Nitrate and Phosphate Growth Experiment

[jraker]

I have recently been having trouble finding what the optimal levels and ratios of nitrate and phosphate are to grow macros. I am also breaking down my old freshwater 29 gallon tank, so I saw the opportunity to start my own experiment.

I am going to be measuring how different species of caulerpa in different substrates grow with different levels of nitrate and phosphate.

I am going to use a mars aqua 165w led light, and old HOB filter for nothing but flow and surface movement.

I am going to test these different parameters:

Control (regular saltwater)
Nitrate at 10, 15, 20ppm
Phosphate at 0.1ppm
Nitrate:Phosphate at 100:1, 150:1, and 200:1

I am also going to put baffles in the tank, to divide it into thirds, because I am also going to use different substrates. One will be sand, the next will be sand with mud, and the last will be bare bottom. I thought this would work well because I will get to have the same nitrate and phosphate, but test different substrates with each parameter.

I will be testing caulerpa this first time around, because they grow quickly. When I put them in, I will trim them all to the same length, so the starting length is a constant. Each time I change a parameter (such as 10ppm nitrate to 15ppm), I will measure how each one has grown, then I will trim them back to the same length I originally had them in. I will be using Caulerpa prolifera, mexicana, and maybe paspaloides and serrulata. I will allow each to grow in a constant parameter for 2 weeks, and then I will change it.

So, how am I going to keep the parameters constant? I am going to buy some test kits for nitrate and phosphate (PO4 is going to be hanna, and NO3 is going to be red sea pro, because hanna didn't have anything for under $300) and test them everyday to see how much of a nutrient I lost. I will then use this calculator to see how much of what chemical I need to use to bring the nitrate or phosphate back up to the level I want it to be at. This means testing and adding nutrients every single day. I am going to use potassium nitrate and potassium phosphate to add NO3 and PO4. I can easily measure how much I need to put in the tank every day.

I think I addressed pretty much everything I am planning to do. Pictures are coming soon. I might not start to test different parameters right away in order to let the tank mature, so I don't have any nuisance algae interfering with my results. Please ask questions and make suggestions. I am open to some new ideas also.

[Michael Hoaster]

Great idea! Experiments are great for figuring stuff out.

So you're really testing nitrate to phosphate ratios, keeping phosphate constant at .1 ppm. If you are going to test all four simultaneously, you will need four separate vessels. If one at a time, one vessel works.

If you are sticking to the scientific method, only one variable at a time should be tested. Using substrate gives you two. Having two variables means you never know for sure which variable had an effect. So I'd recommend dropping substrate from this experiment. Since macros don't have roots, substrate isn't really important, except how it affects bulk water nutrient levels, which is a second variable.

The median C:N:P atomic ratio of benthic marine macroalgae and seagrasses is about 550:30:1. So your macros will vary a bit, but should be in that ballpark. So rather than 100, 150 and 200 to 1, maybe you should test ratios of 40, 30 and 20 to 1, or something like that.

I would think that each macro tested would have a different ideal ratio. That would be very useful information! For example, say your caulerpa's ideal ratio is 40-1, and your grasilaria's is 20-1. If you wanted to favor the grasilaria, you go 20-1, accelerating its growth, while slowing down the caulerpa.

[jraker]

Thanks for the tips. I guess the substrate really isn't that important, but I will still divide the tank with baffles because I might need to put substrate in if I want to do this experiment with seagrasses or mangroves.

I am only testing one at a time, for two weeks each. At the end of every two weeks, I change the parameter. I will test for lower ratios, like 20:1 and 30:1, and depending on my data, I will do higher or lower ratios.

[JZinCO]

So I haven't said it before i any other post, but I am a terrestrial plant ecologist.

You mentioned testing substrate and nutrient ratios. But it sounds like you are mixing up crossing and nesting. I suggest your factorial design be either one of three ways: 1) A one-way analysis of variance, 2) A two-way analysis of variance, 3) A two-way analysis of variance with interaction

In 1, you test just one factor. If you have three levels of nutrient ratios, you treat everything but nutrient ratios the same to try and reduce error caused by other factors. (3 vessels: Ratio1, Ratio2, Ratio3)

In 2, you test the additive effects of nutrient ratios and substrate. You have three levels of nutrients, and for one level of nutrients you have three substrates. this is called nesting. (5 vessels: Ratio1 with Substrate1, Ratio1 with substrate2, Ratio1 with Substrate3, Ratio2 with Substrate1, Ratio3 with Substrate1)

In 3, you test the additive and multiplicative effects of nutrient ratios and substrates. You have three levels of nutrients and for each level of nutrients you have three substrates. This is called crossing (9 vessels: R1+S1, R2+S1, R3+S1, R2+S1, R2+S2, R2+S3, R3+S2, R3+S1, R3+S3)

With 1 you can make statements like "When N:P ratios increase 5x, % biomass accrual increases 10x". With 2 you can make statements like "When N:P ratios increase 5x, % biomass accrual increases 10x in sand AND mud substrates have 2x more growth than sand substrates". Wth 3 you can make statements about interactions such as "When N:P ratios increase 5x, % biomass accrual increases 10x in sand AND 20x in mud."

Easy peasy? Depends on how much time, vessels, and equipment to control water quality you got.
Also, the independent factor should be growth; That is if on week 1 the algae weighs 10 g and in week 2 the algae weighs 15 g than you would be writing down 50%, or 0.5 g growth/1 g of biomass.

You can make this a legit usable study by keeping a log such as:

Substrate N:P ratio Study day Growth
Sand 100:1 15 50%
Sand 150:1 15 60%
Sand 200:1 15 40%
Sand 100:1 25 55%
Sand 150:1 25 40%
Sand 200:1 25 50%
......

Also, to emphasize don't do length. Why? What if the algae in one of your vessels decides to grow laterally or shoot off a new part instead of growing the original? It's probably best to use growth (accrual of biomass). I would also normalize by biomass as I described above because 1 g of growth from 1 g of algae is much more productive than 1 g of growth from 1 kg of algae

[Michael Hoaster]

So, you got that?

[jraker]

I got that. I will only be using 1 vessel, and I will be changing the ratio every 2 weeks, keeping everything but the nutrient ratio the same. I really don't have room for anything else. I see what you mean by measuring in mass and not weight, and I will also order an electronic scale. Thanks for the response.

[JZinCO]

Originally Posted by jraker

I got that. I will only be using 1 vessel, and I will be changing the ratio every 2 weeks, keeping everything but the nutrient ratio the same. I really don't have room for anything else. I see what you mean by measuring in mass and not weight, and I will also order an electronic scale. Thanks for the response.

No prob. Google "classic garden experiment" if you want to know more about design.
What you described just now would work and might be easier. Google "repeated measures design" to learn more.

[cHRoNzZe 619]

Following ........

Interested to see growth and the death .....

[Michael Hoaster]

Hey jraker, sorry for all the advice. I know you asked for input, but I think I may have given too much. It's your experiment. Do it however you like! I'd hate to see you get bogged down, under a heap of "help". Good luck! A lot of us are curious to see your results.

[jraker]

Thanks, all your help is good. I wanted some other opinions on this anyways, and I knew I was going to tweak it. I have ordered my light, and test kits, so the experiment should start to get underway as soon as I get the tank emptied and refilled again.

[jraker]

I got the tank cleaned up today, and I put it where I want it to go. All I need to do is glue the baffles in, which will be done tomorrow or tuesday.



I started receiving some things in the mail. I got my hanna test kit and my scale, but I am still waiting on the nitrate test kit, my light, and the hanna reagents.



The tank should get up and running pretty soon.

[jraker]

More stuff came.


Potassium phosphate


Potassium nitrate

[Subsea]

FYI

The major nutrients in marcoalgae, as in land plants, are potassium, nitrate and phosphate.


Chemical dry analysis for Gracilaria Parvispora:

Nitrogen @ 2.59%
Phosphorus @ 0.082%
Potassium @13.54%
Calcium @ 0.555%
Magnesium @ 1.163%
Sulfur @ 4.81%
Zinc @ 139 ppm
Iron @ 107 ppm
Manganese @ 20 ppm
Copper @ 7.0 ppm

[jraker]

I filled the tank up the other day. Everything is ready to go, I just need to start adding some plants.




I changed up the parameters I will be testing. I will be testing ratios:

20:1 (N:P)
30:1
40:1
50:1
... and possibly more based on my results. I will be testing based on a fixed amount of nitrate (change the phosphate), then a fixed amount of phosphate, where I change the nitrate. For example, 5ppm nitrate : 0.25ppm phospate, 5ppm nitrate : 0.16ppm phosphate, etc. That would be the fixed amount of nitrate, where I only change the phosphate, and I will do another round where I only change the nitrate and keep the phosphate at a constant.

One question, when measuring the mass of the plants on the scale, should I use the dry weight (let plant dry out, therefore killing plant), or can I just use the plant when it is wet, so I can reuse it?

[HippieSmell]

As subsea pointed out, you are also dosing potassium, so you've got three variables you're testing, not two.

[JZinCO]

Originally Posted by jraker

One question, when measuring the mass of the plants on the scale, should I use the dry weight (let plant dry out, therefore killing plant), or can I just use the plant when it is wet, so I can reuse it?

Wet is fine because the moisture content doesn't vary within an individual much. It does vary across species e.g. corralline is <50% but Caulerpa sp. is >90% water by weight.

I wouldn't worry about K as a factor unless you are varying K levels across your different N:P ratios.

Are you going to have multiple individuals? You should have at least 3 individual plants for each N:P ratio. Weigh them separately so you have multiple reps.

[jraker]

Sorry for the lack of updates on this project. I chose three species: C. prolifera, racemosa, and sertularioides. I have two specimens of each, each one weighing 0.7g. That amount wasn't too small or too large. I put each of them in, and there was a little dieoff, but then they began to grow, some more rapidly than others. This is 9 days into the control experiment, so I have 5 more days to go before I start testing ratios.

Pic from day 1:


Day 9:

[jraker]

I completed the control, and it all went well, except for one thing. I decided to test the parameters tonight to see how much nitrate and phosphate I needed to add. I tested the PO4 first, and it came up on the hanna checker as 1.48ppm. I thought that it might have been inaccurate, so I tried it again with the salifert test kit. Still around 1ppm phosphate. I didn't have a chance to test nitrate tonight, but I assume it is pretty high as well. I think this may be because I didn't really give the tank a chance to establish, and I did see a diatom outbreak about a week into the project. I think this means that I will need to do the control again, then continue on with the other parameters. I will give the tank a week or two so I can do water changes and use some GFO media to get the parameters down. Then once I get the parameters down to what a normal reef tank would look like (this is what the control is for, to test how the algae would grow in a normal reef environment with no change) I will do the control again and carry on with the experiment like normal.



This is the growth in 2 weeks. Pretty massive. I haven't had a chance to weigh it yet, but I am going to guess that the largest plants have quadrupled in size. Maybe when this experiment is over I will use the tank to farm macro. Who knows.

One more thing, the parameters I will test will be as follows:

N:P

5 : 0.25
7.5 : .25
10 : 0.25
12.5 : 0.25

10 : 0.5
10 : 0.33
10 : 0.25
10 : 0.2

[JZinCO]

This is really exciting and if it goes well will be sticky worthy

[Ron Reefman]

jraker, I'm late to the macro algae interest. I just started a display refugium with some macro alga that is connected to my 125g mixed reef.

My question is, how did you attach the algae to the rocks at the start?

I'll be following along and I hope you enjoy doing this as much as some of us are interested in your results! Thanks.

[jraker]

In this tank I placed pieces of rubble on the macro just to hold it down. BRS sells rubble by the pound and I used that to hold it down. In my 65 gallon tank I also used the rubble, but I took the macro and glued it to the rubble so I could easily move it around. I also placed some of the macro in the cracks of rocks and allowed their holdfasts to grow and attach to the rock. Both have worked pretty well so far.

[Ron Reefman]

Thanks. I glued a few macros to rocks and about a 1/3rd of them have let go over the last 2 weeks. That's why I asked.

[jraker]

It was tricky to get them on. I figured out that if you dab some glue at the base of the macro (where you want to glue it to the rock), it will dry and give a better surface for you to put on a little more glue to attach the macro to the rock. All the ones I did like that stayed glued down.

[Ron Reefman]

Originally Posted by jraker

It was tricky to get them on. I figured out that if you dab some glue at the base of the macro (where you want to glue it to the rock), it will dry and give a better surface for you to put on a little more glue to attach the macro to the rock. All the ones I did like that stayed glued down.

Excellent idea, thanks.

I should have thought of that. I've done something similar to leather coral frags I cut off the main colony. The coral is full of mucus type fluid, so I squeeze the leather a bit and get some of the fluid out. Then when I release the pressure the cut end of the coral, held upside down, stays dry and I can 'coat' the cut end with super glue. Let it harden and then do a second coat and hold it against the rock you want to attach it to, and 9 times out of 10 it holds. BTW, I've never seen any damage done to the coral either.

[Michael Hoaster]

That's a great idea! A question: Are you leaving the whole plant out of water while the first coat dries?