I recently had an epiphany about how to grow Bryopsis more efficiently.  In retrospect, it was pretty obvious, and I wonder why it took 2 years to get to this point.

It seemed as though culturing adequate Bryopsis was under control.  However, there’s nothing like 6+ weeks of travel to turn things upside-down.  I had hired a service to come in and keep things going while I was away, but their primary task was to prevent biological meltdown, or worse, a big salty disaster that would have me forever on the naughty list of the facilities people.  I am happy to say, there were no smelly or wet disasters.

Unfortunately, I was not there to give the algae cultures the kind of attention they need, and by the time I was back in the office, the system was overrun with Derbesia, and the slugs had devoured the Bryopsis that I had left for them.  The 20-gallon slug tank and both algae culture tanks were full of felty, green hair algae.  In retrospect, I should have taken photos, but I was more focused on cleaning up the mess and getting ready to teach a summer class.

Over a week or so, I pulled out the algae tanks off the system, cleaned out at least two pounds of green glop, salvaged the remaining Bryopsis, and set the tanks  back up.   Somewhere along the line, I came across a few posts about “algae reactors,”  cylindrical chambers with water flowing through and some sort of light source.  My first thought was “maybe I should buy one of those things.”  Then I remembered that I had two media reactors sitting idly in the basement.  They are clear cylinders, designed to have water flow through them, which is exactly what I wanted.

The amount of Bryopsis remaining was so small that it didn’t seem worthwhile to have two algae tanks.  Instead, I shut down the 10-gallon tank and stuck some of the remaining algae into a reactor. The reactor was hooked up to one of the valves, and connected to the drain, and sat under the grow light where the 10-gallon tank had been.

Two Little Fishies media reactor, with a small wad of Bryopsis held in place by eggcrate. 7/12/17

Within a week or so, it became clear that the experiment was working, so I added the second reactor.  I had enough Bryopsis to harvest some for the slugs, and the culture in the first reactor had seeded a sponge that I could use to start the second reactor.

Second reactor, with a sponge seeded from the Bryopsis in the first. 7/21/17

It was working.  Time to make things a little less clumsy.  I built a rack from 3/4″ PVC, setting the reactors at an angle to optimize the connections to the input valves and drains.

Bryopsis reactors, in their improved configuration. 7/26/17.

Things went so well, I begged a few unused reactors from local aquarists.  This one is from Alan (of unidentified algae fame), and I have one more waiting in the wings.

Three reactors. 8/1/17.

At this point, it was clear that my former method of rearing Bryopsis in aquaria was not very efficient.  Raising Bryopsis in reactors allows me to play with growth parameters, like flow and nutrients, much more easily.  Further, keeping multiple separate cultures will make it much easier to eradicate unwanted algae.  At some point, it should be straightforward to maintain cultures free of unwanted algae and invertebrate pests (I am all for biodiversity, except when it eats slug larvae) by UV sterilizing the water going into the chambers.

As far as I can tell, this was a successful experiment, so I converted the 15 gallon algae tank into the second slug tank, shutting down the 10-gallon slug tank.

When I walked in today, the system had two slug tanks (top and middle left), and one remaining algae tank (top right)

Elysia system before rearrangement. 8/5/17.

Five hours of cleaning and rearranging later, there were two slug tanks and three algae reactors.  In the process, the system now has two fewer circulation pumps and one fewer light fixture.

Current slug system arrangement. Two tanks for slugs and and their food on top, three reactors in the middle, and room to expand. 8/5/17.

In the future, you can expect a few more reactors.  Now it’s time to play with parameters to maximize algae growth.  Maybe we’ll finally see some consistent egg production from the slugs.

To many, this post is akin to “how to give fleas to your dog,” or “how to grow ragweed.”  Bryopsis is a benthic (substrate-associated) genus of algae that most aquarists dread seeing in their tanks.  Bryopsis can thrive in reef aquaria, to the point that it can smother and kill corals.  Many methods of eradication have been explored, including near-toxic levels of magnesium, and the anti-fungal medication fluconazole.

E. clarki feeding on Bryopsis; the red alga, Botryocladia, is in the background.

On the other hand, I really want the stuff.  As far as I can tell, it is the favorite food of Elysia clarki.  E. clarki will eat other macroalgae, such as Penicillus, Avrainvillea or Halimeda, but, given the choice, they will go straight for Bryopsis and stay there.  Further, they have consistently produced eggs only at times when they have had unlimited availability of Bryopsis.  One of my primary goals is to maintain a self-sustaining colony of slugs, so I very much want a constant, adequate supply of algae.

I’d be perfectly happy if I could count on the outbreaks of local aquarists to supply my needs.  Unfortunately, sources have been spotty.  My primary source, Justin, who had a beautiful outbreak in a 500-gallon tank, unfortunately managed to get his infestation under control.  He swears the controlling factor for him is the level of phosphate; if PO₄ levels creep up, he gets an outbreak.  Others have provided quality material as well, but not as consistently. Yet others have offered species that turn out not to be Bryopsis.  The take home lesson is that people who are trying to eradicate something are not the most dependable source.

The whole gang enjoying the new batch of algae provided by a local retailer. 4/17/16.

How hard can the stuff be to grow?  It is a pest, after all.  Like any aquatic plant, it needs light, nutrients, and circulation.  It gets a little more difficult when you try to provide for the plants’ requirements on a consistent basis.  As far as I can tell (and PLEASE email or comment if I am wrong), nobody has published specific requirements of Bryopsis for PAR (photosynthetically-available radiation), macronutrients (e.g., carbon, nitrogen, phosphorous), or micronutrients (like iron, copper and cobalt).  When marine aquarists have outbreaks, much of the nutrient load is derived from fish food (well, really fish poop), which is not measured.  In fact, most reef aquaria don’t have Bryopsis outbreaks, which suggests that the unlucky few have managed to combine all the right ingredients.  For a constant supply of Bryopsis, I need to provide for all of its needs.

If I wanted to grow phytoplankton, the single-cell, free-floating relatives of Bryopsis, I would be in luck.  Because of the important role of phytoplankton in our oceans, and because there is a growing industry using them to generate hydrocarbons from sunlight, growing conditions have been explored and published at length.  The most popular formula is Guillard’s F/2, so named because it consists of his formula F diluted to 50% (Guillard & Ryther, 1962, Can. J. Microbiol. 8:229), is readily available through suppliers such as Florida Aqua Farms, or even Ebay.  The formula provides a balanced blend of nitrogen and phosphorous (with an option to add silicon if you want to grow diatoms), along with a mix of trace elements and vitamins.  Mix it up, add a starter of the phytoplankton of your choice, bubble in some CO₂ as a carbon source, and you’ll soon have a thriving culture of green water.

I started dosing the Florida Aqua Farms version (“Plant Fuel Too”) almost a year and a half ago, using the complete dry mix (minus silicates).  The Bryopsis started to look darker and happier pretty quickly.  Perhaps unsurprisingly, there was also a phyoplankton bloom that turned the water green and generated a layer of green scum on the surface of the water.  Also, the added nutrients seemed to increase levels of nuisances like cyanobacteria (a.k.a., red slime algae). That would have all been tolerable, but the biggest sticking point was that Bryopsis would go through cycles of boom and bust, which is not conducive to long-term management of a slug colony.  I needed to get the right balance of nutrients that would get the Bryopsis to grow maximally and out-compete the species I don’t want.

With all of the possible factors (e.g., light, circulation, temperature, nutrients) to be within the right ranges, there were a lot of variables to play with:

• Levels of light seemed to be within the range I observed in tanks with steady outbreaks, so I left that alone.
• Bryopsis always grows well in the areas with the strongest water flow (e.g., pump outlets).  I am not sure why, maybe it keeps other algae from settling on it, but I increased the flow to ridiculous levels in the 15-gallon primary culture tank by combining a power filter Aquaclear AC70), powerhead (Hydor 2450) and wavemaker (MaxSpect Gyre XF130) that were all rated for tanks 3-5 times the size.
• I plunged back into the literature to try to find papers examining the nutrient requirements of benthic macroalgae, rather than phytoplankton.  After a little digging, I found that species related to Bryopsis demand much more carbon and nitrogen, and are not as dependent on phosphorous (e.g., Atkinson and Smith, 1983, Limnol. Oceanogr. 28:568; Pedersen and Borum, 1997, Mar. Ecol. Prog. Ser. 161:155).

In order to separate the variables a bit better, I started using Florida Aqua Farms original “Plant Fuel,” which contains all of the trace elements and vitamins, but no nitrogen or phosphate. That would allow me to play with levels of trace elements, N, and P separately. I also added a source of carbon.  Because white vinegar is cheap, always available, and is readily broken down into CO₂ by bacteria, it seemed like a good choice.

By adjusting the relative input of all four ingredients (C, N, P and trace elements), combined with very high flow in the algae culture tank, I was able to get significantly better growth.  The final improvement was something that I had not expected to have such an impact: rebuilding the system.  Within about a week of the big move, the algae was thriving on the racks of plastic eggcrate I was using as a substrate.  I suspect that the 50% water change and improvements to circulation in the new system played a large part in the improved growth.

Bryopsis growing on eggcrate in 15 gallon algae culture tank. 3/2/17

By growing on plastic racks, Bryopsis cultures can be moved to slug tanks to be consumed, and then removed for regrowth.

Bryopsis-covered plastic rack in 20L slug tank. 3/23/17

So we’re making forward progress.  We are controlling the growth of food algae to the point where it seems to be sustainable.  As a side benefit, the other macroalgae are also thriving.  For example, the three deep green Penicillus in the photo below are new sprouts from the ratty old plant in the center.

Three new Penicillus plants surrounding the faded parent taken from the wild. 4/11/17

At the moment (and likely to change) the system receives major elements at the (molar) ratio of 150:30:1 C:N:P.  This is roughly in the range reported for other macroalgae, and does not support much growth of cyanobacteria or phytoplankton.  In other words, it looks like I am providing nutrients at about the rate at which the are being consumed by the desired algae.

• N: dosing KNO₃ (13.9% N); stock = 80 grams/liter (g/l), diluted to 20 g/l; dosing 80 milliliters/day (ml/d); that equals 0.222 g/d of N, or 0.0159 moles/day
• P: Dosing KH₂PO₄ (22.8% P); stock = 11.6 g/l; diluted to 1.45 g/l; dosing 50 ml/d; 0.0165 g/d or 0.00053 mol/d
• C: from acetic acid: 5% solution (50 g/l); 48 ml/d; C = 40%; 0.96 g/d; 0.08 mol/d
• K: from KNO₃ AND KH₂PO₄:  0.64 g/d or 0.021 mol/d
• Guillard’s F/2 trace element and vitamin solution: diluted 1:20, dosing 100 ml/d;
• I have also started adding calcium hydroxide (CaOH₂, kalkwasser) to the water used to make up for evaporation to maintain Ca.

There is still plenty of room for improvement.  The priority for the moment is eliminating or reducing dinoflagellates in the algae culture tanks.  They do not seem to cause too much trouble in the slug tanks, but they form a slimy film on the Bryopsis in the culture tanks, presumably reducing growth, competing for nutrients, and producing toxins.

Dinoflagellates at low magnification. They can be seen as strings of brown dots surrounding the green algae branches. 4/11/17

At higher magnification they are kind of pretty.  Based on the shape, size and general appearance, it looks a lot like Ostreopsis ovata (Faust and Gulledge, 2002, Contr. U.S. Natl. Herb. 42: 1-144).  Like many dinoflagellates, O. ovata produces toxins, some of which are toxic to mice (Nakajima et al., 1981, Okinawa Bull. Jpn. Soc, Sci. Fish. 47: 1029).  The effects on slugs or other invertebrates is not known.

DInoflagellate from Bryopsis tank, 400X magnification. Gold color is from densely packed chloroplasts.  4/6/17.

At the moment, I have reduced the photoperiod (the time the lights are on), and am rinsing the Bryopsis racks in clean artificial seawater periodically to reduce the population.  At some point, it might be necessary to use harsher methods, such as antibiotics (metronidazole seems to affect dinoflagellates somewhat specifically) or starting a clean culture of Bryopsis and preventing entry of other species using UV sterilization.

For now, there is plentiful food for the slugs, which is a step forward.