Effects of live sand & mud on the microbial communities in my tanks (updated with new data)

AquaBiomics

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[updated Feb 17, 2020 to include data from an additional tank]

One of the most common questions I get is "If there's something I don't like about my aquarium's microbiome, what can I do about it?" In this thread I'll describe my experience with this question on my own home tanks.

The punchline is this: my experience suggests that live sand and mud provide a very low-cost way to adjust the microbial communities in established reef tanks.

Since this describes my home display tanks, which differ from each other in several ways, this is more of an anecdote than a formal experiment. But I figured since it provides a realistic example of what a hobbyist can do with this in a real world setting, it would be worth sharing anyway. (I'll update the thread in the future as I continue to monitor the communities in these tanks)

What I didn't like in my initial tests
The microbial communities in several of my home display tanks were not as diverse as I wanted, and were dominated by different bacterial families than the typical pattern I'd seen in healthy reef tanks. I wanted to change that.

I have four systems in my home (each includes 2 or more tanks so I count by systems instead of tanks). Three of them showed communities dominated by Alteromonadaceae. This was a pattern I've seen in many tanks I've sampled. It's one of the most common ways for a tank to deviate from the "typical" community (i.e., the average of healthy reef tanks I've sampled) -- a bloom of Alteromonadaceae. I don't have evidence showing this group causes a problem. I just know it's a difference from the community I wanted in most of my tanks.
initial tests v2.jpg


Except Tank B. It has the highest levels of Alteromonadaceae. But it is also an unusual tank that is finally behaving like I want it to, after much work. Its unusual (among my systems) because it has no macroalgal refugium. Instead, I have lots of macroalgae in the display. Its mostly Gracilaria hayi with some other assorted reds, I'm terrible at algal ID. The tank is macroalgae, soft corals, and fish. Most of the display tank has low to moderate light and low to moderate flow... I call it the lagoon tank (Tank B, here).

I used to struggle with cyano problems in Tank B, which after a month or two of nitrate dosing to 5 ppm finally went away. Since then I've monitored nitrates and dosed as needed to maintain 5-10 ppm. Whatever microbial community is growing in that algal lagoon is working. I didn't want to disturb it, so I made that tank one of the controls, and didn't change anything.

I decided to experiment with the microbial communities in the other two tanks (A and D) to see how I could adjust them to make them more like the typical reef tank profile. I wanted higher diversity, less Alteromonadaceae, and more Pelagibacteracea and Flavobacteriaceae. Consider it purely personal preference, since they are my personal tanks. And I deliberately didnt change the other two systems, to serve as controls.

What I added to the tanks
For a variety of reasons I wanted to experiment with low-cost options for these tanks. 1) I'm naturally a cheapskate / bargain hunter in just about all aspects of life. 2) especially when it comes to optional expenses for a hobby. 3) when I've advocated live rock others have rightly ask, "this stuff is expensive -- aren't there any cheaper options?"

Here is what I found: live mud and sand at FloridaPets.com, collected in the Florida Keys. Please note I am not affiliated with this product in any way other than as a happy customer. I'm sure there are plenty of other suppliers, and I haven't done any kind of comparison between sources. It would be very interesting to compare sources... I am sure they all have different collection sites.

I bought 1 cup of the Live Keys Sand and 1 cup of the Live Mud for a grand total of $12.99. The material was shipped USPS Priority and looked and smelled good on arrival. I mean, the mud smelled like typical marine mud, but a normal smell, not a nasty decaying smell. I'm sorry I don't have a vocabulary like wine tasters' to describe this. I've just spent a lot of time around marine mud and the material looked and smelled reassuringly like normal ocean-smelling mud and sand. So in it went.

I split it in half, and added half to each of two tanks. I added it to the drain chamber of my sumps, which flows downward, providing the best chance of letting it settle out instead of blowing around the tank. I lowered it under the water surface to the bottom in a little tupperware container, then emptied the mud and sand into the chamber gently, to minimize mixing. That was it. Took about 5 minutes total. It created a little cloudiness when I added it that night, which was cleared up by morning.

Changes I observed in the tanks' microbiomes

I tested the tanks again about a week and a half after adding the sand and mud. The treated tanks initially ranged from low-diversity to very low diversity. After the addition of live sand & mud, these tanks ranked among the most diverse of tank I've sampled so far.


Treatment (Tank name)

Diversity Before (Percentile)

Diversity After (Percentile)
Control (Tank B)151 (0.06)360 (0.57)
Control (Tank C)156 (0.08)332 (0.47)
+Mud (Tank A)201 (0.15)544 (0.88)
+Mud (Tank D)72 (0.00)576 (0.92)
[edit: I've re-analyzed all samples so that the percentiles are calculated relative to the current database. A careful reader may notice that some percentile values have changed as a result.]



However, the untreated tanks also showed a smaller increase in diversity, showing typical diversity in the later sample. This remained well below the high diversity levels of the +Mud tanks.

Since I transfer small amounts of water between tanks daily during feeding, I speculate that this increase in diversity in the control tank may reflect transfer from +Mud tanks (remember, I treated this one like a hobby rather than an experiment. My tank habits predate my interest in aquarium microbiology!)

The changes in communities were even more striking. The systems treated with live sand & live mud had obvious reductions in Alteromonadaceae. Both of them also showed increased levels of Pelagibacteraceae, and Tank D also showed increased Flavobacteriaceae.

Sampled collected from the control tanks remained similar to the initial samples. In both control tanks, the dominant family remained unchanged (Alteromonadaceae for tank B, Pelagibacteraceae for tank C). The relative stability of communities in the control tanks increases our confidence in concluding that the changes in treated tanks were caused by the addition of live sand and mud.

live sand effect v2.jpg


One of the most interesting findings is a recent addition. In revisiting the new data for tank C I realized I had previously overlooked an important benefit of this treatment. Live sand & mud increased the amount of nitrite-oxidizing bacteria (NOB) in the treated tanks. See the following figure, which simply extracts the NOB data from standard AquaBiomics tests before and after the change in each tank.
live sand NOB.jpg

It's a pretty dramatic change: tanks A and D had no detectable NOB before the addition of live sand & mud, but had normal to high levels after adding this material. The untreated tanks showed no such increase during the same time -- in fact, one of them (B) was initially present at low levels and undetectable in the later sample.

Tank D also showed a large increase in ammonia-oxidizing microbes (AOA & AOB). This change (not shown) was less impressive visually because all samples had detectable levels of AOA & AOB before the addition of live sand & mud.

Conclusions
It probably wouldnt surprise anyone that adding live sand and mud to the aquarium affects the community. I doubt anyone could have predicted exactly which changes would occur from adding this material. I sure couldn't have.

I've summarized the effects of these treatments in the following table, to let the reader draw their own conclusions.
UntreatedLive sand & mud
Tank BTank CTank ATank D
Change in dominant familynonoyesyes
Alteromonadaceaeincreasedincreaseddecreaseddecreased
Pelagibacteraceaedecreaseddecreasedincreasedincreased
Nitrite-oxidizing bacteriadecreasedunchangedincreasedincreased
I conclude that the treatments accomplished got exactly the kind of changes I was hoping for -- I got lucky.



Did they improve the health of the tanks? That's another question and a more difficult one. The tanks are so different it would be hard to compare them in terms of benefits. I will say that both tanks have showed marked reductions in problem algae. Tank A had cyano on the sandbed, and the frag tray in Tank B had a variety of problem algae. Subjectively, without any measurements, both have improved. But I lack controls for that effect (i.e. untreated tanks with algal problems) so we can't attribute those benefits to the treatment. Certainly no harm done, and subjectively both tanks are doing well and arguably better than before.

I make no strong claims about any benefits on the general health of the tanks. Its too soon to say. But I was happy with the effects on the microbial communities themselves, and thought I'd share.
 
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Mastiffsrule

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Evening

You just did my older established tank and came up with interesting results. You found a pathogen that may or may not be a factor in newly added fish death. Really interstellar stuff.

I have a question more along the lines of the article here if I read it correctly. You used the live harvested sand and mud to help the diversity in you established tanks. What if a tank was started with the sand and mud from the ocean? Would it maintain it long term, or would it be expected to thin out? I just set up my tank 100% live sand and rock from Tampa bay. would It maintain its diversity ?
 
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AquaBiomics

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Evening

You just did my older established tank and came up with interesting results. You found a pathogen that may or may not be a factor in newly added fish death. Really interstellar stuff.

I have a question more along the lines of the article here if I read it correctly. You used the live harvested sand and mud to help the diversity in you established tanks. What if a tank was started with the sand and mud from the ocean? Would it maintain it long term, or would it be expected to thin out? I just set up my tank 100% live sand and rock from Tampa bay. would It maintain its diversity ?
I'm really interested to know the answer too. Since directly testing it requires monitoring the microbiome of a single tank for, say, 10 years... I don't have a perfect answer.

I can say this. Based on my observations in the live rock study, it appears that a tank started with live rock increases over the first 2 weeks or so, reaching a relatively high diversity comparable to mature reef tanks (300-400) which remained stable for the next 2 weeks.

I can also say that in young tanks (1-2 years) we see the widest range of diversity scores, including the highest scores. Old tanks tend to converge on low to moderate diversity (150-200ish).

One possible explanation for this pattern is that diversity decreases with time. If diversity decreases with time, and if diversity is generally beneficial (neither of which been formally demonstrated for aquariums) .. then perhaps we would benefit from adding new material perhaps every few years.

I operate my tanks under the working theory that diversity is beneficial at least up to a point, and aim to increase my tanks' diversity until they are similar to the most diverse tanks I've tested. It appears that this latest addition has bumped them up to that level. It will be interesting to continue monitoring these tanks to see if the changes persist.

Tank A that I described in this article is my oldest tank thats still up (5 yr), and Tank D is my newest (<1 yr). So with my limited sample size it appears that both old and new communities can be affected by addition of these materials.

--

I'd love to hear more about your recent fish deaths! Several people whose samples contained Photobacterium damselae told me after testing that they had recently experienced unexplained fish mortality. What kind of fish, and did you observe any symptoms?
 

brandon429

why did you put a reef in that
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One day it would be great to see measures from someone's home that contains marine and fw tanks roughly same age to see if any groups are shared between them or if sampled communities are 100% different between established fw and marine systems

any dualists out there would be fascinating to catch on file or exclude at least in cursory initial check
 
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AquaBiomics

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One day it would be great to see measures from someone's home that contains marine and fw tanks roughly same age to see if any groups are shared between them or if sampled communities are 100% different between established fw and marine systems

any dualists out there would be fascinating to catch on file or exclude at least in cursory initial check
I like the idea of testing FW tanks alongside SW tanks, maybe I should start sampling FW tanks of existing clients to build a FW db and enable this comparison.

Id guess there would be some overlap at the family level, with a few family presence/absences.. ranging down to little if any overlap at the level of specific types (within species). Many groups are very sensitive about salinity, so this transition would be tough but perhaps there is something that can make it across. Interesting question...
 
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I wanted to add this description of how the material was stored and collected, that I received by email from John at FloridaPets.

John said:
I normally like to collect the 'Live Mud' the day of or no more than a week of being shipped. Typically when I collect it fresh like that, there will be larger critters in it. The live mud I shipped to you on the 20th of Dec. was collected about two weeks prior to being shipped. It was stored in a styro foam fish box and just kept submerged. It was kept in a cool location, which likely preserves the micro fauna well. The Keys sand and mud are collected from various locations in the Keys and is only collected about four times a year. It is kept in 5 gallon buckets and kept submerged as well. The main difference between the local sand/mud and the Keys sand/mud, is the mineral composition. Some of the Keys mud can also tend to be very, very fine in nature.

I'm adding this in the hopes it is more useful to readers than my subjective impression that it "smelled like normal marine mud" :)

Again, very happy with the product. I consider most of my tanks to have enough live rock (in terms of surface area for microbes and space to fit the rock), so it's not always convenient to supplement the microbial community by adding live rock. Live sand and mud (real, collected in the Keys, sand and mud) appear to be a really effective way to supplement the community in tanks like mine that don't need or have much room for additional live rock.
 

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"One possible explanation for this pattern is that diversity decreases with time. If diversity decreases with time, and if diversity is generally beneficial (neither of which been formally demonstrated for aquariums) .. then perhaps we would benefit from adding new material perhaps every few years." Perhaps this is why replacing live rock with fresh live rock every few years was advocated years ago.
 
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"One possible explanation for this pattern is that diversity decreases with time. If diversity decreases with time, and if diversity is generally beneficial (neither of which been formally demonstrated for aquariums) .. then perhaps we would benefit from adding new material perhaps every few years." Perhaps this is why replacing live rock with fresh live rock every few years was advocated years ago.
Yeah, my impression is that over the years a variety of practices for establishing or maintaining the microbial community have been popular for a while then fallen out of favor.

I've heard of refreshing your live rock every few years... another old practice I've heard about is mixing and swapping sand with your local club. I wonder what other 'old-fashioned' reef keeping practices like this I'm missing?

It will be interesting to revisit some of the old reefkeeper practices with DNA sequencing.
 

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Very well written. I absolutely love reading about your experiences. Thank you for sharing your passion for marine aquariums with us. How many folk are wanting to add live sand to their systems?
I had to remove 90% of my sand due to amphidinium dinos. I have a strong desire to add sand back. The original sand was just reef sand tropic eden reef flakes because I wanted to avoid sand storms. I am thinking add live sand or add bottled bacteria to the sand in a smaller tank for a week before adding it to my DT.
 
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Very well written. I absolutely love reading about your experiences. Thank you for sharing your passion for marine aquariums with us. How many folk are wanting to add live sand to their systems?
I had to remove 90% of my sand due to amphidinium dinos. I have a strong desire to add sand back. The original sand was just reef sand tropic eden reef flakes because I wanted to avoid sand storms. I am thinking add live sand or add bottled bacteria to the sand in a smaller tank for a week before adding it to my DT.
Thanks for the kind words. I feel kinda bad because it looks like the vendor I recommended has been sold out for a while now. So I am not sure how practical my recommendation was!

Still, plenty of other live sand vendors out there in principle, and I know that FloridaPets does restock periodically, I've seen their inventory update perhaps weekly.

In terms of sandstorms, I should clarify that one of the tanks I treated here is bare bottom display, with live -ish rock and now live sand and mud in the refugium. I prefer sand bottoms in display tanks but this is a frag tank system (2 frag tanks with a sump) and I prefer to run those without sand in the display. So the effects of live sand/mud don't require having sand in the display.
 

lexinverts

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Thanks for the kind words. I feel kinda bad because it looks like the vendor I recommended has been sold out for a while now. So I am not sure how practical my recommendation was!

Still, plenty of other live sand vendors out there in principle, and I know that FloridaPets does restock periodically, I've seen their inventory update perhaps weekly.

In terms of sandstorms, I should clarify that one of the tanks I treated here is bare bottom display, with live -ish rock and now live sand and mud in the refugium. I prefer sand bottoms in display tanks but this is a frag tank system (2 frag tanks with a sump) and I prefer to run those without sand in the display. So the effects of live sand/mud don't require having sand in the display.

He is heading to the Keys on Feb 16th to get some more.
 

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Pretty sure reading a post of yours inspired me to order some Florida live sand recently. Microbial diversity has been a concern of mine as I have used mostly dry rock with only a seed of LR when upgrading tanks in the past. I started my first tank with LR bought at a fish store that was closing. But, hard to define "live" in that sense. Cured would probably be a better term.

I have noticed a reduction, maybe even elimination in algae growing on the sand. Purely anecdotal though as I've been trying all sorts of new things. Certainly not controlled lol!
 

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Very well written. I absolutely love reading about your experiences. Thank you for sharing your passion for marine aquariums with us. How many folk are wanting to add live sand to their systems?
I had to remove 90% of my sand due to amphidinium dinos. I have a strong desire to add sand back. The original sand was just reef sand tropic eden reef flakes because I wanted to avoid sand storms. I am thinking add live sand or add bottled bacteria to the sand in a smaller tank for a week before adding it to my DT.
I like this idea a lot and, on the surface , don't see a down side. What if you mixed reef flakes, live sand and bottled bacteria in a new set-up. Would that be a witches brew or a useful substrate?
 

brandon429

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In my opinion the bottle bac would not be beneficial when using true live sand or live rock. It would be waste of $ to use single species nitrifying bac when paying for the shipment costs of wet marine surfaces loaded with diversity, maybe one day bottle bac makers will allude to such redundancy on the label but probably not. They’ve managed to seat their product so far into reefing psyche I must acknowledge their salesmanship


bottle bac makers have a vested interest in redundancy




it will be neat to see the microbiome analyses of aquariums that have gone through very harsh actions like sandbed replacements or rip cleanings/full water changes of the whole system. I appreciate getting to see dna measures as time unfolds, we will get data from both the typical care ways and the atypical ones for comparison. Will be handy to see biome measures just after before a complete substrate change, then a week after, then a year after.
 
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One of the most common questions I get is "If there's something I don't like about my aquarium's microbiome, what can I do about it?" In this thread I'll describe my experience with this question on my own home tanks.

The punchline is this: my experience suggests that live sand and mud provide a very low-cost way to adjust the microbial communities in established reef tanks.

Since this describes my unreplicated home display tanks, which differ from each other in several ways, this is an anecdote, not a formal study of any kind. But I figured since it provides a realistic example of what a hobbyist can do with this in a real world setting, it would be worth sharing anyway. (I'll update the thread in the future as I continue to monitor the communities in these tanks)

What I didn't like in my initial tests
The microbial communities in my home display tanks were not as diverse as I wanted, and were dominated by different bacterial families than the typical pattern I'd seen in healthy reef tanks. I wanted to change that.

I have four systems in my home (each includes 2 or more tanks so I count by systems instead of tanks). Three of them showed communities dominated by Alteromonadaceae. (The fourth system, not shown here, had a more typical community not dominated by Alteromonadaceae). This was a pattern I've seen in many tanks I've sampled. It's one of the most common ways for a tank to deviate from the "typical" community (i.e., the average of healthy reef tanks I've sampled) -- a bloom of Alteromonadaceae. I don't have evidence showing this group causes a problem. I just know it's a difference from the community I wanted in most of my tanks.


Except Tank B. It has the highest levels of Alteromonadaceae. But it is also an unusual tank that is finally behaving like I want it to, after much work. Its unusual (among my systems) because it has no macroalgal refugium. Instead, I have lots of macroalgae in the display. Its mostly Gracilaria hayi with some other assorted reds, I'm terrible at algal ID. The tank is macroalgae, soft corals, and fish. Most of the display tank has low to moderate light and low to moderate flow... I call it the lagoon tank (Tank B, here).

I used to struggle with cyano problems in Tank B, which after a month or two of nitrate dosing to 5 ppm finally went away. Since then I've monitored nitrates and dosed as needed to maintain 5-10 ppm. Whatever microbial community is growing in that algal lagoon is working. I didn't want to disturb it, so I made that tank one of the controls, and didn't change anything.

I decided to experiment with the microbial communities in the other two tanks (A and D) to see how I could adjust them to make them more like the typical reef tank profile. I wanted higher diversity, less Alteromonadaceae, and more Pelagibacteracea and Flavobacteriaceae. Consider it purely personal preference, since they are my personal tanks. And I deliberately didnt change the other two systems, to serve as controls. (my test of the second control failed so I will be retesting that sample and updating with those results later)

What I added to the tanks
For a variety of reasons I wanted to experiment with low-cost options for these tanks. 1) I'm naturally a cheapskate / bargain hunter in just about all aspects of life. 2) especially when it comes to optional expenses for a hobby. 3) when I've advocated live rock others have rightly ask, "this stuff is expensive -- aren't there any cheaper options?"

Here is what I found: live mud and sand at FloridaPets.com, collected in the Florida Keys. Please note I am not affiliated with this product in any way other than as a happy customer. I'm sure there are plenty of other suppliers, and I haven't done any kind of comparison between sources. It would be very interesting to compare sources... I am sure they all have different collection sites.

I bought 1 cup of the Live Keys Sand and 1 cup of the Live Mud for a grand total of $12.99. The material was shipped USPS Priority and looked and smelled good on arrival. I mean, the mud smelled like typical marine mud, but a normal smell, not a nasty decaying smell. I'm sorry I don't have a vocabulary like wine tasters' to describe this. I've just spent a lot of time around marine mud and the material looked and smelled reassuringly like normal ocean-smelling mud and sand. So in it went.

I split it in half, and added half to each of two tanks. I added it to the drain chamber of my sumps, which flows downward, providing the best chance of letting it settle out instead of blowing around the tank. I lowered it under the water surface to the bottom in a little tupperware container, then emptied the mud and sand into the chamber gently, to minimize mixing. That was it. Took about 5 minutes total. It created a little cloudiness when I added it that night, which was cleared up by morning.

Changes I observed in the tanks' microbiomes

I tested the tanks again about a week and a half after adding the sand and mud.

The treated tanks initially ranged from low-diversity to typical diversity. After the addition of live sand & mud, these tanks ranked among the most diverse of tank I've sampled so far.


Treatment (Tank name)

Diversity Before (Percentile)

Diversity After (Percentile)
Control (Tank B)151 (0.33)360 (0.68)
+Mud (Tank A)201 (0.50)544 (0.90)
+Mud (Tank D)72 (0.06)576 (0.95)
However, the one untreated tank for which I have data also showed an increase in diversity, although it remained well below the level of the +Mud tanks. Since I transfer small amounts of water between tanks daily during feeding, this increase in diversity in the control tank may reflect transfer from +Mud tanks (remember, I treated this one like a hobby rather than an experiment. My tank habits predate my interest in aquarium microbiology!)



The changes in communities were even more striking. The systems treated with live sand & live mud had obvious reductions in Alteromonadaceae. Both of them also showed increased levels of Pelagibacteraceae, and Tank D also showed increased Flavobacteriaceae.



Conclusions

It probably wouldnt surprise anyone that adding live sand and mud to the aquarium affects the community. I doubt anyone could have predicted exactly which changes would occur from adding this material. I sure couldn't have.

In this case, I got exactly the kind of changes I was hoping for. Did they help? I will say that both tanks have showed marked reductions in problem algae. Tank A had cyano on the sandbed, and the frag tray in Tank B had a variety of problem algae. Subjectively, without any measurements, both have improved. But I lack controls for that effect (i.e. untreated tanks with algal problems) so we can't attribute those benefits to the treatment. Certainly no harm done, and subjectively both tanks are doing well and arguably better than before.

I make no strong claims about any benefits on the general health of the tanks. Its too soon to say. But I was happy with the effects on the microbial communities themselves, and thought I'd share.

How biodiversity changed during nitrate dosing would have been a useful observation, or nitrate vs ammonium chloride vs amino acids. How biodiversity is impacted by Chemiclean and fluconazole would be interesting. Does H2O2 have a significant effect? What part of the bacterial community is being skimmed out? Stimulated by carbon dosing? Ethanol vs acetic acid? How does Vibrant and WasteAway change biodiversity? What happens to biodiversity with large water changes? How is biodiversity impacted by driving nitrate and phosphate to undetectable levels? How does pore water biodiversity differ from tank water biodiversity? How does biodiversity change during cyanobacteria, dinoflagellate and diatom blooms? How does biodiversity of cyanobacteria mats change with time, from initial growth to mature mats?

So many questions. I hope you tackle them soon :)
 

Do you have a frag tank, frag rack or some other place for coral frags currently?

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