Dinoflagelates. A disruptive treatment

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Beuchat

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THE PROBLEM

As is well known, dinoflagellate pests in the reef aquarium are perhaps the worst that we will face during the life of our system. These pests are quite difficult to eradicate because dinoflagellates have a great capacity for reproduction, propagation and survival. In addition, measures that are effective for one species are not equally valid for others. To complicate matters, aquarium history and environmental variables, e.g., existing nitrate and phosphate concentrations or, more specifically, the variation of these, play an extraordinarily relevant role.

Table 1 shows a list of measures, which alone or in combination, are commonly used by aquarists facing this problem. The table indicate the effectiveness, to the best of my knowledge and experience, of each of them. As we can see, none of the measures have a high degree of effectiveness in all cases, being necessary several of them, and a lot of patience, to reach a successful conclusion.

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Table 1. Common measures to fight dinos.

Fortunately, as we will see throughout this article, we have been able to verify that the gradual addition of an external source of organic carbon has a great effectiveness in solving the problem, most likely for all species of dinoflagellates. This treatment has been tested and verified during an experiment conducted in 11 reef aquariums over a period of 7 weeks, in the city of Madrid. In all cases the result has been the eradication of the pest. Let's see the details.

DINOFLAGELLATES. ¿WHAT ARE?

Dinoflagellates are small unicellular organisms belonging to the group of protists, which cannot be categorized as neither animals nor plants, since they have characteristics of both. They move by means of a rotary motion, using their flagella to propel themselves. For example, the well-known zooxanthellae that house corals, anemones and giant clams, are dinoflagellate protists, although they have given up their extensive mobility to have a safe environment within the tissues their host, sheltered from predators

Unwanted dinoflagellates in the aquarium are often are often classified as "algae" because they are photosynthetic protists, although many are mixotrophic organisms, capable of feeding also on simple organic matter. This dual feeding strategy contributes to their great capacity for survival. Many of them release very potent toxins into the water, e.g., Ostreopsis, Gambierdiscus and Prorocentrum species, which generate compounds similar to the palytoxins produced by Zoanthids. As an example, I will describe the life cycle of Osteropsis to understand what we are dealing with.

The cells of Osteropsis ovata have a diameter at their largest part, about 40 micro meters, which, if we compare it to the diameter of a cyanobacteria cell (2-3 micro meters), gives us an idea of its relative size. Ostreopsis, like many other harmful dinoflagellates, secretes a large amount of mucus to build extracellular matrices, macroscopically characterized by the presence of strands and filaments, in which oxygen bubbles resulting from photosynthesis are trapped. These matrices settle on any existing substrate, sand, rocks, glasses, etc., suffocating the existing micro fauna.This can be observed, for example, when the aggregates formed on the substratum are siphoned off, revealing areas of sand with a dark gray color, indicating anaerobic bacterial activity, resulting from the depletion of oxygen produced by the layer of dinoflagellates settled on top. The extracellular matrices also serve to immobilize potential predators such as amphipods and copepods.

Ostreopsis and other noxious dinoflagellates have a life cycle characterized by the presence of cysts or resistance forms, which are formed as a result of the union of gametes, although also derived from asexual reproduction. When environmental conditions are not appropriate, for example, a shortage of nutrients or light, these cysts anchor to the substrate and remain "dormant" for months, until the situation improves.

This implies that when we use live rock in the aquarium, we are introducing these cysts as part of the resident micro fauna. It is important to note that the use of inert rock does not prevent the propagation of dinoflagellates, which are unintentionally introduced with the coral frags. In fact, aquaria started with inert rock are noted for a higher incidence and duration of this type of pests, because the lack of microorganism competition. I have been able to verify this point through microscope specimens and interviews with hobbyists. As an example, Figure 1 shows the progression of a dinoflagellate pests in an aquarium set up with 50% inert rock (left) and 50% live rock (right). The difference is remarkable.

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Figure 1. Progression of a dinoflagellate pest depending of life rock (right) or inert rock (left)

In the ocean, dinoflagellates produce toxic tides coinciding with periods of rising temperatures and dissolved inorganic nutrients. These blooms, are initiated when the cysts are activated and begin to divide, each cell generating two new individuals and so on. This reproduction generates an exponential pattern that subsequently stabilizes into a flat pattern. After several weeks the population density decreases until the bloom subsides. In the aquarium, the pest behaves in the same way, and can last for months.

DINOFLAGELLATES IN THE REEF AQUARIUM

Dinoflagellates are present in almost all aquaria, forming part of the food web and, therefore, contributing to the general mechanism of nutrient recycling, as they consume light, CO2, nitrate, phosphate, trace elements and dissolved organic matter. If the resident population is limited to a few small colonies and cysts, it does not pose much of a problem. However, it is absolutely necessary to avoid conditions that may favor a bloom, since, once started, it is quite difficult to control and eradicate.

The most favorable scenario is related to young aquaria with very low nitrate and phosphate concentrations, for example, below 0.02 phosphate and 2 ppm nitrate. There is also some probability that a bloom will be triggered when there is a fast reduction in phosphate concentration, which can happen, for example, after the use of lanthanum chloride or GFO. In all aquaria it is good practice to maintain a balanced equilibrium between the two nutrients.

So why, when there is too little nitrate and phosphate in the water, or there is a sudden reduction, is there a greater likelihood of triggering the pest? The answer is neither simple nor immediate, but there are some evidences that help us understand the problem. The key is not in the dinoflagellate itself, since a reduction in the concentrations of nutrients, both organic and inorganic, is not at all favorable to them. What happens is that other competing species, which, among other things, use nitrate and phosphate for food, get starved, and dinoflagellates take advantage of them.

BALANCE, MATURITY AND BIOLOGICAL NICHE

All microorganisms living in a reef aquarium, whether they are bacteria, protists (dinoflagellates, ciliates, coccolithophores, foraminifera, radiolarians), phytoplankton (diatoms, cyanobacteria) or zooplankton (amphipods, copepods) are in continuous competition for light, space or nutrients. They all exchange nutrients with the water. Each organism has a place where it spends most of its day, where it inhabits and performs its main primary functions: feeding, grow and reproduce. Every square cm of a marine aquarium is occupied by an infinite number of these tiny organisms, living in a constant equilibrium. This is what called a biological niche.

When the aquarium reaches a significant maturity (as a reference around 1 year after set-up), the stability of its environmental factors (light, water chemistry, nutrient flow, etc.), favors biodiversity in the populations of all microscopic species, since the greater the stability, the better the conditions for diversity. In this scenario it is more unlikely that one species to dominate over the rest and generate a pest, as most of them have everything they need.

However, when a change occurs, the desired stability is disturbed. The organisms that used to occupy a niche may disappear, die, or simply migrate to find a better place. It is then that the "gap" they leave behind is quickly colonized by other opportunistic and fast-moving organisms. This is the case of unwanted dinoflagellates.

COMPETITION BETWEEN BACTERIA AND DINOFLAGELLATES. THE SURPRISING KEY TO THE SOLUTION.

As we can see, it is practically impossible to eliminate the existence of the different unwanted dinoflagellates in a reef aquarium, since they are introduced with the live rock, invertebrates or frags. The optimal situation is that their populations are limited through natural competition from other organisms such as phytoplankton, zooplankton, or bacteria. As is well known, one of the strategies used to eradicate a dinoflagellate pest caused by maintaining low nitrate and phosphate concentrations is precisely to increase those concentrations. However, the dinoflagellate uses nitrate and phosphate for feeding, so increasing these concentrations will favor it as well.

The rationale for this measure is that the new availability of nutrients will also favor competing organisms, being able to displace dinoflagellates. This strategy is colloquially referred to as "dirty warfare" and its rate of effectiveness is quite low, often favoring the expansion of other unwanted algae such as cyanobacteria or hair algae. However, this mechanism of feeding competing organisms works remarkably well with heterotrophic bacteria. These bacteria aggregate in biofilms within the aquarium substrate, on rocks and other available surfaces, feeding on organic carbon, nitrate, phosphate and trace elements.

It is important to note that all reef aquaria are usually limited in the amount of dissolved organic carbon available to bacteria (DOC), so such bacteria do not grow well until we provide an external carbon source. When adding organic carbon artificially, we get the heterotrophic bacterial communities to displace dinoflagellates.

As simple as this, the reader may ask? The answer is YES.

And why aren't hobbyists using this technique as a pest remediation? The answer is, as in many other cases, fear. The fear comes from the recommendation that all hobbyists follow not to let nitrate and phosphate concentrations fall to undetectable levels, precisely to avoid the risk of cyanobacteria and dinoflagellates blooms. However, in this essay we have verified that, when such low concentrations are the result of the gradual addition of organic carbon, there is no risk of dinoflagellate blooms, but rather, on the contrary, there is a significant regression of the of the existing population, until it disappears completely. As the reader can see, this treatment goes against all recommendations, but it works remarkably. Anyway, it is important to highlight that the risk of cyanobacteria bloom is still there, so we shall not let the inorganic nutrient concentration be reduced too much.

THE TREATMENT

Two brands of organic carbon additives were used in the study Xepta NP out and Red Sea NO3 : PO4-X. After some weeks we got to the conclusion of Xepta being faster in producing the dinoflagellates regression. Xepta NP out is composed of methanol, toluene, glucose, and acetic acid, while Red Sea NO3 : PO4-X contains methanol, ethanol and acetic acid. In the 11 aquaria where this treatment has been applied, the dinoflagellates have receded significantly with either Xepta or Red Sea.

The initial dosage was 1 ml of organic carbon per 100 liters of aquarium water, increasing to an additional 1ml/100 liter per week in cases where dinoflagellates are slow to recede. Up to 3 ml/100 liters have been used in some aquariums. It is important to perform the dosage when the aquarium lights have been turned off, in this way we avoid that the dinoflagellates from taking advantage of the organic carbon. Dose shall not exceed 3 ml/100 l of aquarium water, as we have not tested beyond.

Any sudden excess of organic carbon in aquarium water alters the equilibrium of the coral holobiont, as the heterotrophic bacteria that live within the coral tissues, receive an abnormal dose of food, which can be detrimental to the coral. It is very important that the dosage be as gradually as possible and, at any negative sign in fish, corals and invertebrates, reduce the dosage to half, maintain the treatment, and then increase it again some days after, once the aquarium has self-regulated.

Some side effects detected during the study are depicted below:

- Some corals and tridacnas that have closed more than usual.

- Cloudy water due to bacterial bloom and excessive growth of biofilms inside the return pump pipes.

- Increased export rate from the skimmer. Mostly bacterio plankton and remains of dead organisms.

For cases where there is a large number of dinoflagellates, prior to start the treatment, it is convenient to siphon them out without water change. To do this, a sock is used to retain the dinoflagellates while the siphoned water is returned to the sump. As a consequence of the addition of organic carbon, nitrate and phosphate concentrations will drop significantly, even to the point of being undetectable. We do not want this to happened, because organic carbon addition favors the growth of unwanted cyanobacteria.

ACTION MECHANIMS

The biological mechanism underlying is the combined effect of two factors that cause the dinoflagellate populations to be reduced.

1- Bacteria "steal" food from the dinoflagellates. Due to the addition of organic carbon, the heterotrophic bacteria that are housed in the sand, the rocks and other surfaces of the aquarium, start to reproduce in an extraordinary way, removing nitrate, phosphate and trace elements from the water. They form biofilms that spread on all available surfaces, displacing dinoflagellates.

2- Bacteria directly inhibit the growth of dinoflagellate cells through allelopathy (chemical warfare). The survival strategy of bacteria, protists and phytoplankton, includes a line of defense, which consists of releasing chemical compounds toxic to other species. For example, as a result of copepod predation on diatoms, the algae produce polyunsaturated aldehydes (PUAS), which are noxious to copepods and some phytoplankton species, such as urchin larvae. These PUAS are also produced by other phytoplankton species, such as cyanobacteria, cryptophytes, primnesiophytes, and synurophytes. In addition, PUAS have a potent inhibitory effect on the growth of the dinoflagellate Ostreopsis ovata, altering its DNA structure and disrupting its development.

Figures 2 to show the evolution of 2 of the of the 11 aquaria where the essay was carried out. The addition of organic carbon, allows the heterotrophic bacterial communities to reproduce and generate their allelopathy compounds, which inhibit the growth of dinoflagellates. In the following scientific study: https://www.frontiersin.org/articles/10.3389/fmars.2015.00100/full, it is explained how the dinoflagellate Alexandrium tamarense is inhibited by allelopathy compounds generated by marine bacteria Brevibacterium, Thalassobius, Alteromonoas, Rhodobacteracea, Pseudoalteromonas, Vibrio and Halomonas. According to the article, there is a regression of the cells of Alexandrium tamarense after 10 min, 30 min and 24 h, because of the introduction of the bacteria. The aforementioned study has been kindly shared by Postdoctoral Researcher. Mrs. Esther Garcés, who is a scientific authority in the field of dinoflagellates.

Aquarium no. 1.

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Aquarium no. 2.

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We can see, therefore, that many organisms are capable of interfering with the growth of others through competition for space and nutrients, but also directly through allelopathy. In an aquarium, where the volume is so extraordinarily limited compared to nature, these chemical warfare compounds are of decisive importance. For example, soft corals emit steroids, diterpenes and sesquiterpenes. which are poisonous to many animals, including hard corals. Anemones also make use of allelopathy in the aquarium, which is proven when, after the introduction of new specimens, resident anemones that were thriving without major problems, begin to undergo swelling cycles, that sometimes lead to their death.

CONCLUSIONS

In the aquarium water, when there is a shortage of, whether nitrate, phosphate, trace elements or organic matter, many of the organisms in the food web, will not be able to complete their metabolic processes and, therefore, to emit their allelopathic compounds or even compete for light and space. This is the perfect scenario for dinoflagellates to spread, as they will not be inhibited by allelopathic compounds and have the capability of growing with ultra-low nutrient concentrations. Dinoflagellates begin to reproduce and spread over all available surfaces, using their flagella, with extraordinary mobility.

Using an external source of organic carbon, heterotrophic bacteria reproduce in an exponential rate, grouping together in biofilms that cover all available surfaces: sand, rocks, pumps, aquarium and sump walls, etc. These biofilms "suffocate" and "intoxicate" dinoflagellates, conveniently deactivating them.

One of the most important conclusions of the test carried out in the 11 aquariums is the following: maintaining very low or undetectable concentrations of nitrate and phosphate in a reef tank, does not pose a risk for the emergence of dinoflagellates, as long as it is a consequence of the addition of an external source of organic carbon. It is important to highlight that the risk of cyanobacteria is still there, as they are favored by organic carbon and not affected by the chemical warfare compounds released by the heterotrophic bacteria.

My recommendation is to always maintain detectable concentrations of both nitrate and phosphate, so that there is a small amount of "leftover" nutrients available for all the organisms, avoiding the growth limitation due to lack of nutrients. During the dosing of organic carbon, it is very important to go progressively, monitoring the status of fish and corals to stop or decrease the dosage at any negative sign. It is necessary to be attentive to fish breathing, water cloudiness and possible symptoms of STN in corals.
 
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Dan_P

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Thank you for sharing this study. The article was easily understood and fascinating. I have a few questions.

How old were the 11 treated aquaria?

Was the bloom organism identity confirmed by microscopy in each of the 11 systems?

Does every dinoflagellate species bloom disappear with carbon dosing at roughly the same rate?

Did the bloom in every aquarium with the same species of dinoflagellate disappear at roughly the same rate?

Which aquaria developed visible cyanobacteria growth?

Thank you,

Dan
 

sixty_reefer

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Thank you for the write up.

heterotrophic bacteria can become limited if N C or P becomes limited, allowing for dinoflagellates and Cyanobacteria to take over as this particular bacteria can’t multiply due to lack of nutrients, Cyanobacteria and dinoflagellates being opportunist of the nutrients situation will start to multiply under those events as they utilise mainly N-Doc and organic nutrients wile heterotrophic bacteria needs Doc and inorganic nutrients to multiply.
The reason I believe it’s always hard to outcompete these species without taking any measures could be due the opportunistic species being constantly depleting organic nutrients and by effect depleting the availability of inorganic nutrient that are needed to strengthen the heterotrophic population.
Adding organic carbon is a genius move although I got some questions if that’s ok?

you mentioned that dosing Doc has worked well during the experiments aiding the growth of heterotrophic bacteria that seems to be the line of defence in our system against opportunistic species like dinoflagellates and Cyanobacteria. I agree although if doc becomes abundant and inorganic nutrients become limited due to the sudden availability of Doc wouldn’t that cause the heterotrophic bacteria to become limited again? Or do you recommend for during Doc treatment to dose nitrates and phosphates to keep inorganic nutrients detectable?

I I’m under the impression that if Doc is increased in the presence of dinoflagellates and Cyanobacteria it could reach to a event were inorganic nutrients will become depleted and only organic nutrients ( nitrogen and phosphorus) will be available that could again be a limiting factor for heterotrophic bacteria and a abundance factor for dinoflagellates and Cyanobacteria.

your article makes perfect sense to me just wondering around the effects of excess Doc In a system.
 
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efilho

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After fifteen years of "piloting" (dosing, changing, treating etc etc) my 120g, I "found peace" making partial water changes twice a month in order to bring NO3 and PO4 close to the 16:1 Redfield Ratio. My tank usually runs on avg 30ppm NO3 and 2ppm PO4 (big fishes): when the ratio goes below 16:1 (seldom) I feed more, going up (usually) I make a water change to the same percentage as I need nutrients to come down (calculate for nitrate, phosphate normally follows it)... No cyanos or dinos since I decided to do just that: only greens that my tangs and cleanup crew (2 mitrax for any bubble algae that sneaks in) keep under control. Works for me and for my LPS and a few very tolerant SPS. Dosing carbon was frustrating because it never worked for long. Interesting, though, because of the expectations (until frustration sat in...).
 
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jrmailo

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In my personal experience, I was able to finally beat my dinoflagellate outbreak (ostreopsis and prorocentrum) with a similar method outlined in this article. That is organic carbon dosing.

There is a lot of conflicting information about dinoflagellates regarding nutrients level and how 0 N/P can trigger an outbreak, but I argue that dinoflagellates rely on N/P for survival and growth just like other photosynthetic organisms in our tank and that low nutrients is not the real cause of these dinoflagellate outbreak. This is backed up by many scientific literatures that saw significant growth of dinoflagellates in high nutrients environment with the growth curve inverse to N and P concentration.
 

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I've been battling dinos for a year and would like to give this a try. I'm not following the instructions clearly. Is it recommended to dose "1ml per 100L" per day or per week? When should I increase the amount?
You dose every day and SLOWLY ramp up weekly.

There are risks associated with organic carbon dosing and you should have a good grasp and understanding of the method before you start dosing.

You can find many instructions on organic carbon dosing in reef aquaria by a simple google search.
 

sixty_reefer

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I've been battling dinos for a year and would like to give this a try. I'm not following the instructions clearly. Is it recommended to dose "1ml per 100L" per day or per week? When should I increase the amount?
Per article is 1ml per day and increase 1ml per
week for more persistent species.

Week 1: 1ml per day
Week 2: 2ml per day
Week 3: 3ml per day

not exceeding the 3ml dose

the only thing I may be missing is if nutrients need to be detectable at all times, theoretical you can dose and increase bacteria even without the presence of N and P although imo this could lead to coral bleaching if nutrients remain undetectable for a long period of time.

edit: just re read, nutrient need to stay detectable at all times as suspected, therefore if you have low nutrients you may need to dose nitrates and phosphates first and then add organic carbon to the system, keeping a close eye on your nutrients during treatment to avoid them from bottoming out. If they do heterotrophic bacteria won’t be able to outcompete the dinoflagellates as they will become limited in nutrients to grow and dinoflagellates may come back stronger.

this method goes in line with using mb7 and waste away as product to aid the fight in dinoflagellates as the products contain heterotrophic bacteria with the addition important nutrients such as Doc and phosphates
It also goes in line with live phytoplankton dosing that have worked for so many in the past as live phytoplankton dosing will increase Doc and inorganic nutrients as a result.
 
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How old were the 11 treated aquaria?
7 of them young aquaria lees than 1 year age
Thank you for sharing this study. The article was easily understood and fascinating. I have a few questions.

How old were the 11 treated aquaria?

Was the bloom organism identity confirmed by microscopy in each of the 11 systems?

Does every dinoflagellate species bloom disappear with carbon dosing at roughly the same rate?

Did the bloom in every aquarium with the same species of dinoflagellate disappear at roughly the same rate?

Which aquaria developed visible cyanobacteria growth?

Thank you,

Dan

Dinos were not identified on the microscope, only visual identification. Regression of dinos was not equally in different cases, buy I think it depends on other factors, more than on species. Non of the aquaria developed cyano, but two of them developed hair algae. As we all know, frequently, when a dominant species is receding, it is swapped by a new one, as the biological niche is available
 

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Per article is 1ml per day and increase 1ml per
week for more persistent species.

Week 1: 1ml per day
Week 2: 2ml per day
Week 3: 3ml per day

not exceeding the 3ml dose

the only thing I may be missing is if nutrients need to be detectable at all times, theoretical you can dose and increase bacteria even without the presence of N and P although imo this could lead to coral bleaching if nutrients remain undetectable for a long period of time.

edit: just re read, nutrient need to stay detectable at all times as suspected, therefore if you have low nutrients you may need to dose nitrates and phosphates first and then add organic carbon to the system, keeping a close eye on your nutrients during treatment to avoid them from bottoming out. If they do heterotrophic bacteria won’t be able to outcompete the dinoflagellates as they will become limited in nutrients to grow and dinoflagellates may come back stronger.

this method goes in line with using mb7 and waste away as product to aid the fight in dinoflagellates as the products contain heterotrophic bacteria with the addition important nutrients such as Doc and phosphates
It also goes in line with live phytoplankton dosing that have worked for so many in the past as live phytoplankton dosing will increase Doc and inorganic nutrients as a result.
Sounds like Tropic Marins bacto-balance would work nicely, as it is a carbon dosing solution that adds No3 and Po4, all in one addition.
 

Dan_P

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7 of them young aquaria lees than 1 year age


Dinos were not identified on the microscope, only visual identification. Regression of dinos was not equally in different cases, buy I think it depends on other factors, more than on species. Non of the aquaria developed cyano, but two of them developed hair algae. As we all know, frequently, when a dominant species is receding, it is swapped by a new one, as the biological niche is available
Thank you for the additional information.

I have been studying the growth of algae under various conditions and found some of my older cultures become infested with dinoflagellates. I am going to try carbon dosing on those cultures and observe the effect.
 

sixty_reefer

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Sounds like Tropic Marins bacto-balance would work nicely, as it is a carbon dosing solution that adds No3 and Po4, all in one addition.
The only issue I see with bacto balance is that it adds organic nutrients that from the experiment results leads me to think that might favour dinoflagellates, heterotrophic bacteria seems to favour inorganic nutrient over organic nutrients as a source of energy.
 

sixty_reefer

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7 of them young aquaria lees than 1 year age


Dinos were not identified on the microscope, only visual identification. Regression of dinos was not equally in different cases, buy I think it depends on other factors, more than on species. Non of the aquaria developed cyano, but two of them developed hair algae. As we all know, frequently, when a dominant species is receding, it is swapped by a new one, as the biological niche is available
Was the hair algae due to heterotrophic bacteria limitations? It’s normally observed rapid growth of pest algaes wend this bacteria becomes limited at the nutrient level, it stops being able to recycle ammonia leaving the GHA as a dominant ammonia consumption organisms in a system.
 
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