments in your sample. But the locations of the peaks are highly dependent on the solvent, and don't represent the absorbance peaks of those pigments in cell - aka the real world.

Here's where the peaks are in raw tissue: simply pulverized and diluted in water, and a little light data cleaning to try to remove some scattering noise.

Ignore the relative heights - too much background scattering to trust, this is just to show the "real" locations of the peaks.
The black arrows here indicate how much the peaks are further to the right from where they were in the acetone above.

My takeaways:

• Derbesia is Booming in Chl B relative to Chaeto. Is that because Derbesia is growing under my heavy blues in the tank, and Chaeto is in my Red/Blue/6500k mix in the fuge? Or is it more about the algaes themselves?
• The "green gap" is undeniable, but there's a lot of action in the forgotten wavelengths from the high 400's to the low 600's, if you look for it carefully.
• On the red end of the spectrum, Chl B and C peaks sit almost perfectly in the gaps of each other. Relevant in our sytems?
• I expect to see more spectra like T-iso when I look at coral (and maybe pest) dinoflagellates and diatoms, as they also have Chl C and carotenoid/xanthophyll accessory pigments.

Next I've got my eye on a little patch of red cyano, and maybe later some zooxanthellae. Shout out if you have ideas or interesting stuff to try.

What do you the results would be for other macro-algaes such as Galleria (sp?) red colored pigments?

 

[taricha]

What do you the results would be for other macro-algaes such as Galleria (sp?) red colored pigments?

Gracilaria is red algae, AKA rhodophyte. I don't have any rhodophytes in my tank, except for the one that everybody has - coralline algae.
Reds apparently acquired their photosynthetic machinery from a cyanobacteria. So the reds also contain phycoerythrin phycocyanin etc.
These will make them pink.
Other pigments to round them out are Chl A, some fucoxanthin that serves similar role to peridinin in dinoflagellates such as in our corals, (some weird dinos stole their photo machinery from red algae - which stole theirs from cyano, and so those dinos have fucoxanthin instead of peridinin. Craaaazy.) and reds should also have some carotenoids probably.
Which is all to say, if you see cyano that matches the color of your red algae, they are almost certainly using nearly the same pigments.
To more directly answer your question: the lighter red/ brighter pink the red algae, the more dominant the phycoerythrin with its strong absorption in the 500-560nm range. The darker / browner the red is, the more it'll have broad absorbing pigments like Chl A with its blue and red absorption and carotenoids which absorb well in the 450-500 range.

 

[AltitudeAquarium]

Gracilaria is red algae, AKA rhodophyte. I don't have any rhodophytes in my tank, except for the one that everybody has - coralline algae.
Reds apparently acquired their photosynthetic machinery from a cyanobacteria. So the reds also contain phycoerythrin phycocyanin etc.
These will make them pink.
Other pigments to round them out are Chl A, some fucoxanthin that serves similar role to peridinin in dinoflagellates such as in our corals, (some weird dinos stole their photo machinery from red algae - which stole theirs from cyano, and so those dinos have fucoxanthin instead of peridinin. Craaaazy.) and reds should also have some carotenoids probably.
Which is all to say, if you see cyano that matches the color of your red algae, they are almost certainly using nearly the same pigments.
To more directly answer your question: the lighter red/ brighter pink the red algae, the more dominant the phycoerythrin with its strong absorption in the 500-560nm range. The darker / browner the red is, the more it'll have broad absorbing pigments like Chl A with its blue and red absorption and carotenoids which absorb well in the 450-500 range.

My thought was, do I need to consider the spectrum specifically for the chaeto (the dominant macro algae) when choosing the refugium lights or do I need to consider the correct spectrum for Gracilaria? If so, maybe this influences my outcome of cyanobacteria bloom in the aquarium. Thoughts?