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

Wookii

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I'm so embarassed by my hand writing! Did the C. dubia survive the trip alright?
Lol - it’s better than my handwriting!

Yes, the C Dubia survived just fine, they are in the vase to the right, with two Chlorella cultures set up in the middle:

26DCCE57-939C-4A80-ABD2-B8951FCDFFBB.jpeg
 

MirandaB

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Annoyingly the Biobizz has turned up damaged with the outer top partially smashed off,does anyone know if I can get the inner top open without it if I break the rest off?
A padded envelope is pretty rubbish packaging for something of that size and weight :rolleyes:
 

Wookii

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Not sure - it’s a safety cap, so pressing the outer part down allows you to unscrew the inner cap.

If you can’t get it off you may have to drill the cap and decent it into another bottle.

Annoyingly the Biobizz has turned up damaged with the outer top partially smashed off,does anyone know if I can get the inner top open without it if I break the rest off?
A padded envelope is pretty rubbish packaging for something of that size and weight :rolleyes:
 

Wookii

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My Chlorella cultures seem to be getting a little greener now, with some green at the top, even though my lighting of them isn’t ideal.

But I seem to be getting a lot of green sediment at the bottom, despite them both having decent sized air stones in. I keep giving it a good mix around but it settles back in no time.

Is this the clumping you referred to @louis_last ?

0A5C9FA4-A24E-4A5E-BFD6-DB7982794787.jpeg
 

louis_last

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This looks fine to me, I'm growing it in 2L and 5L bottles with ridged sides and it sort of does this in the ridges, especially at the start of the culure. If you have lids for those containers and can give them a shake that will help get it back into suspension. It should sort itself out though as long as there's water movement. It seems to go back into suspension as it consumes the nutrients in the fish mix. The two big 5L cultures I mentioned setting up the other day did this for two days, I shook them both once each day and now they're back to normal. This is basically what my original culture from blades biological arrived like but in clear water.
I took the airstones off the airline running into the 5L because it looks like the larger bubbles without them disturbs the algae more and created slightly more turbulence.
 

louis_last

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Actually seems it's most likely a defense mechanism in response to some other bacteria present in the mix. Once the chlorella outcompetes it the growth returns to normal.
This temporary defensive strategy might be the result of increased carbohydrate production and secretion induced by macrophyte competition. After removal of macrophytes, colony cells reverted to individual cells and the accumulated carbohydrates in the colony cells could provide sufficient energy for rapid development of the algae.
This quote is obviously talking about plants but it's a general defense mechanism.
 

louis_last

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In an ideal world you'd find a way to shine the LED on the side of those containers too. I'm using an LED bar so I can stand it up upright next to the bottles and I've built a little box lined with tinfoil that goes around them. It's so much faster than when I had them on the windowsill, you can see the green getting noticeably more intense by the end of each day.
 

Wookii

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This looks fine to me, I'm growing it in 2L and 5L bottles with ridged sides and it sort of does this in the ridges, especially at the start of the culure. If you have lids for those containers and can give them a shake that will help get it back into suspension. It should sort itself out though as long as there's water movement. It seems to go back into suspension as it consumes the nutrients in the fish mix. The two big 5L cultures I mentioned setting up the other day did this for two days, I shook them both once each day and now they're back to normal. This is basically what my original culture from blades biological arrived like but in clear water.
I took the airstones off the airline running into the 5L because it looks like the larger bubbles without them disturbs the algae more and created slightly more turbulence.
Actually seems it's most likely a defense mechanism in response to some other bacteria present in the mix. Once the chlorella outcompetes it the growth returns to normal.

This quote is obviously talking about plants but it's a general defense mechanism.

I'll keep giving them a stir - I do have lids, but removing the air stones etc would be a bit of a pain.

In an ideal world you'd find a way to shine the LED on the side of those containers too. I'm using an LED bar so I can stand it up upright next to the bottles and I've built a little box lined with tinfoil that goes around them. It's so much faster than when I had them on the windowsill, you can see the green getting noticeably more intense by the end of each day.

Yes, the way I've set them up was a bit of a rush job, and I need to sort it out better. There arguably isn't enough light on them at the minute.

How do you know when to start using the Chlorella culture - when the brown colouration from the liquid is completely gone?

Do you think its possible to maintain a Chlorella culture on an ongoing basis - e.g. if you ran it in a, say, a 20 litre bucket, once its ready, take a litre out to feed, and add a litre of clean water back in with a little more BioBizz mix in it, and keep going like that - rather than draining a culture and restarting? Or does the water turn bad after a while?

I also read this article - it applies specifically to daphnia, but may be relevant to Moina also:


6.1.4.3. Autotrophic system​

Autotrophic systems on the other hand use the addition of cultured algae. Green water cultures (105 to 106 cells.ml-1) obtained from fish pond effluents are frequently used but these systems show much variation in production rate mainly because of the variable composition of algal species from one effluent to another. Best control over the culture medium is obtained when using pure algal cultures. These can be monocultures of e.g. algae such as Chlorella, Chlamydomonas or Scenedesmus, or mixtures of two algal cultures. The problem with these selected media is that they are not able to sustain many Daphnia generations without the addition of extra vitamins to the Daphnia cultures. A typical vitamin mix is represented in Table 6.1.

Table 6.1. A vitamin mix for the monospecific culture of Daphnia on Selenastrum, Ankistrodesmus or Chlamydomonas. One ml of this stock solution has to be added to each litre of algal culture medium (Goulden et al., 1982).



Nutrient
Concentration of stock solution (µg.1-1)
Biotin
5​
Thiamine
100​
Pyridoxine
100​
Pyridoxine
3​
Calcium Panthothenate
250​
B12 (as mannitol)
100​
Nicotinic acid
50​
Nicotinomide
50​
Folic acid
20​
Riboflavin
30​
Inositol
90​

To calculate the daily algal requirements and to estimate the harvesting time, regular sampling of the population density must be routinely undertaken. Harvesting techniques can be non-selective irrespective of size or age group, or selective (only the medium sized daphnids are harvested, leaving the neonates and matured individuals in the culture tank).


Mass cultivation of Daphnia magna can also be achieved on cheap agro-industrial residues, like cotton seed meal (17 g.l-1), wheat bran (6.7 g.l-1), etc. Rice bran has many advantages in comparison to other live foods (such as microalgae): it is always available in large quantities, it can be purchased easily at low prices, it can be used directly after simple treatment (micronisation, defatting), it can be stored for long periods, it is easy to dose, and it has none of the problems involved in maintenance of algal stocks and cultures.

In addition to these advantages, there is also the fact that rice bran has a high nutritional value; rice bran (defatted) containing 24% (18.3%) crude protein, 22.8% (1.8%) crude fat, 9.2% (10.8%) crude fibre, and being a rich source of vitamins and minerals. Daphnia can be grown on this food item for an unlimited number of generations without noticeable deficiencies.

Defatted rice bran is preferred above raw rice bran because it prevents hydrolysis of the fatty acids present and, consequently, rancidity of the product. Micronisation of the bran into particles of less than 60 µm is generally carried out by treating an aqueous suspension (50 g.l-1) with a handmixer and filtering it through a 60 µm sieve, or by preparing it industrially by a dry mill process. The suspension is administered in small amounts throughout a 24 h period: 1 g of defatted rice bran per 500 individuals for two days (density: 100 animals.l-1). The food conversion ratio has an average of 1.7, which implies that with less than 2 kg of dry rice bran approximately 1 kg wet daphnid material can be produced (with a 25% water renewal per week; De Pauw et al., 1981).

Key points for me were:

a) the suggestion that adding pure Chlorella may require additional vitamin supplementation - have you come across this?
b) The suggestion of 'defatted rice bran' as an optional food source. Though I have no idea where one would source 'defatted rice bran' from - the suggestion that it can "prevent . . . rancidity" presumably means its less likely to foul the water, and "this food item for an unlimited number of generations without noticeable deficiencies"
 

Wookii

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Now the my Moina culture has been running a little while I am noticing that I do get a die off of the adults a day or so after they reach a larger size. I don't think its a crash per se, but I'm unsure what the issue is. Criticlally though I don't seem to be getting a consistent increase in population, as the visible amount of individuals indicates, which would suggest I'm getting similar numbers of births and deaths, and not a lot to harvest off. I've managed one feed in 5 days.

I know they don't have a very long lifespan anyway, but I am wondering if the lack of minerals in the water was causing an early death once mature. Following the previous discussion in this thread, I did the water change partially with tap water this week, with no apparent ill effects, so hopefully this will help with any mineral issues, or at least exclude it as a factor.
 
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louis_last

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How do you know when to start using the Chlorella culture - when the brown colouration from the liquid is completely gone?
What I find is that once it's a nice opaque emerald green, if you turn the airpump off and let the culture settle a bit, for some reason especially in tall bottles the top inch or so of water especially around the edges seems to clear. When there's still a lot of nutrients left from the fishmix this water still has a fairly brown tint but once all the fishmix has been consumed it's actually clear with just a green tint to it.
Do you think its possible to maintain a Chlorella culture on an ongoing basis - e.g. if you ran it in a, say, a 20 litre bucket, once its ready, take a litre out to feed, and add a litre of clean water back in with a little more BioBizz mix in it, and keep going like that - rather than draining a culture and restarting? Or does the water turn bad after a while?
I think you almost certainly can, at least for a while, because I re-dose fishmix into most of my bottles once they've reached a nice density and appear to have consumed all the original fishmix and they get even more green. Eventually I assume you might need to replace the water though. I find it's easier once a bottle is ready to just pour a little off to start a new culture and then feed directly from that bottle while the new one is brewing. I've had no issues removing them from the light and just keeping them on a cool shelf once they're ready, lasts for at least a week this way and I just give it a shake before feeding. I think it would probably even be ok in the fridge, I was thinking about decanting it into squeezy ketchup bottles.
I also read this article - it applies specifically to daphnia, but may be relevant to Moina also:




Key points for me were:

a) the suggestion that adding pure Chlorella may require additional vitamin supplementation - have you come across this?
At least for moina specifically I can 100% confirm that you can produce many generations feeding solely on Chlorella however I do still give them a little bit of yeast sometimes. I think this may be more relevant to some species than others, I've got a few cultures going under different conditions of the ceriodaphnia dubia I sent you for example and it's beginning to appear that they might do better on a mixed diet of algae and yeast. There's been no difference in production I could testify to yet but the ones fed only on chlorella are a slightly different colour and look a little bit sort of washed out. The same is not true for the moina, if anything the opposite seems to be the case. I also consider the possibility that the organic fish mix contains a much wider array of minerals than a basic chemical formula and therefore algae grown on it may be more nutritious but that's pure speculation. I've actually been wondering whether you could feed the fish mix itself to moina, I have certainly given them chlorella from a culture that hadn't used up all the fish mix before with no ill effects.
b) The suggestion of 'defatted rice bran' as an optional food source. Though I have no idea where one would source 'defatted rice bran' from - the suggestion that it can "prevent . . . rancidity" presumably means its less likely to foul the water, and "this food item for an unlimited number of generations without noticeable deficiencies"
Not sure about defatted rice bran but I found this today and thought you might find it interesting. I'm going to try and get ahold of some dero worms to experiment with. I managed to suck a few out the water feature in my vivarium with a pipette but only about 10.
co culture of daphnia/moina and dero worms
 

louis_last

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Now the my Moina culture has been running a little while I am noticing that I do get a die off of the adults a day or so after they reach a larger size. I don't think its a crash per se, but I'm unsure what the issue is. Criticlally though I don't seem to be getting a consistent increase in population, as the visible amount of individuals indicates, which would suggest I'm getting similar numbers of births and deaths, and not a lot to harvest off. I've managed one feed in 5 days.

I know they don't have a very long lifespan anyway, but I am wondering if the lack of minerals in the water was causing an early death once mature. Following the previous discussion in this thread, I did the water change partially with tap water this week, with no apparent ill effects, so hopefully this will help with any mineral issues, or at least exclude it as a factor.

It could be something to do with minerals. I use water from my tank for all the water changes and it presumably had minerals present from the aquasoil, rooibos tea etc.
There's something very wrong if you're getting a similar number of births and deaths with Moina though and it probably implies that at least some are dying long before they reach adult size as with each moult on the way to adult size they should be releasing several young. You're definitely using bakers yeast and not brewers yeast? maybe check whether there's any weird ingredients listed on the packets, there could be some weird stabiliser or something that accumulates in your water.
If I was you I'd isolate a few and try and get a culture going in a small jar, I really think there might be something weird going on with the size of your container and relatively low population density. Let me try and get a video of a small culture I have going in a tub that I subdivided last week and have fed only on chlorella so you can compare.
 

Wookii

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What I find is that once it's a nice opaque emerald green, if you turn the airpump off and let the culture settle a bit, for some reason especially in tall bottles the top inch or so of water especially around the edges seems to clear. When there's still a lot of nutrients left from the fishmix this water still has a fairly brown tint but once all the fishmix has been consumed it's actually clear with just a green tint to it.

Thanks, that's useful as always.


I think you almost certainly can, at least for a while, because I re-dose fishmix into most of my bottles once they've reached a nice density and appear to have consumed all the original fishmix and they get even more green. Eventually I assume you might need to replace the water though. I find it's easier once a bottle is ready to just pour a little off to start a new culture and then feed directly from that bottle while the new one is brewing. I've had no issues removing them from the light and just keeping them on a cool shelf once they're ready, lasts for at least a week this way and I just give it a shake before feeding. I think it would probably even be ok in the fridge, I was thinking about decanting it into squeezy ketchup bottles.

At least for moina specifically I can 100% confirm that you can produce many generations feeding solely on Chlorella however I do still give them a little bit of yeast sometimes. I think this may be more relevant to some species than others, I've got a few cultures going under different conditions of the ceriodaphnia dubia I sent you for example and it's beginning to appear that they might do better on a mixed diet of algae and yeast. There's been no difference in production I could testify to yet but the ones fed only on chlorella are a slightly different colour and look a little bit sort of washed out. The same is not true for the moina, if anything the opposite seems to be the case. I also consider the possibility that the organic fish mix contains a much wider array of minerals than a basic chemical formula and therefore algae grown on it may be more nutritious but that's pure speculation. I've actually been wondering whether you could feed the fish mix itself to moina, I have certainly given them chlorella from a culture that hadn't used up all the fish mix before with no ill effects.

That's a good point, the molasses and fish extracts alone probably contain all of the vitamins mentioned, I didn't think of that.

Not sure about defatted rice bran but I found this today and thought you might find it interesting. I'm going to try and get a hold of some dero worms to experiment with. I managed to suck a few out the water feature in my vivarium with a pipette but only about 10.
co culture of daphnia/moina and dero worms

That is really interesting. I have wondered it was worth adding a sponge air bubble filter to the tank on the basis that a mature filter might provide additional bacteria to the water column for the Moina to feed on, and also help deal with any ammonia present - I just didn't know whether the Moina would end up getting sucked into the filter and trapped in the sponge?

Those worms however would seem to be ideal if they essentially feed the Moina directly and also provide another live food source at the same time. Will you be trying to source any? I see a couple of sources on eBay. I'm surprised they aren't mentioned more on this forum - I wonder if @dw1305 has cultured them?
 

louis_last

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20210330_155743.jpg

Hard to get the lighting right to show you what I'm talking about but you can see here a little bit of sedimentation in the ridges of the bottles similar to what you're seeing in your own. There was more a few days ago but it quickly sorts itself out as the algal cells use up the fish mix. These are the two bottles I mentioned setting up last thursday evening as a side by side comparison. It turned out I don't have the dry ferts I thought I did for the comparison so these are both just fish mix but you can get a sense that probably by around friday these will be ready to either use straight away or add more fishmix for an even denser culture. One of these bottles will feed all my cultures for a couple of weeks.
 

louis_last

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Those worms however would seem to be ideal if they essentially feed the Moina directly and also provide another live food source at the same time. Will you be trying to source any? I see a couple of sources on eBay. I'm surprised they aren't mentioned more on this forum - I wonder if @dw1305 has cultured them?
I'm holding off on buying the ebay ones from Denmark until I've asked around on here incase anyone has them. Seems like too much potential for brexovid shenanigans ordering from Denmark.
 

Wookii

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It could be something to do with minerals. I use water from my tank for all the water changes and it presumably had minerals present from the aquasoil, rooibos tea etc.
There's something very wrong if you're getting a similar number of births and deaths with Moina though and it probably implies that at least some are dying long before they reach adult size as with each moult on the way to adult size they should be releasing several young. You're definitely using bakers yeast and not brewers yeast? maybe check whether there's any weird ingredients listed on the packets, there could be some weird stabiliser or something that accumulates in your water.
If I was you I'd isolate a few and try and get a culture going in a small jar, I really think there might be something weird going on with the size of your container and relatively low population density. Let me try and get a video of a small culture I have going in a tub that I subdivided last week and have fed only on chlorella so you can compare.

Yes, its definitely bakers yeast. This:

Amazon product
Do you ever see any dead Moina in your cultures then?

I'm holding off on buying the ebay ones from Denmark until I've asked around on here incase anyone has them. Seems like too much potential for brexovid shenanigans ordering from Denmark.

These ones are in the UK:

 
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