Phyto Culturing and Sanitization

[reefinnewb]

I'm getting ready to split my first culture. For the first one I used Starsan (brewing sanitizer) to sanitize the container, then used 2 mil bags inside to house the culture. I am going to be splitting it in the next day or so and would like to take a 1/3rd of it and put it in a bottle for later use, can I use the Starsan and then rinse with RODI or is there a better way? How does everyone do it? I've seen peroxide and Isopropyl. Wondering if there are other ways.

Thanks!

 

[NeonRabbit221B]

Bumping. I have had two culture crashes and can't figure out why. On my latest batch I used now bottles and boiled the SW to sterilize.

I would suggest rinsing it another few times until you think it would be absurd to rinse it again. Like 6x. I think by trying to clean the bottles and reuse caused a few issues for me.

 

[Mr. Mojo Rising]

I use vinegar to sterilize my phtyo container (usually a 2 litre pop bottle), then rinse well with tap water and final rinse with rodi water, I haven't crashed a culture yet. I always toss the bottle when I'm done and start with a new one.

 

[fryman]

I use bleach. I believe this is commonly recommended by professionals (which I am most definately not)

You can also use bleach to sterilize the water then sodium thiosulphate to deactivate it.

https://shellfish.ifas.ufl.edu/wp-content/uploads/Phytoplankton-Culture-for-Aquaculture-Feed.pdf

 

[reefinnewb]

Thanks. I was trying to avoid bleach only because I inevitably get it on myself. That article mentions sterilization of the medium but not the vessel (that I saw). Would you use a capful a bleach per gallon of water in the vessel or should it be more concentrated than that?

 

[cryptodendrum]

So I used to get unexplained crashes when I ran my phytofarm indoors under artificial lights. Not often, but it would still happened. Getting religious about sterilisation of the bottles only seemed to partially make a difference; I was never really convinced it improved it that much. When I went from 5000K CFL's to 6000K LEDs, I noted it did seem to also lesson the frequency of crashing.

Then, by chance, I learned about something and decided to do things fundamentally differently.

I now grow my Nannochloropsis the same way the Dutch farmers do it for the American Healthfood market - outdoors under natural sunlight. About the only difference is, where they have giant pools in the ground for this in greenhouses, I still use my 1.5L recycled Dr. Pepper bottles. But after that switch to sunlight, all my crashes stopped. I used to be very strict about bleaching my bottles, now I just hit them with 60C+ hot water and vinegar and a bottle scrub brush, and they're good.

Further, I've checked even with a microscope; there's been no contamination or supplementation by some other algaes; it's still Nanno, and we're rolling over onto year 5 this summer I've been going at it this way and not a single crash since moving outside under natural sunlight.

The bottles sit in a saddle like recycled toolbox with a handle in the middle. I use T-5 reflectors set on top of the bottles to keep rainwater from getting inside the bottle tops (I drilled holes in the caps for the ridged air pipette, but rainwater could still seep in through that hole, since it's not air-tight). One of the bottles has a digital temp probe that links to my aquarium controller, so I can monitor for freezing temp conditions (-1.5C). The recycled toolcase makes it easy to quickly and temporarily relocate the whole farm indoors for those rare winter days it get's that cold here.

 

[fryman]

Thanks. I was trying to avoid bleach only because I inevitably get it on myself. That article mentions sterilization of the medium but not the vessel (that I saw). Would you use a capful a bleach per gallon of water in the vessel or should it be more concentrated than that?

I used to carefully measure out I think 1.5 ml 5% bleach solution per gallon (I use 5 gal buckets) because I had to use a precise dose of sodium thiosulphate to neutralize it. But then I bought a UV sterilizer so I mix the culture water ahead of time and just let it circulate through the UV sterilizer until needed. Since I do not have to deal with bleach in the culture water anymore I just pour excess bleach into the buckets with tap water, let it sit a day or so, dump it and dry.

I have had issues with bacteria before. I think my rodi filter setup grows stuff somewhere but I don't really know what the problem was. TDS measures 0 and ICP test looked fine.

 

[fryman]

So I used to get unexplained crashes when I ran my phytofarm indoors under artificial lights. Not often, but it would still happened. Getting religious about sterilisation of the bottles only seemed to partially make a difference; I was never really convinced it improved it that much. When I went from 5000K CFL's to 6000K LEDs, I noted it did seem to also lesson the frequency of crashing.

Then, by chance, I learned about something and decided to do things fundamentally differently.

I now grow my Nannochloropsis the same way the Dutch farmers do it for the American Healthfood market - outdoors under natural sunlight. About the only difference is, where they have giant pools in the ground for this in greenhouses, I still use my 1.5L recycled Dr. Pepper bottles. But after that switch to sunlight, all my crashes stopped. I used to be very strict about bleaching my bottles, now I just hit them with 60C+ hot water and vinegar and a bottle scrub brush, and they're good.

Further, I've checked even with a microscope; there's been no contamination or supplementation by some other algaes; it's still Nanno, and we're rolling over onto year 5 this summer I've been going at it this way and not a single crash since moving outside under natural sunlight.

The bottles sit in a saddle like recycled toolbox with a handle in the middle. I use T-5 reflectors set on top of the bottles to keep rainwater from getting inside the bottle tops (I drilled holes in the caps for the ridged air pipette, but rainwater could still seep in through that hole, since it's not air-tight). One of the bottles has a digital temp probe that links to my aquarium controller, so I can monitor for freezing temp conditions (-1.5C). The recycled toolcase makes it easy to quickly and temporarily relocate the whole farm indoors for those rare winter days it get's that cold here.

That's awesome I may try it out. I have left culture buckets out in the sun during good weather before and it grew fine. I have had issues with LEDs and nanno crashing, I think too much light because when I switched back to cfl it seems ok. My LEDs give 500 PAR at the water surface which I think is too much for anything but tetraselmis.

 

[cryptodendrum]

Just a quick photo which supplements my description above.... this was last month right after a harvest. I harvest about 80% of the bottle, and use the remaining 20% for seeding a new culture. Each bottle get's about 2.5 ml of F/2 and I aim salinity at around 1.010-1.015 and just let it grow to the right density.

BTW, my bottle cleaning protocol for the last 3 years has been:

Fill bottle with 60+C Water - let sit 5 minutes
Scrub inside of bottle with bottle scrub brush, hitting the entire surface 2-3 times over (takes about 5 minutes)
Rinse bottle
1/5th full of vinegar, put cap on, shake vigerously 5 minutes & let soak. Repeat shake and soak 2-3 times.
Rinse & fill with 60+C Water and let set 10 minutes
Empty and use bottle.

These are the same bottles I've been using for about 3 years now, but that's because we don't really drink sodas in our home very often anymore.

 

[reefinnewb]

Right now I have the culture sitting on a window sill and it's been doing great. I'll probably split this weekend so I'll update everyone.

 

[cryptodendrum]

BTW....just made a photo of same cultures today. Nice and thick & ready to harvest today, despite getting a lot of rain and cloud cover these last 3 weeks. I could / should have harvested it a week ago, but life got in the way. But that's what I've noticed (repeating self now) is that under sunlight, if I'm late with a harvest, I don't see decreased density or crashes of the cultures outside under sunlight if I push the harvest date back a week or two. Under artificial light, this was a no-no.

 

[Clownfish2]

I'm getting ready to split my first culture. For the first one I used Starsan (brewing sanitizer) to sanitize the container, then used 2 mil bags inside to house the culture. I am going to be splitting it in the next day or so and would like to take a 1/3rd of it and put it in a bottle for later use, can I use the Starsan and then rinse with RODI or is there a better way? How does everyone do it? I've seen peroxide and Isopropyl. Wondering if there are other ways.

Thanks!

I culture isochrysis Galbana in Classico Spaghetti sauce glass jars. Sterilize them in the dishwasher. I clean the plastic tubes that generate the air bubbles under tap water and a little dish soap. To keep debris out and stop evaporation, I cover with clear plastic food wrap.

 

[el aguila]

I really like the idea of having this outside. I’m assuming that our winter climate in Charlotte, NC is somewhat similar to yours.

Im going to have a couple of months where the cultures will be inside more than outside because of leaving early in the morning when temperatures are below freezing.

Do you supplement light during longer cold snaps? Have you had your cultures freeze?

Just a quick photo which supplements my description above.... this was last month right after a harvest. I harvest about 80% of the bottle, and use the remaining 20% for seeding a new culture. Each bottle get's about 2.5 ml of F/2 and I aim salinity at around 1.010-1.015 and just let it grow to the right density.

BTW, my bottle cleaning protocol for the last 3 years has been:

Fill bottle with 60+C Water - let sit 5 minutes
Scrub inside of bottle with bottle scrub brush, hitting the entire surface 2-3 times over (takes about 5 minutes)
Rinse bottle
1/5th full of vinegar, put cap on, shake vigerously 5 minutes & let soak. Repeat shake and soak 2-3 times.
Rinse & fill with 60+C Water and let set 10 minutes
Empty and use bottle.

These are the same bottles I've been using for about 3 years now, but that's because we don't really drink sodas in our home very often anymore.

 

[cryptodendrum]

I really like the idea of having this outside. I’m assuming that our winter climate in Charlotte, NC is somewhat similar to yours.

Im going to have a couple of months where the cultures will be inside more than outside because of leaving early in the morning when temperatures are below freezing.

Do you supplement light during longer cold snaps? Have you had your cultures freeze?

No supplementation of light at all. In fact, in the peak of winter, the bottles only get about 1-2 hours of sunlight, if at all. The backyard patio tiles and surrounding walls are all white-ish in color, so it does get lots of reflected light year round, and long overcast cloudy days also do not really impact growth rates.

during the 4 to 6-ish months of winter, if I really need a set amount daily of phyto, I just scale up my crop with extra bottles to compensate for the longer times to harvest in winter months. That's really all I do anymore for the winter time.

I use 20-25% of a harvested bottle to seed a new bottle. During summer, I can do harvests about every 7 days. During winter, it's about 3 weeks.

Never had the cultures freeze, even when the bottles were caked in snow. The snow stuck for days and I'd have to go out every day and shovel snow off the bottles (1-2 minutes, top) but the temps never went (or were predicted to go) below -1.0C (below 0C), and with the salinity of the bottles at what I run at, the cultures stayed alive and never froze (freezing point was -1.5C). So I rode that winter out without ever bringing them inside.

My home automation is also my aquarium controller, and I have monitors to watch weather forecasts below the freezing point of the phyto cultures & it will raise alarms if it's forecast, so I know before hand, I'll have to bring them in.

Additionally, I have a RaspberryPi nearby, so I dropped in a DS18b20 waterproof temp probe connected to the RPI into one of the bottles to also monitor the culture temp & also raise alarms if the forecast is ever wrong.


This past winter was the only time I've used artificial light on the cultures. The hard freeze lasted 3+ weeks, so I just setup the bottles inside my fishroom in a dry frag tank & used the lights on that frag tank to illuminate the cultures for those 3 weeks.

 

[ChaosAquaculture]

I'm getting ready to split my first culture. For the first one I used Starsan (brewing sanitizer) to sanitize the container, then used 2 mil bags inside to house the culture. I am going to be splitting it in the next day or so and would like to take a 1/3rd of it and put it in a bottle for later use, can I use the Starsan and then rinse with RODI or is there a better way? How does everyone do it? I've seen peroxide and Isopropyl. Wondering if there are other ways.

Thanks!

Everything that comes into contact with the phyto jars being brewed are wiped down thoroughly with isopropyl alcohol to ensure everything’s as clean as possible. It doesn’t take too much bacteria for a phyto culture to crash so cleanliness is very important. If you plan on regularly brewing your phyto, we recommend keeping RO in your storage containers you plan on using to avoid any contamination while they aren’t being used.

 

[reefinnewb]

So an update. My first phyto culture went great! I split it up and then tried again. Some went into my copepod culture. The two splits both crashed, the copepod tank, grew a ton of phyto and pods! LOL Doing some changes with the reef and organization. Going to revisit this again once I'm done.

 

[NeonRabbit221B]

I have about a 30% success rates with splits. Trying with 1/2 gallon brewing jars...

 

[cryptodendrum]

So an update. My first phyto culture went great! I split it up and then tried again. Some went into my copepod culture. The two splits both crashed, the copepod tank, grew a ton of phyto and pods! LOL Doing some changes with the reef and organization. Going to revisit this again once I'm done.

That awkward moment you create a co-culture and find success. I've also done this intentionally with pods and rotifers. The trick is to keep the pods and rotifers at population levels that don't out perform the phyto. In turn, the phyto feeds off the waste nutrients of pods and rotifers. I could keep them going for awhile but mostly for only 2-3 months using my recycled IO salt buckets. There's some tutorials on YouTube for this.

That also prompts me to ask - did you fertilise your split cultures when you split them? And if so, with what? If you didn't, it's very possible you starved them to death. I've had the best success with using f/2 formulas. You can find the recipe online and make it yourself or buy it from some shops.

Also, how much air are you turning your phyto cultures over with? And what species of phyto are you culturing?

 

[reefinnewb]

I found it odd. It was really a "Huh, look at that!" moment.

I did fertilize with f/2. I have a pretty good bubble rate going, not to the point of creating a foam head but a light boil. I was culturing nanno.

 

[cryptodendrum]

I found it odd. It was really a "Huh, look at that!" moment.

I did fertilize with f/2. I have a pretty good bubble rate going, not to the point of creating a foam head but a light boil. I was culturing nanno.

When you harvest your ready culture and make a seed culture, do you filter that seed culture? I've been using brown paper coffee filter No. 4's to filter each new seed culture. I originally started that when I was still culturing indoors & accidentally cross-contaiminated brine shrimp cysts into a few cultures. I just kept it up thinking since I'm culturing outdoors, any tiny mites or spiders that somehow make their way into the bottle, and any other chunky bits can be filtered out every harvest.