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HOW TO: Clean, easy and highly nutritious greenwater culture for Daphnia and Moina.

Wookii

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This is exactly the sort of thing I was hoping to see when I sent out these cultures. Please let us know how this works out, if you do get good growth of the chlorella in here you could try adding chicken manure with the moina to act as fertiliser for the chlorella and a source of various fungi etc. for the moina.

That’s a good idea, I might do that. I think I can get small bags off Amazon - I don’t want to be stuck with 25Kg of the stuff! 🐓💩
 

Wookii

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I'm still very curious what results you might get with your dry ferts and the chlorella too @Wookii, in an ideal world the fish mix could smell slightly better when you first start running air through it.

I will give that a go at some point, though I’d need to figure out how to add the additional vitamins in too.

I harvested some Moina last night, which the fish were very pleased about, however I haven’t added any more Chlorella in the last couple of days, yet the tank isn’t clearing, it’s actually getting decreased visibility every day. It now looks like this:

B6424288-13F9-4B06-9A34-23B764E11CE1.jpeg


I don’t know if it is the Chlorella multiplying faster than the Moina can eat it in the relatively nutrient rich tap water and new very high output light, or if there is a bacterial bloom from the 5% water left from the water change (which would still have had yeast in it). Moina numbers seem to be getting higher as far as I can tell, as there are clouds of them in all corners of the tank now. I was just expecting the water to be clearing rapidly, not going the other way?

Any thoughts?
 

louis_last

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I will give that a go at some point, though I’d need to figure out how to add the additional vitamins in too.

I harvested some Moina last night, which the fish were very pleased about, however I haven’t added any more Chlorella in the last couple of days, yet the tank isn’t clearing, it’s actually getting decreased visibility every day. It now looks like this:

View attachment 166513

I don’t know if it is the Chlorella multiplying faster than the Moina can eat it in the relatively nutrient rich tap water and new very high output light, or if there is a bacterial bloom from the 5% water left from the water change (which would still have had yeast in it). Moina numbers seem to be getting higher as far as I can tell, as there are clouds of them in all corners of the tank now. I was just expecting the water to be clearing rapidly, not going the other way?

Any thoughts?
I'm not sure really, it's weird that it doesn't look more green but it could well be growing from potential nutrients in the tapwater, or maybe unconsumed nutrients left from the fish mix? I've never had a bacterial bloom in a moina or daphnia culture really, not of free floating bacteria anyway because they tend to get eaten by the moina. I suppose there must be some species they couldn't eat though. Does it pass the sniff test? If the water smells at all bad I'd do a big water change.
On the other hand if it is just the chlorella growing then I'd say that's a good thing, the moina population will boom until it catches up and you have to start feeding again.
I often add enough chlorella that the water stays opaque for a few days and when it's like this it can be harder to see the tiny babies when they first hatch so it's not as easy to get a sense of the real numbers.
I was messing around with the chlorella today and managed to 'flocculate' a batch with a product derived from squid called chitosan. It counteracts the negative charge that keeps the chlorella cells in suspension and causes them to stick to each other and fall to bottom of the container. I poured the water off and made some gel food mixing it with a bit of bee pollen, garlic and engevita yeast flakes. It worked pretty well, I used gelatin rather than agar because it doesn't need to be heated to as high a temperature. Seems like by putting it in agar I'd cook/kill the chlorella whereas this way I can just pour a little warm gelatin into the cold chlorella and it's actually alive when the shrimp eat it. I need to look at some of the other plant based alternatives.
I found this paper which seems to demonstrate, that daphnia magna at least, get much more nutritional value out of the living algae. There are also a lot of references online to the loss of nutritional value for humans caused by the freeze/spray drying methods used to process spirulina and chlorella at the industrial scale.
Freeze-dried Chlorella vulgaris as food for Daphnia magna Straus in toxicity testing
The shrimp and snails seem to like it, not more than any other gel food, and I'm not sure it's really going to have any noticeable impact on their health longterm that would justify the effort for most people, but I think it could be really useful for shop starved otocinclus in particular and maybe baby shrimp in very clean tanks.
Also, I did a big water change on my moina culture today and noticed that the water is actually pretty tannin stained because the fresh water is coming from my aquarium that I add rooibos and blackwater extract to. It reminded me of this other paper I read about an experiment involving moina and leaf litter leachates that were basically blackwater extract.
Leaf litter leachates have the potential to increase lifespan, body size,and offspring numbers in a clone ofMoina macrocopa
xcept of P. abies, exposure to the leachates reduced this antioxidant capacity by approximately 50%. Leachate exposures, except that of Quercus, increased body size andextended lifespan; furthermore, particularly the leachates of both Picea species significantly increased the off spring numbers. This unexpected behavior of exposed Moina may be based on food supplements (e.g., high carbohydrate contents) in the leachates or on yet to be identified regulatory pathways of energy allocation. Overall, our results suggest that the potentially adverse effects of litter leachates can be over ruled by either bacterial-growth supporting fractions in the leachates or an internal compensation mechanism in the Moina individuals.
This was what originally made me start adding leaves to my cultures and I think the botanicals coming from the boraras tank might help too. You can also find papers online that suggest tannin specifically acts as a stressor on some daphnia though so who knows, but I hadn't really noticed how tinted the water in my culture is before.
 

Maf 2500

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Maybe just a tortoise (Moina) and hare (Chlorella) situation. A race can be dynamic and not always follow the same path as previous races, particularly if the racecourse has changed..

(Very much enjoying the thread BTW, informative, educational and interesting.)
 

Wookii

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I'm not sure really, it's weird that it doesn't look more green but it could well be growing from potential nutrients in the tapwater, or maybe unconsumed nutrients left from the fish mix? I've never had a bacterial bloom in a moina or daphnia culture really, not of free floating bacteria anyway because they tend to get eaten by the moina. I suppose there must be some species they couldn't eat though. Does it pass the sniff test? If the water smells at all bad I'd do a big water change.

It doesn’t have any odour as far as I can tell. I’ll just have to keep an eye on it, if the population seems to decline, I’ll do another water change.


I was messing around with the chlorella today and managed to 'flocculate' a batch with a product derived from squid called chitosan. It counteracts the negative charge that keeps the chlorella cells in suspension and causes them to stick to each other and fall to bottom of the container. I poured the water off and made some gel food mixing it with a bit of bee pollen, garlic and engevita yeast flakes. It worked pretty well.

Good stuff - nice that you’ve been able to use a natural product too.


I used gelatin rather than agar because it doesn't need to be heated to as high a temperature. Seems like by putting it in agar I'd cook/kill the chlorella whereas this way I can just pour a little warm gelatin into the cold chlorella and it's actually alive when the shrimp eat it. I need to look at some of the other plant based alternatives.

Because I add things like Bacter AE to my gel food, I always let the Agar Agar cool first. I heat it to dissolve the powder ofcourse, and just before it boils take it off the heat and set the pan in a sink full of cold water.

I don’t add any ingredients until it’s cooled to around 40 degrees. I’m not sure if that is low enough to prevent the Chlorella dying, but I would have though so? Especially as I decant it into the plastic mould straight away, and it cools very quickly then.

That said, I always freeze most of what I make, so I’m not sure if that would kill the Chlorella anyway? Either way, alive or recently deceased, it’s got to be more nutritious than the dried stuff?
 

louis_last

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Because I add things like Bacter AE to my gel food, I always let the Agar Agar cool first. I heat it to dissolve the powder ofcourse, and just before it boils take it off the heat and set the pan in a sink full of cold water.

I don’t add any ingredients until it’s cooled to around 40 degrees. I’m not sure if that is low enough to prevent the Chlorella dying, but I would have though so? Especially as I decant it into the plastic mould straight away, and it cools very quickly then.
That's actually really good to know. I thought agar set too much at a higher temperature than that, THIS paper confirms that 40 degrees isn't a significantly fatal temperature for the chlorella. As long as the chitosan doesn't affect it setting I'm going to try switching to agar, I knew gelatin would be ok because they mix it with chitosan to construct organic scaffolds during the repair of some human injuries.
That said, I always freeze most of what I make, so I’m not sure if that would kill the Chlorella anyway? Either way, alive or recently deceased, it’s got to be more nutritious than the dried stuff?
I know chlorella can definitely be frozen and then thawed and revived so it doesn't necessarily kill it but I don't know how technical you have to get. On agar plates it can be stored for a long time in the fridge in the cold and total darkness. I have one in my fridge right now, still green, and I still think I could start a culture from it.
 

louis_last

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The Ceriodaphnia are really starting to take off, these were tricky to get to this point but if anyone else wants a starter culture to experiment with just give me a shout. Being even smaller than moina they are really great for fry or tiny fish, my Boraras maculatus and scarlet badis love them. Their behaviour in the tank is very different to moina, they immediately swim down and hide in the plants whereas moina tend to remain swimming freely in the water column.
The population in this culture has grown in proportion to the amount of biofilm and 'aufwuchs' you can see here. In fact these structures are made from filamentous algae covered in miscellaneous biofilm and provide some sort of refuge for the young ceriodaphnia, if I shine a bright torch on the blobs thousands of tiny ceriodaphnia all emerge from them at once. I think that when the young first emerge they might need to feed on cells that are smaller than either yeast or chlorella as I've never been able to get the numbers this high in a 'clean' culture feeding with either or both.
Interestingly it seems possible to co-culture them with moina as they are now present in almost all my moina cultures due to cross contamination from using the same pipette to harvest from all the tubs. Neither the moina or ceriodaphnia seem affected by the presence of the other and because they have different degress of phototropism they tend to naturally segregate themselves into swarms but in different locations within the culture. I actually originally isolated these from one of my moina cultures.
 

Wookii

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So the weirdness in my Moina culture continues.

It’s got even cloudier today. In the last couple of days, having not fed the tank with Chlorella due to the cloudiness, I started to notice all the Moina hanging around at the very bottom of the tank.

Tonight when I have gone to check the tank, I see that the green layer of snail pop has almost completely disappeared!

48C1E0E3-14DB-423E-A520-1AC97F354093.jpeg


I can only deduce that the Moina have eaten it. I have no idea how they managed it, as I assumed they were filter feeders? But it’s definitely been hoovered up in short order.

So as they are that hungry, I decided to add some more Chlorella. Within about a minute they all rose up like a cloud from the bottom up into the water column.

I can only deduce therefore, whatever is clouding the water, isn’t edible to the Moina, so I’ll water change tomorrow to try and get rid of it. Luckily I don’t see any dead Moina anywhere as yet.
 
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louis_last

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So the weirdness in my Moina culture continues.

It’s got even cloudier today. In the last couple of days, having not fed the tank with Chlorella due to the cloudiness, I started to notice all the Moina hanging around at the very bottom of the tank.

Tonight when I have gone to check the tank, I see that the green layer of snail pop has almost completely disappeared!

View attachment 166580

I can only deduce that the Moina have eaten it. I have no idea how they managed it, as I assumed they were filter feeders? But it’s definitely been hoovered up in short order.

So as they are that hungry, I decided to add some more Chlorella. Within about a minute they all rose up like a cloud from the bottom up into the water column.

I can only deduce therefore, whatever is clouding the water, isn’t edible to the Moina, so I’ll water change tomorrow to try and get rid of it. Luckily I don’t yet see any dead Moina anywhere as yet.
I wouldn't wait until tomorrow. I'd do a big water change tonight if you can as this sounds a lot like a culture that's about to crash real bad. I think it's possible there was too much chlorella at once and after it used up all the nutrients present in the water -, as it began dying, some kind of bacteria that the moina cant eat has taken over and is multiplying from the dead chlorella.
I hope I'm wrong given that the idea here is to avoid complications due to water fouling but I don't want your culture to crash. I think it's more likely that the bacteria has eaten the snail poo than the moina and there's a bad chance that you will wake up to a lot of dead moina in the morning if you do nothing.
I think overall this is probably just a step towards your culture 'maturing' though.
It's also possible that there was some kind of bacterial contamination that caused the chlorella to clump together and come out of suspension as a defense mechanism and that this is largely what was actually settled on the floor of the tank - now as the populations fluctuate maybe the chlorella is returning to suspension in the water column? I never know how to interpret the behaviour of the moina, sometimes they all swarm against the bottom of the tank, sometimes they're very evenly distributed, there doesn't seem to be a recogniseable pattern but they're telling us something with this behaviour. The only thing I know for sure is that every single time I've catastophically crashed a culture they have all formed a very very tight swarm at the surface shortly before hand, every time I've noticed this they've almost all been dead within hours.
I've only had something similarish happen when feeding chlorella once and it was in a smaller backup culture. I think I gave them too much when the population density couldn't process it and the water never became less cloudy, instead it remained opaque and cloudy like when I first added the chlorella but started turning much more yellowish.
I reckon you should stop harvesting them at all for a week or so to let their numbers get up to a decent level in your larger tank. It will allow you ultimately to harvest far more and there's much less scope for overfeeding, whether it's yeast, chlorella or anything else as they are more likely to consume it before any significant water fouling can happen. Remember that these can supposedly maintain populations of multiple thousands per litre. If you have less than 10,000 moina in a 20L culture it's quite unpopulated. It's always hard to tell from pictures but at a glance it looks like I probably have ten times as many in a container less than half the size and I'm not having any problems.
Something else I've realised lately is that I always thought moina began reproducing almost immediately upon being born but now I don't think this is the case at all, at least whatever species I have seems to have several instars before finally moulting and taking on its quite distinct and more spherical adult form at which point they seem to moult and produce young almost every day. Only these adults seem to produce more moina, and very explosively, but it's easy to hinder your population growth by harvesting too many of the adults because they are more prone to swarming than the smaller youngsters. Right now in my large culture there's a big swarm of adults right under the LED bar, a big swarm of ceriodaphnia in one corner, and thousands of babies distributed pretty much evenly everywhere else. It's very easy to always go after the low hanging fruit and just suck the adults up with a pipette but if I turn the lights off and attract them with a torch after introducing food, they all seem to be evenly attracted to the light when there is a high density of food in the water. I even use a plankton seive sometimes to only harvest babies. I think you can accidentally really hobble their population growth by preferentially harvesting reproductive adults unintentionally. I'm not suggesting this is something that you're doing @Wookii, just that it's worth bearing in mind for anyone else reading the thread.
 

louis_last

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One other possibility for the change in appearance of the chlorella by the way is just that it's adapting to the lower light levels in your moina tank and exploiting some other source of carbon in the water, or maybe even the snail poo to produce heterotrophic growth. In a purely heterotrophic culture the chlorella is actually yellow. What we grow with the fishmix is technically a mixotrophic culture but you can also grow it in complete darkness feeding glucose.
Normally in a mixotrophic culture it goes the other way with primarily heterotrophic growth occuring first followed by a transition to more autotrophic growth but there could be something weird going on here. It's possible all the sugars from the fishmix weren't consumed and due to a reduction in light the chlorella has reverted to more heterotrophic growth?
 

Wookii

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Thanks @louis_last - OK, so I’ve done a 95% water change this morning - the colouration of the water didn’t seem to have affected the Moina population to be honest - there are literally thousands of them now I can see them in the clear water.

You are probably right in not harvesting too early - it’s difficult to know what the right population is - it’s a bit unnatural to see so many individuals in such a small amount of water. I do use a fine brine shrimp net to harvest though, so I definitely a get mixture if individuals from adults to babies.

It’s definitely not lack of light on the Chlorella - the AI Prime I have over it is a light cannon, and it’s running at 100%. I probably wouldn’t run it at that level on a 400mm deep planted tank. It’s probably 10 times the light that was over the original Chlorella culture bottle.

Also, the green layer in the base of the tank wasn’t settled live Chlorella, it was there before I started adding the live Chlorella. It was mainly snail poo - I’ve seen the snails ‘producing’ it in long strands. There may have been some of the dried Chlorella that had settled too, from when I was feeding that with the yeast.

It’s perfectly plausible that the bacteria - if that is what the bloom was - consumed it, but it’s still pretty amazing that it disappeared in the space of two days.

I have the sponge from the new bubble filter currently ‘maturing’ inside my main tank canister filter. I’m not sure how long I should leave it in there though? However once it’s ready, I’ll add the filter into the Moina tank to see if that adds some bacterial stability.
 

louis_last

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Thanks @louis_last - OK, so I’ve done a 95% water change this morning - the colouration of the water didn’t seem to have affected the Moina population to be honest - there are literally thousands of them now I can see them in the clear water.

You are probably right in not harvesting too early - it’s difficult to know what the right population is - it’s a bit unnatural to see so many individuals in such a small amount of water. I do use a fine brine shrimp net to harvest though, so I definitely a get mixture if individuals from adults to babies.

It’s definitely not lack of light on the Chlorella - the AI Prime I have over it is a light cannon, and it’s running at 100%. I probably wouldn’t run it at that level on a 400mm deep planted tank. It’s probably 10 times the light that was over the original Chlorella culture bottle.

Also, the green layer in the base of the tank wasn’t settled live Chlorella, it was there before I started adding the live Chlorella. It was mainly snail poo - I’ve seen the snails ‘producing’ it in long strands. There may have been some of the dried Chlorella that had settled too, from when I was feeding that with the yeast.

It’s perfectly plausible that the bacteria - if that is what the bloom was - consumed it, but it’s still pretty amazing that it disappeared in the space of two days.

I have the sponge from the new bubble filter currently ‘maturing’ inside my main tank canister filter. I’m not sure how long I should leave it in there though? However once it’s ready, I’ll add the filter into the Moina tank to see if that adds some bacterial stability.

I was probably over reacting and the strong light was just causing the chlorella to bloom in the culture, I'm glad there were no signs of a crash.
To be honest the disappearing layer of snail poo is really mysterious, I can't quite figure out what's going on there. Maybe the moina did eat it somehow but it's weird either way.
By all accounts the sponge filter will be a big help. I'm curious how it will work out because I don't have much experience with air driven sponge filters and it's always seemed to me like, in order for there to be enough water flowing through for it to be an effective biological filter, it would suck up baby moina. Plenty of people say it's made their cultures much more stable though, it's just not an option in the current container I use because of how shallow and flat it is but if you get good results I might upgrade to a bigger tub with a sponge filter too.
 

louis_last

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@Wookii you were right about the agar, much better results. The gelatin batch turned into a sloppy disgusting mess when the room temperature spiked by a couple of degrees so I made some with agar today. Messed up the first one by trying to be smart and boiling a teaspoon of agar in 100ml of water than adding that to 400ml of flocculated algae but obviously the agar just set the minute it touched the cold algae and didn't mix. Second batch I warmed the algae to 40c while the agar was cooling from 100c and when they met roughly in the middle I mixed them and it set just fine. I'm going to set some aside in the fridge for a long while and see if I can still start a culture from it after maybe a month. In the meantime the shrimp love it.
 

Wookii

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@Wookii you were right about the agar, much better results. The gelatin batch turned into a sloppy disgusting mess when the room temperature spiked by a couple of degrees so I made some with agar today. Messed up the first one by trying to be smart and boiling a teaspoon of agar in 100ml of water than adding that to 400ml of flocculated algae but obviously the agar just set the minute it touched the cold algae and didn't mix. Second batch I warmed the algae to 40c while the agar was cooling from 100c and when they met roughly in the middle I mixed them and it set just fine. I'm going to set some aside in the fridge for a long while and see if I can still start a culture from it after maybe a month. In the meantime the shrimp love it.

Nice one, I’m glad it worked. I didn’t think about the flocculated algae cooling the agar agar, I’ve only ever added dry ingredients. You can add assorts into the agar agar, I tend to add:

Ground Bee Pollen
Bacter AE
Chlorella
Spirulina
Astaxanthin
Ground Shrimp Mineral
+ a few ml of Seachem Nourish vitamins
 

Wookii

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I was probably over reacting and the strong light was just causing the chlorella to bloom in the culture, I'm glad there were no signs of a crash.
To be honest the disappearing layer of snail poo is really mysterious, I can't quite figure out what's going on there. Maybe the moina did eat it somehow but it's weird either way.
By all accounts the sponge filter will be a big help. I'm curious how it will work out because I don't have much experience with air driven sponge filters and it's always seemed to me like, in order for there to be enough water flowing through for it to be an effective biological filter, it would suck up baby moina. Plenty of people say it's made their cultures much more stable though, it's just not an option in the current container I use because of how shallow and flat it is but if you get good results I might upgrade to a bigger tub with a sponge filter too.

No, I don’t think you were overreacting, it was a valid concern, and still is. I’m still not convinced that the clouding was Chlorella, given how all the snail poo was consumed, and how all the Moina were at the bottom of the tank. Every time I’ve fed them Chlorella since, they all cloud up near the surface to feed.

If the clouding was bacteria, I can’t understand why the Moina weren’t feeding on it and clearing the water given their high numbers - as far as I understand it they are indiscriminate filter feeders and can’t pick and choose what they eat.

I was hoping @dw1305 would have a theory as our resident bacteria and water quality expert?

As to the sponge filter, I’ll have to try it an see if it sucks in baby Moina - I’ve never used one before either - I suspect the flow won’t be strong enough, and the sponge is a very fine type. I imagine the quantity of ammonia generated is fairly low, so a slow flow through the filter sponge should be fine (hopefully).
 

louis_last

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as far as I understand it they are indiscriminate filter feeders and can’t pick and choose what they eat.
I'm not sure about this, I don't have a link to hand right now but I found a study where they demonstrated that at least they have the ability to sort and preferentially consume or discard particles based on size. They fed them two types of algae with different average cell sizes but that they knew were both capable of sustaining the moina. Ultimately all of the algae was consumed but they ate the smaller cells first or something like that.
 
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