A Spectrophotometer view of Photosynthetic Organisms in our Tanks

[taricha]

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?

If I were trying to grow gracilaria, I'd use a spectrum that accommodates it, and let the chaeto which is tougher and more flexible adjust to what was provided.
I'd consult the magroalgae pros for what light configuration they use with successful gracilaria.
Cyano may become a concern, but I'd deal with it in ways other than light.

 

[Dana Riddle]

I'll toss these into the mix:

 

[AltitudeAquarium]

I'll toss these into the mix:

Great information capturing what I was thinking about. Thanks

 

[Genetics]

Best thread I’ve read in a long while!

 

[taricha]

Coralline Algae time! This is a crustose coralline rhodophyte, as the sceintific papers would describe it. Our coralline algae are probably mostly Lithothamnion, I would guess.

Some fluoresce an orange color very nicely when the proper excitation source is used.

My coralline fluoresce orange under actinics only when dying.

Phycoerythrin again making a big show. This form of Phycoerythrin is a little different, R-Phycoerythrin (R-PE), and it has a big absorption spike at 495nm in addition to the broad absorbance at ~550.

check out what acetone soluble - left (mostly chlorophyll) vs water soluble - right (mostly phycoerythrin) looks like.

The water soluble phycobilins on the right are very pink - absorb green light, and the acetone soluble chlorophyll+carotenoids on the left are green, absorbing red and blue.
I should note here, that this little visual undercounts the water soluble pigments. A look at coralline can tell you that the pink pigments are the more dominant ones.

The rhodophyte action spectrum Dana posted above is for a fleshy red macroalgae, and maybe the coralline reds use blue light a little better.
This paper got an absorption spectrum for coralline algae (see fig 3) and it is pretty similar to the above graphic I got, they didn't do an action spectrum though, so hard to say how well these corallines actually use the blue light they absorb - if they outperform their fleshy red cousins in that department. What is clear, is that though blue may be king in corals, there's a bunch of stuff that lives in my system that uses cyan and green light more than I expected.


One final note: this comment of mine is a mess of inaccuracy.

some weird dinos stole their photo machinery from red algae - which stole theirs from cyano, and so those dinos have fucoxanthin instead of peridinin.

This paper has the full story.
The correction is relevant here because there's no fucoxanthin in the red algae. Their pigments are very simple. Chl A, the Phycobilins, a handful of carotenoids, and that's it.
That kind of amazes me because the red algae diverse to levels of absurdity, like 4,000-6,000 species. So it's impressive they do so well with such a small pigment set.

 

[Dana Riddle]

Only when dying... Interesting, I wonder if chlorophylls mask the fluorescence in healthy coralline species, much as healthy leaves' chlorophyll mask the pigments that are only apparent in the Fall?

 

[taricha]

Only when dying... Interesting, I wonder if chlorophylls mask the fluorescence in healthy coralline species, much as healthy leaves' chlorophyll mask the pigments that are only apparent in the Fall?

yep. when I see orange fluorescing under actinics, it's coralline patches that were either air-exposed, or sand-buried and later it becomes apparent under normal light they died.
the fall leaves analogy makes a lot of sense. I can get strong fluorescence from either the Cl A or the PE from these organisms, but not in the same sample. Like one is totally blocking the other.
Another really interesting idea I ran across was this one:
"We measured fluorescence emission spectra during bleaching of cell suspensions in a number of cyanobacterial cultures. A near instantaneous shift from fluorescence originating from Chl a around 685 nm to increased phycobilisomal fluorescence was observed. The phycobilisomal fluorescence was first typical of allophycocyanin or energy flow in intact phycobilisomes (>650 nm), then shifted to shorter wavebands corresponding to phycocyanin fluorescence."

That makes it sound like the excitation-flourescence handoff of energy cascades from pigment to pigment to pigment down toward red end, until you break the chain chemically, mechanically or with solvents or whatever. Then you see the fluorescence at the broken link in the chain.

 

[Rybren]

yep. when I see orange fluorescing under actinics, it's coralline patches that were either air-exposed, or sand-buried and later it becomes apparent under normal light they died.

I've observed something similar in my tank. I also get the orange fluorescence after soaking pumps, etc in vinegar. Any residual coraline on the items tend to fluoresce orange.

 

[oreo54]

Organic luminescence stems primarily from the incorporation of naturally luminescent humic acids, the major constituent of dissolved organic matter (DOM) in the sea [28]. Inorganic luminescence comes from the chemical properties of the carbonate skeleton. An intrinsic blue luminescence has been observed in carbonates, including aragonite, but it is weak and overshadowed by extrinsic luminescence when activator elements are incorporated into the crystal [29]. Transition metal ions occupy the Ca2+ site of the carbonate and this results in a strong yellow luminescence [30]. Such inorganic luminescence occurs in corals when trace elements are incorporated in the coral skeleton (such as Mg, Mn, Zn, Sr and Cu). Though not luminescent, ferric iron absorbs strongly in the UV and can also be a common trace element in coral skeletons [31].

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2776492/

just throwing it in..
And this for fun..
https://www.nature.com/articles/ismej2010152

 

[Dana Riddle]

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2776492/

just throwing it in..
And this for fun..
https://www.nature.com/articles/ismej2010152

Fluorescent banding in coral skeletons often coincidences with the wet seasons, where terrestrial runoff carries fluorescent-inducing agents.

 

[taricha]

I've observed something similar in my tank. I also get the orange fluorescence after soaking pumps, etc in vinegar. Any residual coraline on the items tend to fluoresce orange.

I forgot about vinegar. Seen that as well. Might play around with that later and see if anything interesting comes up.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2776492/

just throwing it in..

That's cool. To clarify, the orange fluorescence from coralline we can be sure is from the R-Phycoerythrin - water soluble, absorbance and emission spectra match perfectly.
However, a good while back I was trying to look for spectral effects of coral skeleton (absorbance, fluorescence) and I didn't find anything useful. This is excellent.
the yellow fluorescence looks pretty weak. I doubt I can observe it, and I don't have any source that far in UV.

 

[taricha]

Ok, one more weird thing with a bunch of phycoerythrin before I go back to symbiotic dinoflagellates.
I found some pure spirulina - a type of cyanobacteria - growing as a deep maroon red in my sump.

it's pretty weird to watch. It spirals like a corkscrew to move around. Video I took a while back here.


In addition to the Phycoerythrin it has the strongest phycocyanin absorbance (around 630nm) I've encountered yet.

Pretty strong absorbance literally across the entire spectrum. Only Yellow & Red have notably less absorbance than the others.

(Up next I may have gotten an interesting result from aiptasia I think - most blue absorbing Chl A heavy symbiodinium spectrum I've seen yet. I'll need to double-check it.)

 

[taricha]

Here's the Aiptasia spectrum I was talking about. It's the first thing I've seen where there's variation in the symbiodinium absorbance like in the paper talked about in post #65. In the paper there seems to have been a pigment difference between the massive coral brain types and the thin branchy types.

the sample was taken from the aiptasia tentacles - as they are totally packed with zoox.
Chl A (and C) are more significant and the peridinin + other carotenoids is the smallest of the symbiodinium I've looked at.

For comparison, look at the spectrum overlaid with the profile of zoox from a neon green sarcophyton toadstool (faded).

If geometry-influenced light levels within the coral tissue is indeed the reason for the difference the authors of the paper found between the brains and the branches, then something similar may be in effect here...
Aiptasia is essentially transparent, and the tentacles are extremely small in cross-section, so the zoox is basically all getting surface light, like in the thin branchy corals. meaning more zoox ought to see more short wavelength blue light.
The sarcophyton is quite big and fleshy and rounded with some zoox at depth below the coral surface - like in the brain corals - those cells would see less of the shortest wavelength blue and so they may be adapted to longer wavelengths.

The geometry argument just a guess, there are many other reasons why the pigment profiles of zoox from a sarcophyton and aiptasia may be different.
Overall, I wasn't sure if I would find symbiodinium with different pigment profiles in my system - or if it would all be the same, and this certainly seems to answer that question.

 

[taricha]

I'm messing around with hard corals now, and I'm working toward getting an absorption spectrum of the zooxanthellae separate from that of the coral animal itself. Because I suspect that the bright, colorful specimens in our tanks have enough pigment in the coral tissue that the overall absorption is not a great guide to what the zoox are actually absorbing and using.

So far I'm finding bright fluorescent pigments from the coral tissue that are water soluble and come out from tissue with a little encouragement in just water, whereas zoox pigments - Chlorophylls, Peridinin, Carotenoids etc are pretty water-insoluble and not much from them goes into the water. I don't know if this is generally true, I also don't know if the colorful non-fluorescent coral pigments are water soluble too. @Dana Riddle got any thoughts?

Coral fluorescence is well known but I still find it endlessly fascinating that I can take two barely colored solutions of slightly orangish yellow water - one from zoox squeezed out of a sarcophyton (left), and the other from the coral tissue (right), hit them both with the same blue light - and one glows like a neon green highlighter.

 

[Dana Riddle]

I'm messing around with hard corals now, and I'm working toward getting an absorption spectrum of the zooxanthellae separate from that of the coral animal itself. Because I suspect that the bright, colorful specimens in our tanks have enough pigment in the coral tissue that the overall absorption is not a great guide to what the zoox are actually absorbing and using.

So far I'm finding bright fluorescent pigments from the coral tissue that are water soluble and come out from tissue with a little encouragement in just water, whereas zoox pigments - Chlorophylls, Peridinin, Carotenoids etc are pretty water-insoluble and not much from them goes into the water. I don't know if this is generally true, I also don't know if the colorful non-fluorescent coral pigments are water soluble too. @Dana Riddle got any thoughts?

Coral fluorescence is well known but I still find it endlessly fascinating that I can take two barely colored solutions of slightly orangish yellow water - one from zoox squeezed out of a sarcophyton (left), and the other from the coral tissue (right), hit them both with the same blue light - and one glows like a neon green highlighter.

There are a couple of chlorophyll-peridinin complexes - one is soluble in water. Should be easy to pick out unless your fluorescent pigments from corals are >650nm (which would be highly unusual.)From my experiences, water-soluble fluorescent proteins usually leach from coral tissues in less than an hour, although a few take overnight. Naturally, de-ionized water is best. But there is a caveat - pH can influence the emission. If DI H2O has a pH of, say, 6, how does that compare to the emission is at seawater pH (8.2 or so?) It would be interesting to use various LEDs to excite the pigments to see if pigments are relaying fluorescence - in other words, is the florescence of one protein used as the excitation source for another protein. Some of the red fluorescent proteins can be excited by blue light - does this indicate a huge - and undescribed Stokes Shift - or are proteins relaying light?

 

[taricha]

There are a couple of chlorophyll-peridinin complexes - one is soluble in water.

Thanks! More details later, but that fits what I'm seeing. I thought I was getting a small amount of Chl A contamination in all my samples no matter how I filtered them, but the Chl fluorescence responded as well or better to light in the peridinin range.

I hadn't thought about pH. What pH do we think these pigments function in within the cells? And if I'm comparing to the charts in your articles on coral coloration, what pH are most of those generated at?

 

[Dana Riddle]

Thanks! More details later, but that fits what I'm seeing. I thought I was getting a small amount of Chl A contamination in all my samples no matter how I filtered them, but the Chl fluorescence responded as well or better to light in the peridinin range.

I hadn't thought about pH. What pH do we think these pigments function in within the cells? And if I'm comparing to the charts in your articles on coral coloration, what pH are most of those generated at?

This article examines regulation of pH within coral tissue and how zoox can influence it. I'd have to look at my database for references on pH and fluorescent proteins..
https://www.pnas.org/content/106/39/16541

 

[SDchris]

Only when dying... Interesting, I wonder if chlorophylls mask the fluorescence in healthy coralline species, much as healthy leaves' chlorophyll mask the pigments that are only apparent in the Fall?

I have a phyto, Proteomonas sulcata, that is fluorescent pink under blue light, red under daylight, orange when nutrients get low and green when it crashes.

 

[Dana Riddle]

I have a phyto, Proteomonas sulcata, that is fluorescent pink under blue light, red under daylight, orange when nutrients get low and green when it crashes.

I wasn't familiar with that cryptophyte - it contains chlorophylls a, c2 and alloxanthin. I need a couple more cups of coffee before I can even begin to think about those color shifts.

 

[taricha]

So here's my first attempt at isolating pigments from a coral and not the zoox. (After that I can subtract those pigments and look just at the zoox absorbance in a coral.)

In blue is what dissolved out of Neon Sarcophyton tissue, and in red is what dissolved out of the zoox in the coral. The tissue solution is dominated by a green fluorescent protein, and the red line is almost a pure Peridinin-Chlorophyll protein (spectrum for comparison). You can see each has a little contamination from the other in it.
But subtract them and what you're left with is this profile of a Green Fluorescent Protein...(with scaled Fluorescence shown)

This is the "neon" in my neon green sarcophyton. Ignoring the ChlA fluorescence, the absorbance/emission sounds kind of like P503 from a carpet anemone in Dana's article, though there are many similar GFPs. It also looks a lot like this stupidly named NowGFP.

(@Dana Riddle What bugs me is the increased absorbance going from 420 to 400nm (I don't trust my stuff below 400nm). The absorbance increase there is probably real - I've seen it in different solutions out of the sarcophyton tissue done different ways. But it doesn't seem connected to fluorescence. There is fluorescence at 502 & 675 in the sample when excited with 405nm, but it's quite weak - much weaker than at 430, etc.
Makes me think most of the 400nm absorbance is an unrelated nonfluorescent pigment. Weird.)