HOB for phyto culturing...?

[creativeballance]

I've been having issues with my phytoplankton culture, and I think that there may be contaminants inside of my air pump. I am wondering if it is possible to use a HOB filter instead? I would remove all inserts.
Culture would be in a 10 gallon tank.

 

[ichthyogeek]

What makes you think of contaminants inside of the air pump? As opposed to contaminants from other sources (hands, the nearby reef tank, etc.)?

I feel that a 10 gallon tank would have too many deadspots from flow unless you did it very carefully. A 10 gallon tub would be better.

 

[creativeballance]

What makes you think of contaminants inside of the air pump? As opposed to contaminants from other sources (hands, the nearby reef tank, etc.)?

I feel that a 10 gallon tank would have too many deadspots from flow unless you did it very carefully. A 10 gallon tub would be better.

I've sterilized the water. Used new plastic bottles. New innoculant disks. New hoses and air stones. I've tried new everything except for a new pump and a new bottle of F1.

I was able to get a good culture going before in the 10 gallon using a pair of airstones, but it gradually stopped working correctly.

I'm trying to rule out the pump or the bottle of F1, and I have an extra unused hob lying around.

 

[ichthyogeek]

Ok, cool! But...what's the contaminant? Or rather, what's the thinking behind "it's a contaminant" aside from an algae crash due to lack of nutrients?

How did the culture stop working correctly?

The filter could work. I run one rated for 29 gallons on a 10 gallon tank, and it doesn't shred the BBS I add in for feeding. I personally think a circulation pump would be better, but ya gotta make do when ya gotta make do.

 

[creativeballance]

Ok, cool! But...what's the contaminant? Or rather, what's the thinking behind "it's a contaminant" aside from an algae crash due to lack of nutrients?

How did the culture stop working correctly?

The filter could work. I run one rated for 29 gallons on a 10 gallon tank, and it doesn't shred the BBS I add in for feeding. I personally think a circulation pump would be better, but ya gotta make do when ya gotta make do.

The culture kept turning white after a couple of days.

 

[ichthyogeek]

So that doesn't sound like a contaminant to me. It could be a bacteria that's causing it...but it sounds like something might be wrong with either the F1, or the algae itself. What strain are you using?

I have another idea, but let me dig up my plankton culture manual first...I'll get back to this.

 

[creativeballance]

So that doesn't sound like a contaminant to me. It could be a bacteria that's causing it...but it sounds like something might be wrong with either the F1, or the algae itself. What strain are you using?

I have another idea, but let me dig up my plankton culture manual first...I'll get back to this.

I've tried a from a bottle of, I think, Nanna. Only goes for about 2 days. Clumps and turns white.
I tried a disk of tetra. It greened up, but after I moved it to a bottle of water and aerated, it turned white.

And thank you for taking an interest in my problem!

 

[dimsim]

If airborne contaminants are a concern you could use a sterile syringe filter on the airline (not sure if these fit on standard 1/4" tubing, might need some kind of luer lock/barb adaptors...).

How are you sanitizing everything? Bleach/thiosulfate?

Not sure how to sterilize f2 medium without degrading the vitamins in it...

Also, I've read that the smaller bubbles produced by airstones may cause issues with foaming/clumping (maybe not creating enough flow as well?). Pretty sure that beyond a certain size culture vessel, magnetic stir bars (at somewhat low rpm though to minimize shear stress on algal cells) are the way to go for agitation. Air should be well adequate for 10g though, probably split in 2 lines to avoid dead spots I'd think be good.
Best of luck!

 

[ichthyogeek]

If airborne contaminants are a concern you could use a sterile syringe filter on the airline (not sure if these fit on standard 1/4" tubing, might need some kind of luer lock/barb adaptors...).

How are you sanitizing everything? Bleach/thiosulfate?

Not sure how to sterilize f2 medium without degrading the vitamins in it...

Also, I've read that the smaller bubbles produced by airstones may cause issues with foaming/clumping (maybe not creating enough flow as well?). Pretty sure that beyond a certain size culture vessel, magnetic stir bars (at somewhat low rpm though to minimize shear stress on algal cells) are the way to go for agitation. Air should be well adequate for 10g though, probably split in 2 lines to avoid dead spots I'd think be good.
Best of luck!

If I remember correctly, there's definitely a size of sterile syringe filter that fits on regular airline. I had to use them each time I had to restart rhodomonas cultures because...rhodomonas.

As an update, I pored through the microalgae section of the PCM, and nothing on the cultures turning white. I know that that means that the cells are dead/dying. But I can't remember what the cause of it is. Only thing that might be useful is the PCM saying that contamination might be due to the source material (the bottle of Nanno/the algae starting disks).

Can you walk us through your protocol step by step? List absolutely everything you do. If we can't find anything from that, then it's an extraneous problem like the air pump, F1, etc.

 

[rhdoug]

Have you tried it without the airstone? I always just used the tube, which I think gives better circulation/water movement than with an airstone, which is what we aim for, no?

 

[creativeballance]

Hey all, thank you for the ideas to check. Here's a rundown of everything I had tried:
First (successful) runs.
New instant ocean water.
Bottle of nano from mercer.
10 gallon tank.
Led grow light strips, on/off for 12 hours
20ml f1 from mercer.
I separated cultures about 3 times and got about a month and a half from this bottle.
1 air pump, with 2 air stones

I was filling a 5 gallon bucket, and topping off with new asw.


After about a month, I started getting most of the phyo falling out after 3 days and getting lighter. So I tried experimenting:

With subsequent batches, I tried the following:

Used stored culture from last successful harvestCleaned the tank and airline with bleach. New air stones. 20ml f1.

Used stored culture from last successful harvest. New air stones, New airline
Used a new clear 2litre coke bottle
2ml f1. 3 days, then fall out.

Used stored culture from last successful harvest. New air line, no stones. New coke bottle. Microwaved old tank water for 6 minutes, then cooled back down to 78f before introducing culture. 2ml f1. Turned white/yellow after 2 days.


New culture disk. New airline, New airstones, New coke bottle. Microwaved rodi water for 6 minutes, then added salt mix after cooling. 2ml mercer. Before adding the f1 or airline, the culture did green up a bit for a day.
Culture turned yellow/white after adding the f1 and airline, after a day.

 

[ichthyogeek]

You used.... 2mL F1 (not F/2) in a 2L bottle? Uhhh.....I think you might be overdosing. I'm looking at some notes I took while optimizing Rhodo culture for a lab, and it says that I was dosing 115 uL for every 300 mL culture. Which translates to around 766 uL/2L culture. And from what I remember, Rhodo likes to be fed heavily (I think it ended up being somewhere around 1.75x the normal dosage for good Rhodo growth). We were using old Rhodo cultures, and from them, we also used MicroAlgae Grow from Florida Aqua Farms. That is...if I'm reading my really bad handwriting correctly. And we were using F/2.

I think what's going on, is that you might be overdosing with the smaller cultures. You added 20 mL into a 10 gallon tank (assuming filled), which translates to 20 mL/37.8L = 2 mL fertilizer/3.78L (aka 1 gallon). And in the 2L bottles, you were dosing 2 mL fertilizer/2L...so a double dosage. And if you were using F1 (and not F/2), that means it was a double dosage as well, so a 4x dosage on the microalgae.

Try the following with the Nannochloropsis; if it still falls out, then something's wrong (me, the microalgae, the f1, ¯\_(ツ)_/¯ ):
1 2L bottle (filled accordingly, with sterilized water), sterilized.
Add .5 mL F/2. If you're using F1 fertilizer (Guillard's?), then dose half that amount (.25 mL).
Add in enough microalgae to tint the water a light green (basically standard protocols).

If it turns green, then we've maybe solved the problem. If not, then we know it's not an overdosing issue.

Also, turns out I have an interview with a lab in lousiana about microalgae culture, so thanks for helping me practice my troubleshooting!

 

[creativeballance]

You used.... 2mL F1 (not F/2) in a 2L bottle? Uhhh.....I think you might be overdosing. I'm looking at some notes I took while optimizing Rhodo culture for a lab, and it says that I was dosing 115 uL for every 300 mL culture. Which translates to around 766 uL/2L culture. And from what I remember, Rhodo likes to be fed heavily (I think it ended up being somewhere around 1.75x the normal dosage for good Rhodo growth). We were using old Rhodo cultures, and from them, we also used MicroAlgae Grow from Florida Aqua Farms. That is...if I'm reading my really bad handwriting correctly. And we were using F/2.

I think what's going on, is that you might be overdosing with the smaller cultures. You added 20 mL into a 10 gallon tank (assuming filled), which translates to 20 mL/37.8L = 2 mL fertilizer/3.78L (aka 1 gallon). And in the 2L bottles, you were dosing 2 mL fertilizer/2L...so a double dosage. And if you were using F1 (and not F/2), that means it was a double dosage as well, so a 4x dosage on the microalgae.

Try the following with the Nannochloropsis; if it still falls out, then something's wrong (me, the microalgae, the f1, ¯\_(ツ)_/¯ ):
1 2L bottle (filled accordingly, with sterilized water), sterilized.
Add .5 mL F/2. If you're using F1 fertilizer (Guillard's?), then dose half that amount (.25 mL).
Add in enough microalgae to tint the water a light green (basically standard protocols).

If it turns green, then we've maybe solved the problem. If not, then we know it's not an overdosing issue.

Also, turns out I have an interview with a lab in lousiana about microalgae culture, so thanks for helping me practice my troubleshooting!

I looked at what I was dosing, and it's actually F2. My mistake.
I will try a lower dose anyway. And I'm glad to be talking to someone who knows what they're doing to help troubleshoot this. Thank you, I'll try and see what happens. I have 1 disc left, so hopefully I get it right this time.

 

[creativeballance]

Thank you @ichthyogeek - I think you may have been right about me overdosing the fertilizer.
Going on day 5, using 8 gallons in a 10 gallon tank. Single culture disk of Tetra.

 

[ichthyogeek]

Awesome! Glad to hear that it's no longer crashing!