New (and Old) Species for Live Feed - Research & Experiments

[Guppysnail]

I often find things happen easier without me than with my best efforts. I flopped 3 ostracod cultures. Not paying attention my snail tank had a massive boom of them 🙄. 
This is great info you provided. Guesstimate on how much sooner CPD fry could consume these than BBS?  

[modified lung]

On 10/21/2022 at 8:54 AM, Guppysnail said:

I often find things happen easier without me than with my best efforts.

Lol same here. I always say most of my discoveries come through procrastination and incompetence.

On 10/21/2022 at 8:54 AM, Guppysnail said:

Guesstimate on how much sooner CPD fry could consume these than BBS?  

I think Chydorus could possibly be used as a first food. I'll have to get some fry going again and find out.

[CornAndCrawlers]

Can you share name/brand of the microscope you are using for these outstanding photos? Or link where I can buy one. this journal is amazing. 

[modified lung]

 

On 10/29/2022 at 8:30 PM, CornAndCrawlers said:

Can you share name/brand of the microscope you are using for these outstanding photos? Or link where I can buy one. this journal is amazing. 

This guy: 

AmScope M150C-I 40X-1000X All-Metal Optical Glass Lenses Cordless LED Student Biological Compound Microscope https://a.co/d/3WGJHDM

Edited October 30, 2022 by modified lung

[CornAndCrawlers]

Thank you for linking that! And how are your getting these great photos? Are you just placing your phones camera lens into the part of the microscope where you would normally be placing your eye?

[modified lung]

On 10/30/2022 at 5:53 AM, CornAndCrawlers said:

Thank you for linking that! And how are your getting these great photos? Are you just placing your phones camera lens into the part of the microscope where you would normally be placing your eye?

Yep, I just use my phone.

[modified lung]

***Freshwater Microalgae (3): "Mixed" vs "Pure" Greenwater***

Although I rarely participate in them, I like to visit other aquarium forums or groups to see what's going on in them. This year I've noticed a trend of trying to keep pure greenwater cultures containing only a single species of microalgae, usually Chlorella, to feed Daphnia, Moina, and other filter feeders. I tried this for a short time but, because I like to keep my home fish related activities as low maintenance as possible, I found it to be a bit too much effort.

To justify my laziness, I started questioning if feeding a single species of microalgae is really a good idea. There are, of course, many exception to this but most animals need a varied diet to stay healthy.

Ceriodaphnia: A Low Effort Experiment

I started testing this with Ceriodaphnia which I previously found reports claiming "mixed" greenwater, usually containing Chlorella and Scenedesmus, being more effective than "pure" greenwater containing only one of these species of microalgae. So I started alternating between feeding greenwater of unknown content and spirulina powder every 4 to 7 day. I saw a very noticable difference with cultures receiving the alternating feed schedule booming much more than anything I had before. However, since this was a low effort experiment, I didn't keep any records or take any photos to show off. 

Moina: A Not As Low But Still Low Effort Experiment

But whether or not mixed species greenwater is better for Ceriodaphnia, that doesn't necessarily mean it's better for anything else. So I did another not as low but still low effort experiment using Moina. I filled 3 jars with different greenwater cultures, added 5 adult and about 15 juvenile Moina to each, and left them sit for a little over a week.

(*Microalgae identification is difficult and confusing so take the following IDs as not seriously as you like.)

(From left to right: Tetradesmus, Chlorella, wild)

Jar 1: microalgae genus: Tetradesmus (a subgenus of Scenedesmus that doesn't form long chains)—grown with snails fed vegetables and old flake food, supplemented with NPK (low P) fertilizer; 0 ppm NH3/NH4+, 0 ppm PO4-

Jar 2:  (skipped for consistency later on)

Jar 3: microalgae genus: Chlorella—grown in recycled Moina culture water (for some reason Chlorella seems to love this); 0 ppm NH3/NH4+, 3 ppm PO4-

Jar 4: 'wild" culture grown in open container outdoors, 7 or 8 difference types of microalgae counted—grown with snails fed vegetables and old pellet food, supplemented with NPK (low P) fertilizer; 1.5 ppm NH3/NH4+, 0 ppm PO4-

(*By "wild" I mean not controlling the types of microalgae that happen to show up)

Results 

In the above photo (from left to right):

Jar 1 (Tetradesmus) cleared out first but much of the microalgae seem to have settled to the bottom.  The Moina reproduced but are low in number and most are very small in size.

(No jar 2)

Jar 3 (Chlorella) didn't seem to clear out at all. The Moina are still in there but they population hasn't seemed to grown much. It's possible they're just hard to see. I'm going to keep this one going see what happens over time. 

Jar 4 (wild) mostly cleared out with minimal settling. The population grew multiple times the amount of Jar 1 including large, fully grown adults.

Conclusion?

So the first "not as low but still low effort" experiment seems to show mixed greenwater is better for Moina as well. But there's a lot of different variables that could have contributed to the results. Each jar had different levels of total ammonia and phosphate for example.

Jar 1 (Tetradesmus) had no NH3/4+ or PO4- so, depending on how long it's been low, it's possible the greenwater was malnourished.

Jar 3 (Chlorella) had no NH3/4+ and excess PO4- which may matter because microalgae tends to store more nutrients in their cells when PO4- is low—meaning the greenwater in this jar may have been less nutritious. Jar 3 was also kind of a double experiment to see if Moina can be grown in greenwater grown in water from a different crashed Moina culture. 

Jar 4 (wild) had excess NH3/4+ and no PO4- meaning the greenwater was likely well nourished and more nutritious than the others.

I do still think it's leaning in the direction that mixed greenwater is better but maybe that's just the laziness bias talking. More experiments to come ...this time including a jar 2 with Scenedesmus.

 

Edited November 18, 2022 by modified lung

[modified lung]

***Freshwater Microalgae (4): Nutrients 

I've been a little obsessed with greenwater again lately.

Look at these two cultures:

Both were started on a week before this photo with only one difference. To the culture on the left I added an NPK fertilizer with a P and K level 25% lower than N. To the culture on the right I added an NPK  fertilizer with a P and K level 25% higher than N. As you can see the low PK fertilizer did much better.

First lesson: use a low P fertilizer; greenwater does not like too much phosphate.

 

Getting a Darker Culture 

Here's the same culture two weeks later: 

They didn't change much. If not for the 10 ramshorn snails in each tank, both of these cultures would probably have died out. But, although the snails sustained the cultures, they were not enough to grow the cultures. 

After the last photos, I added more low P fertilizer but this time to both cultures. Up until yesterday, another week later, the cultures still didn't change much.

But here's a photo taken today:

Wow. The tank on the left, I want to say  twice as dark. Tank on the right, murdered dead. What happened? Yesterday I added a cap full of Seachem Replenish and a few drops of Vitachem to both tanks. The original low PK cultures loved it. The high PK tank, not so much.

 

Water Testing Time

Before adding the second fertilizer, lefty showed 0 ppm NH3/4+ and 0 ppm phosphate on the API liquid kits. Today lefty tested 0.25 ppm and 0 ppm. Before the second fert, righty showed 0 ppm NH3/4+ and 3 ppm phosphate. Today righty tested 8+ ppm and 10+ ppm. 

The rest of righty's parameters were as expected. pH was 7.6 (the ACO strips always read low), 3 KH, 16 GH (raised by the Replenish) with 60 ppm Ca++ which would make Mg++ around 30 ppm. Conductivity was about 820 us/cm.

For lefty, a digital meter showed a pH of 9.8 which seemed to be a bit high for the ACO strip to handle lol. Such a high pH is expected because, not only does microalgae consume CO2, it also consumes carbonate and bicarbonates shown by the 0 KH reading on the ACO strip.

To the part that surprised me. Despite receiving the same amount of Seachem Replenish, lefty tested at only 9 GH. On top of that, lefty also tested at 60 ppm Ca++. That means there's only less than 10 ppm Mg++. Does microalgae eat that much magnesium?

There always seems to be a point where greenwater cultures just quit no matter how much more fertilizer I add. I figured there was a micronutrient being depleted but didn't know what it might be. Interestingly, the fertilizer I use, Vigoro Citrus, Fruit and Nut, has most of the micronutrient required but it has zero magnesium.

Really I don't know yet if it was the Replenish or Vitachem or maybe both that cause the boom for lefty. But so far this seems to point to the magnesium in Replenish being at least a major part of the cause. But I have no idea why they caused the death of righty, may he rest in peace. 

Second lesson: magnesium exists, eat it up.

Edited November 23, 2022 by modified lung

[nabokovfan87]

Very nice work. That information and studying cultures like this will be so useful to hobbyist.  I can imagine using it for amano breeding among other projects.  Have you ever tried to breed any with your outdoor ponds?

[modified lung]

On 11/21/2022 at 10:13 PM, nabokovfan87 said:

Very nice work. That information and studying cultures like this will be so useful to hobbyist.  I can imagine using it for amano breeding among other projects.  Have you ever tried to breed any with your outdoor ponds?

It's amazing how little info is out there about growing good freshwater greenwater cultures at home. I see people claiming success with various brands of guillard's f/2 media but that stuff was really made for marine phytoplankton. I tried it for a while and freshwater microalgae seemed to take off fast but crash faster. The original recipe assumes seawater is being used so it doesn't have any potassium, calcium, or magnesium included. Idk if any of the brands modify it at all.

Amano shrimp? I haven't tried outdoor breeding. Do they have a wide temperature tolerance? I tried it indoors a few years ago. The babies died on the second week after the first water change.

Edited November 23, 2022 by modified lung

[nabokovfan87]

On 11/22/2022 at 5:30 PM, modified lung said:

Amano shrimp? I haven't tried outdoor breeding. Do they have a wide temperature tolerance? I tried it indoors a few years ago. The babies died on the second week after the first water change.

They can go basically 78 or colder without much issue.  I have had mine get really cold, but I can't say how cold. 

On 11/22/2022 at 5:30 PM, modified lung said:

Amano shrimp? I haven't tried outdoor breeding. Do they have a wide temperature tolerance? I tried it indoors a few years ago. The babies died on the second week after the first water change.

Same here.  I didn't feed them often enough, I would think you get your greenwater and then feed in a portion 3-4x a day, then they can sustain. I was using bottles rotifers, but it's so much better to have live foods.

[modified lung]

@nabokovfan87 now my gears are turning.

[nabokovfan87]

On 11/23/2022 at 3:48 PM, modified lung said:

@nabokovfan87 now my gears are turning.

Check this out....  Very awesome microscope stuff showing the whole process.
 

For food, this is the method I've seen used on the larger scale.  daily they would flood the tanks (vats) with the algae / rotifer water.
 

 

Edited November 24, 2022 by nabokovfan87

[modified lung]

***Freshwater Microalgae (5): "Mixed" vs "Pure" Greenwater (2)*** 

I repeated the same test in two separate sets of jars. 15 adult Moina were added to each jar and allowed to grow for six days.

Trial #1

Trial #2

The results in each trail were basically the same:

Jar 1: Tetradesmus

The lowest population growth. Adults in trial #1 grew to medium size and in trial #2 to a small size.

Jar 2: Scenedesmus

Second best population growth. Adults grew to a medium size.

Jar 3: Chlorella (in recycled moina water)

Less population growth than jar 2 but adults grew to be larger.

Jar 4: "wild"

By far the most population growth and the largest sized adults.

Trial #1 was exposed to a good amount of light during the days. Trial #2 was in lower light which is probably why the microalgae in jar 1 and 2 settled much more. It's odd though that jar 4 in trial #2 didn't have any settling. Maybe the Moina population grew fast enough to eat all the microalgae before it settled?

These trials still have the same problems as before which makes the results not as concrete as I'd like. But wait ...there's something else that does point more toward mixed greenwater being better.

 

Oops ...Accidental Trial #3 ...sort of

I also had a 2 gallon jug of recycled Moina water growing Chlorella. I accidentally dropped a few Moina in the jug the same day I set up the other jars. Oh well, I decided to roll with it. I dumped a quart of the jug and refilled with water from the Tetradesmus and Scenedesmus cultures. We now have a mixed culture made from jar 1, 2, and 3.

I actually forgot I dropped the Moina into this jug and a day or two after I added some low P fertilizer to darken up the culture. The culture definitely darkened and also this happened:

The culture tested with 0 ppm NH3/4+ and 0.25 ppm PO4-. We can compare this to jar 3 in the other trials which only had Chlorella, 0 ppm NH3/4+, and 3.0 ppm PO4-. Except we know the greenwater in this jug was recently fed whereas I don't know how long jar 3 sat without any ammonia to eat on—meaning the greenwater in the jug was likely much more nutritious than in jar 3.

If similar results happened just by mixing the three cultures without adding the fertilizer (oops), I think we could say mixed green water is better. Instead right now I have some Moina in a jug of recently fed, dark, healthy Tetradesmus.  We'll see if the Moina population explodes just as fast.

Then in the next experiment I should have some pure and mixed cultures properly fed up. If the mixed culture wins again, then I think we can say for sure whether or not the lazy "wild greenwater" way is the better way. Who am I kidding? The lazy way is better no matter what.

Edited November 24, 2022 by modified lung

[modified lung]

***Freshwater Microalgae (6): "Mixed" vs "Pure" Greenwater (4)*** 

I'm satisfied in saying  feeding wild or mixed greenwater is better when compared to pure, single species  greenwater.

Left jar had pure Tetradesmus. The population growth was good, better than the picture makes it look actually. 

Middle jar had pure Scenedesmus. The population growth was quite bad.

Right jar had a mixed of 7 or 8 different types of microalgae. The population growth was the best in this jar but not by a huge margin.

This time all the different microalgae cultures got a fresh batch of nutrients before being fed to the Moina. I also held a lux meter up to each jar and diluted the greenwater concentration of each until the readings matched.

What's weird is how much more microalgae settled to the bottom in the jars given Tetradesmus and Scenedesmus. Those two microalgaes were also in the wild greenwater culture but did not settle at all. This same thing happened in the previous two trials. I thought it was because the population in the wild greenwater fed jars grew fast enough to not allow settling. But in this trial the population growth on the Tetradesmus jar was not that far behind the wild jar. Even so, much of the Tetradesmus settled to the bottom anyway. Idk what would cause such a difference but the settling could be part of why the pure greenwater doesn't do as well as the wild.

I also did 5 separate jars fed pure Chlorella. Settling was not an issue with any of these jars. All of them did better than Scenedesmus but worse than Tetradesmus and the wild fed jars. Interesting that the greenwater in these jars didn't clear out. Maybe the Chlorella grows faster than it can be consumed? I once read in a study I'm too lazy to find now that Chlorella has an especially tough outer cell and can be difficult for some filter feeders to digest. Maybe Chlorella is filtered much slower than other types of microalgae?

Just thinking out loud here ...maybe something like this could explain the increased settling with the other two jars given pure greenwater. We know that Daphnia filters different types and sized cells at different speeds—microalgae is consumed at almost twice the speed of yeast. Maybe it's possible the jars given wild greenwater didn't see any settling because the mix of small and large celled microalgae allowed for an increased overall feeding rate and thus more population growth compared to when only the larger cells of Tetradesmus and Scenedesmus are present. I'm of course just speculating here though.

Getting back to the Chlorella jars, there was an added difference between each of the 5. Jar #1 was unaltered. Jar #2 recieved enough Seachem Replenish to increase hardness from 8 to 13 GH and two drops of Vitachem. Jar #3 received only the Replenish and Jar #4 recieved only the Vitachem (3 and 4 are out of order in the pic). As an interesting side experiment, both jars that recieved Vitachem had what looks like more than double the population growth compared to the other two Chlorella jars. But these still had less growth than the Tetradesmus and wild fed jars.

Maybe the Chlorella was bad? Draw your attention to the square jar on the left. That one recieved no additives. However, this jar had Ceriodaphnia and not Moina. As you can see, the Ceriodaphnia cleared out the Chlorella to a degree the Moina could not. This seems to confirm the Chlorella in the culture was suitable to eat.

 

Conclusion

so what should we make of all this? Whatever I tell you to, that's what. I think feeding wild or mixed greenwater culture are better than feeding pure, single species greenwater, although, maybe not by a whole lot. However, take into consideration the effort of keeping a pure culture—isolating a single species of microalgae (or the cost of buying a pure starter culture), keeping the culture from being contaminated by anything else, and checking and rechecking if the culture is still pure. Compare that with maintaining a wild greenwater culture—wake up from nap, throw some nutrients in an open bucket, take another nap. Ok ok, maybe I'm under exaggerating the effort of a wild culture just a tiny bit. But still there doesn't seem to be much reason to keep pure, single species greenwater cultures unless you're using it for something that really really needs it, like freshwater rotifers.

[OnlyGenusCaps]

On 12/2/2022 at 10:15 PM, modified lung said:

I thought it was because the population in the wild greenwater fed jars grew fast enough to not allow settling. But in this trial the population growth on the Tetradesmus jar was not that far behind the wild jar. Even so, much of the Tetradesmus settled to the bottom anyway. Idk what would cause such a difference but the settling could be part of why the pure greenwater doesn't do as well as the wild.

I wonder if the wild culture also had hetrotrophs capable of acting as detritavores, which would cycle the nutrients back into use?  I'm an ecologist, it's the way we think about the world.  I could also be misinterpreting what you were conveying, and be totally off base.  That seems likely as well pre-caffeine on a Saturday morning.  😜

[The endler guy]

On 5/8/2022 at 1:11 AM, Torrey said:

I miss having a microscope, looking at hacks on how to turn a cellphone camera into a microscope😁

If you have a local school and thrift store in close proximity some times the school or students will “donate” them

[modified lung]

On 12/3/2022 at 5:25 AM, OnlyGenusCaps said:

wonder if the wild culture also had hetrotrophs capable of acting as detritavores, which would cycle the nutrients back into use? 

I hadn't thought of that. There are a lot of flagellated things swimming around I haven't tried to ID. Nothing worm-like though.

On 12/3/2022 at 5:25 AM, OnlyGenusCaps said:

I'm an ecologist, it's the way we think about the world. 

Sometimes I wish I got more into ecology. I've always been multidisciplinary which of course means I'm a decade behind on each subdiscipline. 🤣 

[modified lung]

Remember the Scenedesmus culture that died out and turned brown on me after adding some Seachem Replenish a couple weeks ago? Last week I inoculated it with some wild greenwater. Here it is now on the right side:

One advantage of mixed greenwater cultures over pure cultures is you don't have to worry about keeping the proper parameters or nutrient ratios for a specific species of microalgae ...which of course leaves more time for naps.

[memorywrangler]

On 5/4/2022 at 5:09 PM, modified lung said:

Moina has been successfully mass cultured with the same feeds as Daphnia. Moina however can be fed fish and canola oils, a higher percentage of suspended bacteria, and cyanobacteria without the initial die off.

Moina is being seriously tested by some hatcheries as a full replacement for baby brine shrimp which has resulted in higher reported fry survival rates. This may be because Moina contains higher levels of a few very important essential nutrients for many fish larvae than BBS.

This is all very excellent information.  Thanks for assembling it.

Do  you have citations for the comments above?  I've looked for more information about culturing moina, but I haven' t found much beyond "Culture Techniques of Moina : The Ideal Daphnia for Feeding Freshwater Fish Fry" by Rottmann et.al.

Thanks.

 

[modified lung]

 

"The Cladoceran as live Feed in Fish Culture: A Brief Review"

http://www.isca.in/AVFS/Archive/v4/i3/2.ISCA-RJAVFS-2016-006.php

"A review on the improvement of cladocera (Moina) nutrition as live food for aquaculture: Using valuable plankton fisheries"

https://www.proquest.com/openview/7b2a351c3c64b3521c729cdecb72c1c8/1?pq-origsite=gscholar&cbl=636374

"Culturing live foods for fish larviculture using non-microalgal diet: The role of waste-generated bacteria and selected commercial probiotics—A review"

https://onlinelibrary.wiley.com/doi/full/10.1002/aff2.33?utm_sq=gs92jbsh0b

 

Enjoy

[modified lung]

Back in July all my Daphnia magna died out. I kept one of the Daphnia buckets in the back yard capped and filled with water. Last week tons of Daphnia started hatching out. Only took half the year.  

NEVER GIVE UP! AND NEVER CLEAN YOUR YARD!

[OnlyGenusCaps]

Your thread here has combined with my burgeoning interest in the saltier side to inspire me to try to create cultures of FW benthic copepods to help with detritus and algal growth in FW aquariums.  On the salty side there is growing appreciation for the role these micro-crustaceans play in holding back unwanted growth and managing detritus.  I'm thinking a good target will be the Canthocamptidae.  This summer I am planning on collecting and testing some.  We'll see how it goes.  Just wanted to let you know your thread here was inspiration for that crazy idea! 

[modified lung]

@OnlyGenusCaps Crazy is the general approach here. I've only seen some of those once. They appeared in my low conductivity, low pH blackwater licorice gourami tank. I don't have an RO unit so there weren't a lot of water changes and a good amount of detritus buildup.

 

[modified lung]

***Freshwater Microalgae (7):  SUCCESS!

We did it, kids. We reached peak greenwater ...or at least the limit of what my RGB analyzer app can detect.

Greenwater cultures on January 25th in window + aquarium light:

Same day under just the aquarium light:

Here's a graph with data from the RGB analyzer app showing the growth of the culture over the last ~5 weeks (the darker red line is surprisingly accurate when compared to readings taken with a spectrometer):

At its peak this culture, the one on the right side, was so green it was almost black in the sun light. At night the aquarium light could barely penetrate the top 25% of the tank's height.

How you say? Although I was having good success with NPK fertilizer, I was getting impatient with the time it takes the fertilizer to dissolve and release the nutrients. On top of that, many of the NPK fertilizers release urea which my ammonia tests can't detect. That means I had no way of knowing if I could safely feed the greenwater to my live food cultures.

So I decided to move away from the fertilizers and go with ammonium hydroxide paired with the Alaska Morbloom PK liquid fertilizer I was already using:

The gallon jug of Ace Hardware ammonia cost only $5 and 1 mL makes 5-6 ppm total ammonia per gallon of water. The Morbloom cost about $10 with 0.25 mL making 4-5 ppm PO4 per gallon. Basically what I'm saying is these two bottles are not very expensive and contain so much nutrients they will last me years.

Nutrient ratios don't seem to matter much using ammonium hydroxide like it seems to using NPK fertilizer. The culture on the right is months old and even completely died and turned brown last November. Somehow the phosphorus levels in the culture has reach unreadable levels. In the past this much extra phosphorus would kill the culture soon after running out of nitrogen. But since switching to ammonium hydroxide, it doesn't seem to matter. I have no idea why.

But it's probably a good idea not to overdo it anyway. Luckily, the two products pictures above come in the perfect amounts. Generally, you want just a little more nitrogen than phosphorus to get your greenwater as nutrient dense as possible. Adding 0.25 mL of the PK fertilizer for every 1 mL of the Ace ammonium hydroxide will not only give you the perfect nutrient ratio, but will empty both bottles at the same time. Convenient.

 

The tank on the left is interesting too. Here's the graph:

The culture on the left started out a little lighter in color than the other. However. It almost completely cleared out due to being contaminated by protists. Probably because I seeded it with greenwater grown outdoors. I decided to let it sit and not a week later the culture began to recover. It's now almost as dark as the culture on the right side. This picture was taken on February 16th:

The conventional advice is that once your culture becomes contaminated, there's no saving it. That doesn't seem to be true. Or maybe it is true because there may not be any protists left but now the culture is contaminated with seed shrimp and rotifers. Oh well.

Anyway, I highly recommend using ammonium hydroxide and liquid PK fertilizer over both NPK fertilizer granules and nutrient media like Guillard's F/2. I haven't been able to get greenwater nearly as dark using NPK fertilizer granules and a small bottle of F/2 costs about the same but will give you far less bang for your buck.