Chapter 17. Maybe it was all a coincidence.

It's four weeks on from Chapter XV and it is becoming clear that heating the AW to 70 C before using it for a water change did not result in an increase in cyanobacteria (possibly Jaagenima see Chapter XVI). Not only that, but the same process of gradual die-back has continued. I have made a slideshow covering the period week 114 through 124 focussing on an Amazon Sword plant. It's available on youtube.

The final two stills cover the period after heating the AW, the tank looks better than ever. For the last two changes (week 125-126) I have been using ordinary tap water (it's good not having the hassle of running a second air-stone and heater), and so far no sign of a return either. There's still cyano in there of course.

Week 125.  Cyanobacteria hunting

Waiting, patiently, the only remaining species 1 mat is on the plastic filter housing (top left). There's a little species 1 in amongst the Java Moss as well (it's obvious if your eye's in) and what looks to me like a mixed (species 1/2) fine mat on the bogwood.

Week 125.  Fix bayonets.

The fact that heating the AW didn't result in any increase in cyano might suggest (if you believe what you read in Chapter XIII) that it wasn't the microbial community in the AW that affected the cyano. But the fact that going back to tap water hasn't caused a recurrence means that it is a formal possibility that the whole thing was a coincidence. I don't think I can conclude anything because the fact is my aquarium has fundamentally changed.

1. The pH has lowered from 7.6+ to around 7.2. 
2. Nitrates now accumulate between water changes.
3. The plants are growing.
4. There is a lot less cyanobacteria.

1. As for the pH dropping, I think I made a big mistake in Chapter XII. I measured a drop in tank pH in week 115 it's true but, looking back at the calender I write all my tank notes on, I see that I first recorded a drop in tank pH in week 111, one week before the first AW change. It seems that this slipped my mind when I wrote Chapter XII. So the statement " It seems maturing my tap water for a week results in a lowering of its pH. And that by using it for water changes I have lowered the pH of my tank" was wrong. I don't know why the pH of my tank dropped and I don't know when because the closest pH reading I can find is 7.6+ in week 37. I gave up measuring the pH back then because it was always the same. Maybe the death of the Cardinal in week 115 suggests the pH drop had been recent. Or maybe the cardinal just died.

2. As I said in Chapter VII, at the height of my cyano problem levels of nitrate dropped between water changes even though I was adding KNO3. At the time (week 50) I gave up testing for nitrate because I had decided that nitrates and phosphates didn't have any effect on my cyano problem. I started testing again in week 115. Levels were close to zero and remained so until week 117, since then they have tended to increase. These last few weeks they have been around 1-2.5ppm. The simplest explanation of this would be that the cyano was using up a lot of nitrate, but that might not be the only reason. 

3. As you can see from the slideshow, the cyano die-off has had a dramatic effect on the plants. They're all growing, but the Amazon Swords are growing well. The mats that covered them were on both sides of the leaves so I'm amazed they survived. Amazon Swords are the ultimate low light plant.

4. I don't bother cleaning the remaining cyanobacteria any longer. Live and let live I say.

Whatever the cause, the change has been dramatic. It's like a totally new aquarium with a different balance of organisms, a new ecosystem if you will. One of the most dramatic changes is the amount of algae. I used to get some green spot algae on the tank walls (especially the back wall). As you can see from the slideshow, the green spot turned orange, then brown and was easily scraped off with a scouring pad on a stick. I haven't seen any green hair algae since I stopped adding nutrients. And yet I'm testing positive for nitrates, so by definition there must be an excess in my aquarium. It seems that the changes in my aquarium that disadvantage cyanobacteria also disadvantage green algae. Of course all this has made my life a lot easier. Cleaning the tank takes about 5 minutes and I can do a water change in 45 minutes. It used to take hours.

However, intellectually this is most dissatisfying. I have no idea what caused my cyanobacteria outbreak, and I am uncertain what solved it. The whole point of starting this blog was to say something sensible about the causes of cyanobacteria problems and to provide some advice that was based on reason. All that is left to me is to speculate.

It's possible it was all a coincidence, but it's also possible that using AW for ten weeks (weeks 112-122) set in motion a chain of events that could not be reversed by going back to tap water. I imagine it's possible that once the ecology of an aquarium changes, once new species become established, the previously dominant species can never naturally take over. Maybe if I'd gone back to tap water after five weeks I would have seen a recurrence. But I got spooked by the Cardinals demise, I didn't realise the pH of my tank had already dropped. There is a way to test my theory. If I have learn't anything about the causes of cyanobacteria in my fish tank, I should be able to trigger an outbreak by dosing with antibiotics. This should convert the tank to a state that previously has favoured the dominance of cyanobacteria after using tap water for changes. I would be very interested to know the effect that would have on tank pH. If the cyano returned I could then test if using heated AW could reduce the cyano, and if it didn't, I would be able to repeat the observation that AW did reduce cyano. It's Christmas eve tomorrow and I am in no mood to start a cyanobacteria outbreak, but in the new year, if I don't see any return after 10 weeks of tap changes, I'll think about it.

Chapter XVI. Retraction of chapter XIV and cyanobacteria identification 4

I know that the the world is an extremely volatile place, but nothing could have prepared me for the turmoil I discovered in the world of cyanobacteria classification. My mistake had been not to check how up to date the taxonomy keys I was using were. I now know that if you want to identify your cyanobacteria with more recent information the book to read is Freshwater Algae of North America: Ecology and Classification. By John D. Wehr and Robert Gordon Sheath. It contains a new key as of 2003. It seems that the previous system didn't consider enough consistent filament features and paid too much attention to features that tended to vary. As a result, during the 1990s/2000s, the thin (less than 3 microns) filamentous, non-heterocystous cyanobacteria were completely re-classified. Oscillatoria splendida and O. amphibia were removed from the genus Oscillatoria and into a new genus Geitlerinema. Not only that but the genus Geitlerinema is one of eight in the family Pseudoanabanaceae and the thin filaments had been distributed amongst them. At first I thought it would be no problem. I would find out the new names for O. splendida and O. amphibia. Then when I searched for information about them I would get the latest. Maybe then I could answer my questions:

1. Has my species 1 been found in cyanobacterial blooms in nature? 
2. Does it produce toxins?
3. Can it fix nitrogen?
4. Is it common in the environment/water supply?

It turns out O. splendida and O. amphibia are now called Geiterinema splendidum and G. amphibium but  one of the defining characteristics of the genus Geiternema is that the filaments exhibit "intense longitudinal gliding". I know what they mean by that because when I first examined the mixed sample from the moss ball (Chapter XIV) the species 2 filaments were moving out and into the field of view like freight trains. The species 1 filaments gently waved at the ends and it looked like there was some rotation to the movement but it was not longitudinal. As a result I can safely say I wrongly identified species 1 in Chaper XIV because filament movement  is one of the new genus defining features. So if it wasn't a Geiterinema sp. which of the other seven genera was it? I was back to square one but I had a new key to follow. I went back to the original photos and looked at them more closely. I'm going to ignore the filaments with one tapered end and one rounded (heteropolar filaments) for now. (Edit 7-9-14 see Chapter 22 for an update on the issue of longitudinal gliding.)

Picture A. Cyanobacteria species 1. Thin (2 microns) filament showing the 'granules'  at the cell junctions (arrows)

Things start off easy enough. Q1-Are there heterocysts? No. Q2-Are the filaments less than 3 microns wide? Yes. Q3-If the trichomes have sheaths, are the sheaths thin with one trichome per sheath? Yes. Q4-Do the trichomes have thin, fine or firm sheaths? I had to give this question a lot of thought. If I look at almost any photo of species 1 I can see a brownish layer round the outside and sometimes a thin diffuse light diffracting layer around that. Many species of cyano produce a sticky layer around their filaments called a mucilaginous layer. It helps then form matts. But is that what I see? or is it a thin sheath? The problem is, if I answer yes I end up at a genus that looks a lot like species 1. Here is a photo of a Leptolyngbya species from a website that is run by scientists so it should be trustworthy. The gaps between the cells in this photo remind me of my filaments and Leptolyngbya are defined in the key by their ability to form mats which mine certainly can. Looking at this photo I have to say that if these filaments have sheaths then maybe so do mine, they look 'as sheathed' if you know what I mean. However, in the description of the linked photo it says "Filaments sometimes lacking sheath material" and "Sheath highly variable in thickness" which contradicts the key as Leptolyngbya are supposed to have 'thin, fine or firm' sheaths.

Picture B. Cyanobacteria species 1 filaments showing  gaps between cells

Here is another Leptolygbya photo, again from academics. The description regarding sheaths is "Sheath thin, colorless, usually diffluent, indistinct, mucilaginous, rarely distinct". So maybe my filaments do have a thin mucilaginous sheath. For me, a sheath has to be a separate structure from the trichome. Here is a photo of species 2 from the moss ball in Chapter XIV

Picture C.  Cyanobacteria species 2 filaments showing the clear sheath

All my other cyano photos are from fresh samples examined within hours of sampling. This photo was taken after the sample had sat in a tube for a week. Because some of the trichome cells have died you can clearly see the sheath. Compare that with species 1

Picture D. Species 1 filaments apparently lacking sheaths but having a thin mucilagenous layer (small arrow). The process of filament fragmentation without necridia may be occurring (large arrow).

The only thing holding these two four celled filaments together is the two dead cells between (large arrow). The thin diffuse layer around the trichome does not extend to the dead cells so that's no sheath in my view. It's a coating that the living cells seem to exude but that doesn't behave as a cohesive structure. So I'm going to answer no to Q4. Q5-Are the trichomes straight/wavy/irregularly coiled? Yes. Q6-Are the trichomes cylindrical and multi-celled without wide mucilaginous envelopes? Yes. Q7-Are the filaments solitary or in fine colonies and are the trichomes constricted at the cross walls, sometimes with polar aerotopes? I don't think I could describe the mats that used to cover my tank as 'fine colonies' and I don't think the trichomes are constricted at the cross walls either. But what about polar aerotopes? An aerotope is a collection of gas vesicles which give some species of cyanobacteria buoyancy. They can regulate how much gas is stored and so move up and down in the water during the day. As you can see from all three of the species 1 photos in this post, the cells in my filaments contain objects.

Enlargement of Picture A. Cyanobacteria species 1

These two enlargements are from Picture A. I think the fact the objects look pale at the top of the filament and dark at the bottom is a trick of the light. Could these be polar aerotopes? They do seem to be mainly at the ends (poles) of the cells. Here is a photo of  Limnothrix redekei a species which has polar aerotopes. Unfortunately you can't zoom in but if you download the picture and have a good look you will see that aerotopes seem to vary in size. I've read that some can take up 50% of the cell. The objects in my filaments all look roughly the same size. So if it's no to Q7 then it's yes to "Trichomes in larger clusters/mats, not constricted at the cross walls. No aerotopes but scattered or polar granules". Q8-Are the filaments motile? No, not if they mean 'intense longitudinal gliding' and I think they do because if I answer yes I get Geitlerinema. So in conclusion, my new best guess is that my tank was overran by a species from the genus Jaaginema. There are very few photos of Jaaginema I can find and some of them I think have been wrongly identified because their cells are thinner than the trichomes are wide and the trichomes look to be >3 microns wide. I find quite a lot of wrongly captioned photos of cyanobacteria, this is why I don't include genus or species names in my photos. Here is a link to a photo of a Jaaginema species that looks similar to my filaments, allowing for the fact that they seem to have used a fluorescent light source for the photo. And here is the description of Jaaginema from cyanodb, a website run by the guys who reclassified these thin filamentous cyanobacteria.

Descriptions:Komárek (1992): Filamentous; filaments usually solitary or freely clustered (tangled and coiled) into small colonies, rarely forming macroscopically visible mats; trichomes always without sheaths, cylindrical, isopolar, usually wawed or coiled, narrow, thin, 0.5-3 μm wide, uniseriate, usually not narrowed to the ends, slightly constricted or unconstricted at the cross walls, always immotile. Cells cylindrical, elongated, longer than wide (up to several times), without aerotopes, sometimes with solitary granules (rarely at the cross walls); end cells rounded_or narrowed, pointed or conical – rounded, always - without calyptra. Cell content pale blue-green, grey, yellowish or olive-green; in some species ability of chromatic adaptation (changeable phycobiline ratio).
Reproduction strategies, life cycles, cell division:Komárek (1992): Cell division by the crosswise binary fission, perpendicularly to the long axis of a trichome, daughter cells grow +/- up to the original size before the next division. All cells capable to divide. Reproduction by the fragmentation of trichomes without necridic cells into immotile hormocytes (indistinct motility was not proved yet).
Ecology, ecophysiology, ecological significance:Komárek (1992): Mainly benthic organisms, growing on the bottom of diverse water biotopes, pools, lakes, reservoirs with rich vegetation of water plants, commonly in metaphyton. Several species known from mineral, thermal or salinic waters.

Most of this info fits with what I see although "always immotile" is a worry as the filaments from the tropical tank and goldfish tanks did wave about as I mentioned in Chapter XIV. Perhaps "rarely forming macroscopically visible mats" suggests that this cyano behaved unusually in my tank if it is a Jaaginema.  I notice "end cells rounded or narrowed, pointed or conical - always without calyptra" means I don't have to worry about filament ends any more (this must be one of the features they stopped using to classify these species). Calyptra are thickened or enlarged tips to a filament, they can be hood-like, lid-like, or cap-like and I can't see anything like that. Mainly benthic means that this genus usually lives on the bottom of water bodies, usually freshwater. Uniseriate just means the cells are in a single chain. Jaaginema is a little known genus with  27 species according to cyanodb. The question is, if I now search for information on these 27 Jaaginema species (and their previous names from the Oscillatoria days) will I be able to answer any of the questions I listed at the start of this post?

Chapter XV. The effects of heating water on pH

I'm taking a break from taxonomy to provide an update on the experiment I described in Chapter XIII.  After writing "This should kill 99% of bacteria etc. but I think leave the water essentially unchanged" it occurred to me that I better look into what the likely effects of boiling water before adding it to an aquarium might be. There is very little information I could find, especially relating to fish keeping. But the always dependable Skeptical Aquarist has an article on using boiling to soften water. The linked article predicts that the water will be depleted in dissolved oxygen and may have lost some of its buffering capacity. I decided to test what the effect of heating my tap water (TW) was. I heated 15 liters of TW to 70 Celsius in a stainless steel stockpot. I measured the pH before heating and after. I then added an air stone and left it for one week, taking daily pH readings. As I described in Chapter XII, leaving my TW for a week results in a slight reduction in pH. I assume this is the result of the aerobic respiration of organisms present (see here for a great article on bio-acidification). If my heated TW had lost buffering capacity then I might expect to see a more dramatic reduction in pH than in the AW as a population of microbes developed. I went for 70C because I didn't wan't the water to bubble and drive off too much dissolved gas but I should still kill most microbes when the time came to repeat  the process for real on the AW.

TW 0h

Heated TW 70 C + 16h

Heated TW 70 C + 6 days

As you can see, either heating the TW reduced its pH or leaving it overnight has (I should have bought two stockpots, filled them both with water and only heated one of them :). There doesn't seem to have been any further drop in pH during the week so no evidence of a reduction in buffering capacity. The drop in pH is about the same as I see in the AW. Given that this heated TW must have been low in microbial life and has dropped in pH within 16 hours, it does make me wonder if bio-acidification is responsible for the reduction in pH I see in the AW. Maybe its a chemical process. To test this I measured the pH of a sample of tap water straight from the tap and filled a saucepan, left it for 30 minutes and measured the pH.

TW 0h

TW 30m

There's a clear difference and the 30 minute sample looks to be about the same pH as the heated TW after six days. I would guess the process is chemical as 30 minutes seems too fast for a biological process. My explanation would be that water for domestic use is pumped around under pressure. When I fill a container with it the water equilibrates to atmospheric pressure and gaseous exchange takes place. In my case this seems to result in a slight drop in pH (which may suggest that CO2 is being absorbed). The pH then seems to remain stable. I haven't been able to find much information on what the likely effect on pH of storing water would be but here is a link where they measure an increase in pH over 12 days. In the discussion they say "The observed increases in pH during storage could be due to the activities of the resident flora and or their death, which results in the release of inorganic substances such as ammonia". This doesn't seem to be happening with my water. My question is: what were the processes in my aquarium that had previously conspired to maintain the tank pH 7.6 or above while the water I was adding naturally drops to a pH below that after adjusting to atmospheric pressure? As I mentioned in Chapter XIII, I had never measured tank pH lower than 7.6 in the entire two years and three months of cyanobacteria. Anyway, I was satisfied that heating my water to 70C doesn't have a dramatic effect on its pH so I repeated the process on AW and used it for a water change today.

AW 6 days

AW 6days 12 hours after 70 C

It took 30 minutes to heat up the 15 litres of AW and eight hours to cool from 70 C - 20 C. To achieve this rate of cooling I left the stockpot outside on a cold day. When I got back from work I added a heater and an airstone for an hour or so to add some oxygen and warm it up to tank temperature (23 C). As you can see, heating the AW doesn't seem to have affected its pH as predicted. After the water change the fish seemed fine. Now I play the waiting game.

Chapter XIV. Cyanobacteria identification 3

Unidentified cyanobacteria 12-11-12

I noticed a new patch of cyanobacteria on the moss ball. It forms a coarser matt than my previous cyano and looks to be a different colour. The matt above took four days to form so it's quite lively. Once again it was microscopy time and, if I do say so myself, I took some great photos.

Three filamentous cyanobacteria species from a freshwater aquarium. The wide filaments may contain necridia (arrowed). Sample taken from a macroscopic matt growing on a moss ball.

(Edit 7/9/14 see Chapter22 for more recent cyanobacteria identification)

There are obviously three types of filament here. Species 1. looks a lot like the cyanobacteria in the unheated tank and that had previously dominated the tropical tank. I'll call the broad filaments species 2 and the small species 3. So back I went to phycokey where I discovered that all three are probably Oscillatoria (filaments, un-branched, un-tapered, no heterocysts, no sheath, not spiral). The two arrowed cells above are probably not heterocysts. They look like this diagram from an algae identification guide I found.

The clear cells are the separation discs (necridia). These are the site of filament fragmentation giving rise to a hormogonium, which is how some species of Oscillatoria reproduce. But not all Oscillatoria it seems as I have never seen necridia in my species 1 (note the single cell 'filament' in photo two Chapter VIII Appendix I).

Species 2 is much more interesting to look at than species 1.
Note how dark the pigment in species 3 is, almost black.

Given that the three species look so different and possibly reproduce differently, I thought maybe I could take a guess at which species I have (contradicting what I wrote in Chapter VI Appendix I) . I found a guide to identifying algae in water supplies that has a section on cyanobacteria and Oscillatoria in particular (thankyou The University of Texas). All I needed to do was measure the width of the filaments and the length of the cells that make up the filaments and It seemed I could follow the key.

Species 1. Filament diameter 2.0 micrometers (2 thousandths of a mm), average cell length 5.3 micrometers (L/W ratio 2.65). So, question one, are the cells half to equal or greater in length than the width of the filament? Answer yes. Question two, are the filaments red/purple? No. Question three, are the filaments blue/green? Yes. Question four, are the cells 2-3 times as long as the filament diameter? Yes. Question five, are the filaments tapered? At this point I am left with two choices. If I say yes I get Oscillatoria splendida, if no O. amphibia. The cell dimensions are correct for both so don't be put off O. amphibia because the filament in the linked photo looks a different colour to mine. Microscope types make a huge difference to the images. So the question is, are species 1 filaments tapered? I will make two points. Firstly I can find photos annotated as O. splendida with non-tapered filament ends (there's one in the main photo from the above link). Secondly, as I pointed out in Chapter VI Appendix I, I observe a mixture of tapered and non-tapered filaments (admittedly non-tapered dominate). I suggested then that I might have a mixture of species, and if being tapered really is being used as a species defining characteristic in the genus, then perhaps I do. However, I have made a disturbing observation.

The filament marked has a tapered and non-tapered end and wasn't the only one I observed, although they are rare. I cannot find a filament that is tapered at both ends in any of my photos so I'm not sure tapering can be used to define these filaments. In summary, my best guess at the moment is that my tank was overrun by O. splendida or O. amphibia but I've got some reading to do to check how consistent tapering is in these species. Also, these might not be the only possibilities. The University of Texas guide doesn't cover all Oscillatoria species, only those found in water supplies. If you follow the O. splendida link above you will see a related species that looks very similar but that is not mentioned in the key. Oscillatoria taxonomy is a contested area and I notice from algaebase that out of 1049 species in the genus, only 60 are accepted taxonomically. I think this will be a work in progress.

Chapter XIII. Theorise!

The results of the pH tests showed that I hadn't thought of all possible reasons why AW might reduce cyano numbers in Chapter XI. With this in mind I now think there are three possible reasons, but I would be interested in other opinions.

1. Ecological. The aging process results in the growth of a population of bacteria/unknown organisms (probably heterotrophic bacteria) in the AW who directly compete with the cyano for space and nutrients after being added to the tank.
2. Nutritional. During the aging process the AW is depleted in a factor (X) that promotes cyano growth. The mechanism could be biological (e.g. uptake by bacteria) or chemical (e.g. precipitation/chelation).
3. Chemical. The aging process results in a change in water chemistry so that it no longer favours the growth of cyano (e.g. pH).

They're all interlinked of course. A drop in pH could increase the rate X precipitates, X could be a factor which discourages the growth of bacteria Y, which compete with cyano etc.

Anyway, I thought of an experiment that might help eliminate pH as a possibility. I could add the minerals I bought before Arthurs intervention to my tap water and then age it. According to the manufacturer this would result in raising the carbonate hardness by 4 dKH, and the general hardness by 7dH. As a result the acidification of the AW should be prevented by the extra buffering compounds (bicarbonate (HCO3) mainly). I haven't tested my tap water for carbonate hardness, but I think the water here is soft. If it wasn't I wouldn't be seeing a drop in pH in the AW. Also, my unheated goldfish tank pH is 6.0 or below before a monthly water change and usually ~6.4 afterwards. This suggests that my water has very little buffering capacity. It has always surprised me that I never took a pH reading below 7.6 from my tropical tank before I started using AW. My plan was to switch to fresh tap water until the cyano came back (assuming it did), this should gradually cause a slight increase in tank pH (~7.0-7.6+) over a period of around three changes. Then if I used the modified AW, I might see a reduction in cyano with no decrease in pH. If not then it would suggest that pH was important. Unfortunately I can't risk it. A fish-keepers primary responsibility is to his or her fish. Also it would take weeks.

As an alternative I will try and eliminate 1. Ecological as a possibility. Of the three, it's the one I think most likely (I will explain why later). What I will do is age the water as usual but before using it, heat it to near boiling point for five minutes. This should kill 99% of bacteria etc. but I think leave the water essentially unchanged. If I measure no difference in pH before and after the heating I will add it to the tank. If i see an increase in cyano numbers in the tank it would suggest that the ecological composition of AW is its important quality in limiting cyano growth rather than it's chemical composition. This is a better experiment because I will hopefully be changing far fewer variables than the comprehensive change in water chemistry required for the pH experiment. One thing I will be doing different is probably adding a very large number of bacterial corpses.

Chapter XII. Safety disclaimer

I know what you're thinking, you're thinking "why don't you switch back to using fresh tap water for water changes and see if the cyano comes back?" It wouldn't be scientific proof (for that I would need eight identical cyano infected tanks in a randomised block design) but it would be reassurance that using AW was the critical factor in reducing the cyano. The reason I have been putting off doing this is because, on Saturday 6th October 2012 (week 115) one of the Cardinal Tetra died. This makes a total of six fish I have lost. The three Otocinclus from Chapter II. One of the nine Glowlight Tetra developed what looked like a tumour sometime in 2011 and died. I don't feel guilty about these deaths as they might not have been my fault. Then in week 85 one of the five Black Phantoms died. This probably was my fault. The death happened five weeks after the fish were netted for the total tank breakdown in Chapter IX. The Phantom had been visibly ill for a while and showed the classic symptoms of a bacterial infection. Swollen eyes and body, totally off his food. I think he probably picked up the infection through a wound inflicted during his netting. The Cardinals death was different, he/she developed the sunken chest and curved spine that I have only ever seen when newly added fish are failing to adjust to tank conditions. This made me suspect that a change in tank pH might be responsible. So two days after I did some pH tests.

AW pH

Tap water pH

Tank water pH

 It seems maturing my tap water for a week results in a lowering of its pH. And that by using it for water changes I have lowered the pH of my tank. I think the Cardinal Tetra that died failed to adjust to this change in tank pH. He/she died four days after the third AW change.

Chapter XI. Arthur intervenes or How I solved my Cyanobacteria problem

Edit 15-8-14
I have not been able to repeat the reduction in cyanobacteria observed in this post using aged water (see Chapter 23).

I had come to believe, that there was something about my tap water, in the context of the biochemistry of my fish tank, that inevitably led to the dominance of cyanobacteria. From my reading of the reef forums I knew that many people had found it necessary to completely control the chemistry of their tank water in order to keep sensitive corals in reef tanks. They used reverse osmosis water or distilled water, in which practically all dissolved substances have been removed. They then add added back all the necessary dissolved buffering substances and trace elements etc. at known concentrations. In this way they didn't have to worry about their tap water affecting their ecosystems. I was surprised that I would have to go to the same trouble as a reef keeper in order to keep assorted Tetra and a moss ball but that's life. I bought a 20 liter camping water container and a product called Tropic Marin re-mineral tropic. Here is the blurb.

Tropic Marin's Re-Mineral Tropic adds all natural minerals and substances which are vital for the well-being of the fish and lush vegetation and the reverse osmosis water gets back all its beneficial natural minerals and substances. At the same time the carbonate hardness is raised and stabilized at its optimal value. Re-Mineral Tropic is also excellent for aquarium conditioning of soft tap water, rain water and other soft water. Pure natural composition. No by-products. no nitrates. no phosphates.

Carbonate hardness determines waters ability to buffer against changes in pH that would occur as a result of the many biochemical reactions that generate Hydrogen (H+) ions. 

I planned to use distilled water from work, heat it to tank temperature with a small 25W heater, add the minerals and use it for water changes. At this point Arthur intervened.

Over the years I have asked for advice from local fish-keepers about my cyano problems. The guy at my local fish shop explained to me that excess nitrates and phosphates were probably the cause and advised me to buy nitrate and phosphate adsorbing media for my filter. A couple of fish-keepers at work explained that direct sunlight was probably the problem or that sometimes you are just unlucky and get a cyano infection. But Arthur said something different, something new. Arthur has kept tropical fish for 40 years and in that time has seen it all in fish-keeping. He made two points. Firstly, that weekly 37% water changes was too often and secondly, not to use fresh tap water for a water change. Instead use water that had been allowed to stand for at least a week. I thought it was an interesting idea because at the time, I had been reading about bacterial blooms in fish-tanks. Anyone who has set a a new tank will tell you that in the first week you can get milky water and a film of bacteria develops on the tank surfaces. Both usually go away naturally as the tank matures. From what I read the blooms were heterotrophic bacteria (bacteria that use organic compounds as food rather than autotrophic bacteria like cyano). It seemed logical to me that adding water that already had an established bacterial population to the tank might change the ecology of the tank and tip the balance away from the cyano. Or that the heterotrophs would deplete the water of mystery substance X and limit cyano growth as a result. In order to encourage bacteria to grow I added an air stone and the heater to 20 liters of water and left it for a week.

The water carrier needs to be supported or spillages can occur, here I'm using a washing-up basin.

Then in week 112 I did a 37% water change as usual but used what I'll call 'aged water' (AW). I stuck with weekly changes because it felt right, my tank needs a clean after a week. I wish I had photos of the tank before I started, but here are a series of photos from three videos shot immediately before AW changes. I posted two of them on youtube.

Week 114. After two AW changes. 50% gravel coverage, cyano on tank walls, bog-wood and plants.

Week 115. 5% gravel coverage, tank walls largely clear, some cyano on the bog-wood , plants heavily coated.

Week 116. Gravel clear, tank walls and bog-wood mainly clear, biofilm on
the plants showing signs of breaking down

As I sit here, the day before a water change in week 120, I can stop writing (variably) in the past tense. Its time this blog went live. Here is a photo I just took.

Until at last I threw my enemy down, and smote his ruin on the mountainside.

It seems that using AW dramatically reduced my cyanobacteria problem within five weeks. It is possible that this was a coincidence and switching to using AW was not the reason for the gradual reduction in cyanobacteria I observed. But it seems to me that it might be the reason and I want to understand why it works. This blog will now be where I explore my ideas about why using aged water reduced the cyanobacteria in my freshwater aquarium.

Chapter X. Measuring relative levels of DOCs

Cyanobacteria always returned to my tank in the same way. First I would see it on plants, then the bog-wood, then the gravel, then the sides of the tank and equipment. The first sign of it on this occasion was on green hair algae growing on some Java Moss. For a long time I thought it would be a milder infestation than I had seen before. I believed that my now scrupulous gravel cleaning and carbon filtration was bound to reduce it, but history repeated itself. It got worse and worse until it was as bad as it had ever been. To be clear what I mean by that. No matter how much cyano was removed at a water change, within seven days it would grow back to cover all tank surfaces. The only place it didn't grow was on the fish.

It may be indicative of my feelings towards my fish tank during this period, that I can find no photos or videos of the tank. From week 86 (when I first noted that cyano had returned) until week 110, I did weekly 37% water changes with gravel cleaning and watched the inevitable march of the cyano. The only explanation I could think of at the time, was that there was something  in my tap water that encouraged the dominance of cyano, I'll call it X. I believed this because, one of the most depressing aspects of my cyano problem was that I always observed the most vigorous growth within the first 12-24 hours after a water change. It seemed to me that this indicated that the fresh water itself stimulated a surge in cyano growth which then declined, perhaps as a result of the cyano depleting the water of X. This is heresy in terms of aquarium fish-lore. The idea that water changes might be the cause of a cyano problem went against everything I had read on the forums. There was another problem with the theory, the cyanobacteria had first appeared during a period where I was doing bi-weekly water changes, which at the time had seemed insufficient as it had caused a green water algal bloom (see Chapter II). But that was over two years ago. Perhaps my tap water had changed and the initial causes of cyano dominance were no longer the cause. I didn't believe X was phosphate because I had never detected phosphate in my tap water. I didn't think it was nitrate as my tap water had never tested >1ppm. But maybe my tap water was high in DOCs.

I needed a test for DOCs and, although I couldn't find a commercial kit, I did find a method that I think answered my question. I got the idea from this site which describes the various uses potassium permanganate is put to in the aquarium hobby. One of these uses is as a crude test for the relative levels of DOCs. The method is based on the fact that potassium permanganate (KMnO₄) solutions change colour as they oxidise DOCs. A good example of the colour changes observed can be found here. So I took a 10 ml sample of my tank/tap water and added five drops of a KMnO₄ stock solution.

Both samples were the same colour to begin with.

Here is a quote from the skeptical aquarist link above.

You can see the reaction happening, as the magenta pink color of unreacted KMnO₄ oxidizes first to a rosy tea color, then to amber and brown; the time it takes to spend itself depends on the concentration of dissolved organic matter. In fact a rough-and-ready field test for dissolved organics measures the time it takes for KMnO₄ to completely oxidize in a water sample.

4 Hours

As you can see, the tank water sample had changed to a rosy tea colour by 21 hours.

21 hours

The tap water sample had remained unchanged, which told me that my tap water was lower in DOCs than my tank. So a water change would be expected to reduce the level of DOCs in my tank, contradicting the idea that X = DOCs. This situation reminded me of Chapter IV when I didn't know if my tap water was high in nitrates and phosphates. Back then, even though I had measured that nitrates and phosphates were low, I went ahead and spent weeks lowering them further. This time I decided a complete change in tank water chemistry was the only way I would be rid of cyano.

Out of Interest

I think the unidentified animal in the final photo from Chaper VIII Apendix I is a Rotifer.

One of over 300 species of bdelloid rotifers

Here is the wikipedia page on rotifers. The rotifer above looks most like the photos of bdelloid rotifers.

Chapter VIII-Appendix I. Cyanobacteria identification 2

Here are the photos of the cyano from the unheated goldfish tank mentioned in Chapter VIII.

Filamentous, un-branched

Un-tapered, no heterocysts, no visible sheath, not spiral, Oscillatoria

I got worms

I got ?

I think it's the same genus as the cyano in the tropical tank, but it might not be the same species. It does make the point that you can't avoid having cyano in your tank. The question is: what are the factors that control how much? In the case of my tropical tank it doesn't seem to be nitrate, phosphate, circulation, direct sunlight or DOCs. I would be very interested to know why cyano doesn't dominate in my unheated tank.

Chapter IX-Cyanobacteria control by reducing dissolved organic compounds

From week 62 on I just did 37% water changes/cyano siphoning/gravel siphoning every two or three days and nothing else. I no longer had any hope that the cyano would be affected, it was just so I could bare to look at the tank (and to reduce the smell). I remember a brief period when I did water changes but didn't remove any cyano with the water (except to scrape it off the tank walls). I think the idea was that the cyano population would grow so large that it would use up whatever it was feeding on and 'crash'. I lasted about 10 days but the tank got so disgusting I couldn't stand it. I also worried that if the cyano did crash and start rotting, it would use up oxygen in the water and harm the fish. I also carried on reading.

I can't remember where I first read about dissolved organic compounds (DOCs), I think it was on one of the reef forums. Reef tanks, and salt water tanks in general, also get cyano problems. Usually the cyano is red so it's often called red slime algae. Once I started searching for DOCs and cyano I discovered a whole new world where  it was well known that DOCs had to be removed or algae and cyano problems would occur. That's what protein skimmers were for, and from what I read they were pretty much universally used in the reef world. Another technique recommended for reducing DOCs was the use of activated carbon.

So what are DOCs? Basically they are a pool of organic matter at various stages of decomposition. Everything that had ever died in my fish tank (bacteria, fungi, algae, plants) and all the fs would have contributed to them as they decayed. Some DOCs are short lived and are broken down to compounds that can be used by plants (e.g. nitrates) but apparantly some degrade slowly and can persist (e.g. tannins) 

It made perfect sense to me, the food source for my cyano was DOCs. Maybe, I thought, cyano could use some of the slowly degrading complex DOCs as a food source but algae couldn't, and that was why cyano always seemed to dominate my tank. I guessed the DOCs were mainly coming from the dirty gravel. I had done a pretty good job of cleaning it but it had apparently not been enough. I also started reading criticisms of under gravel filters (UGFs). The criticism was that UGFs make it harder to keep a tank clean. Because any waste is drawn down into the gravel it is harder to siphon off and, even if you keep the gravel clean, you can get a build up of decaying matter under the filter plate. In week 73 I installed an internal canister filter and left it for a month. The filter had two carbon/wool filter pads, two sponge pads and a compartment for 'biomax' pellets (pellets that encourage the growth of nitrifying bacteria).

Week 74. Still from a video one day after a water change. The canister filter is
installed and the gravel looks clean-ish. I notice there's hardly any Ellodea left. 

Then in week 77 I turned off the UGF. I removed the riser tube, blocked up the hole in the filter plate and carried on with the water changes as before. I hoped that because the tank water was not being drawn through the gravel bed, that less DOCs would build up in the water. Or that maybe the different conditions in the gravel would result in a change in tank chemistry, perhaps due to different types of bacteria living there. I also hoped that the carbon in the filter and mechanical filtration would reduce DOCs to a point where the cyano at least slowed down in it's growth. It didn't. I thought OK, if the gravel is dirty enough then turning off a UGF won't do. Maybe you reach a point where there's so much organic matter in the gravel that you always get sufficient DOCs for a cyano bloom. I decided to start again, but this time to use a fine grained gravel so any fs would just lie on the surface and be easy to siphon off.

In week 80 I siphoned off 20 liters of water and put the canister filter and the fish in the 20 liters. I then removed the remaining plants and treated them with antibiotics before completely breaking the tank down. I removed the gravel and UGF. I should say that the gravel and filter plate were absolutely filthy despite all the siphoning etc. I scrubbed  the tank and fittings (no chemicals) and put in new fine grain black gravel, 34 liters of water then the canister filter and fish with the 20 liters of original tank water. The fish seemed fine after they calmed down. A couple of days later, I washed the plants and put them back in. Weekly water changes as before but I used a new gravel siphon, one of the ones with the wide riser tube that the gravel goes up a few inches while you're siphoning. I found this was the best product I had ever used for gravel cleaning, you could see it siphoning off everything. You may be wondering if cyanobacteria returned to my fish tank and if so how long it took? The answer is it returned in six weeks.

Chapter VIII. Cyanobacteria control by eliminating direct sunlight

It was a desperate time. I can remember using a toothbrush to remove a continuous sheet of cyano from the back wall of the tank in one go, I wish I had filmed it. Honestly, the biomass was awesome.

In chess they say it's better to have a bad plan than no plan at all. I thought I would try cutting off the direct sunlight the tank got ( it only got one or two hours a day but I pinned all my hopes on this at the time) and then dosing with antibiotics. In week 57 I installed blackout blinds over the skylights and in week 58 dosed. I gave up adding nutrients, it had been a waste of time and the plants were losing anyway. I just cleaned the gravel and plants and did weekly water changes. As before, back came the hair algae. It seemed like the algae and cyano were in direct competition because knocking the cyano out always caused the algae to grow well. I'll return to this point in a later post. Below are two stills from a video shot in week 62

Week 62. 

The bogwood is covered in hair algae (and cyano) but you can see the gravel is dirty, it was amazing how much fs had built up. The bubbles on the back wall are trapped by the cyano sheet covering the hair algae, this is the cyano equivalent of 'pearling'. So the cyano came back within four weeks, I thought maybe it was getting resistant to the antibiotics and decided to give them up. It was also clear that direct sunlight was not a factor.

From reading the forums it seems that there is an association between localized cyano patches and direct sunlight. The posts are unusual in that the authors often quote their direct experiences. They say things like "I had cyano in my gravel at the front of the tank and it went away after I put black tape over it" or "it goes away if I clean that patch of gravel regularly". I can back this up.

This is a photo of my unheated golfish tank taken recently, it's in a different room but on the same side of the house as the tropical tank. In that bottom corner against the glass is the unmistakable sign of cyano. And that corner gets the most of the one or two hours of direct sunlight. But this is very different to what I'm talking about. The cyano only grows in this corner of the tank and this is four weeks after a water change. It never takes over like it does in my tropical tank. I will examine this cyano  under a microscope and report back, could be interesting.

Back in week 62 I decided it was time to simply accept cyano as my companion.

Chapter VII. Cyanobacteria control by NO3 limitation

"They say the definition of madness is doing the same thing and expecting a different result." 
                                                                                                Howlin' Pelle Almqvist

There are suggestions on the forums that cyano problems can be caused by nitrates being limiting. The idea is that because some species of cyano can 'fix' inorganic nitrogen (N) and convert it to organic nitrates, they have an advantage over other organisms that can't fix N when nitrates are low. But, given that I had no idea if my cyano was able to fix N,  I was in no mood for such thinking. So I thought I would try knocking the cyano out with antibiotics and then dosing with potassium but no nitrates or trace elements. I thought maybe adding nitrates had been part of the problem because I had noticed something strange about my tank. Levels of nitrate decreased between water changes and often became undetectable. This went against everything I had read on the forums. Nitrates were suposed to increase, it was one of the reasons to do a water change. Where was the nitrate going? Given that the fastest growing species in the tank was cyano, this suggested to me that the cyano was using up a lot of the nitrate I was adding, and the nitrate from the nitrifying bacteria in the bio-filter, and the nitrate from the decomposition of fs and dead plants etc. It didn't seem likely that denitrifying bacteria (bacteria that convert organic nitrate to inorganic N) were responsible for the missing nitrate because my understanding at the time was that the anaerobic denitrifying bacteria (bacteria that can't tolerate oxygen) couldn't function in the oxygenated gravel of a UGF. Here is a summary of the N cycle I found.

Adapted from: Mills, D. The Marine Aquarium. Salamander Books LTD.
8 Blenhein Ct., Brewery Rd. London N79NT; 1987.

I stopped adding trace elements because I had read that they, especially iron, can cause cyano problems. I hoped that making the tank more nitrate limited might have an impact on the cyano. At the least I thought it would give me a good run at cleaning the gravel. After a major cyano removal session in week 50 I dosed again with antibiotics. As before things went well to begin with. The cyano died and the tank became dominated by algae and plants.

Week 51. Cyano free but for how long?

I was doing weekly 37% water changes and adding K2SO4. I haven't made a note of exactly when the cyano came back, but I think it was getting serious again within six weeks.
So it seemed that attempting to control the levels of nitrate and phosphate did not effect the growth of the cyano. I'm pretty sure cyano will use added nutrients if they're there, but it didn't seem to depend on them. I am not convinced that the idea that nitrates and phosphates effect or cause cyano problems, comes directly from peoples experiences of fish keeping. I think it comes from the environmental sciences. When I started searching the internet for information about cyano I found a lot of scientific studies of algal blooms in lakes and coastal waters. It seems there is no doubt that nutrient run-off from agricultural land causes cyano blooms in nature. Phosphate is often cited as the main limiting factor for these blooms, and they have been reduced by managing phosphate inputs. One thing that strikes me about these studies is that the blooms are seasonal. I was six weeks into year two of my cyano bloom and it was not seasonal. Maybe I was missing something simple.

Chapter VI-Appendix I. Cyanobacteria identification 1

The face of evil is always the face of total need
William S. Burroughs

After posting Chapter VI it occurred to me to take a sample of my cyano into work, have a look at it under the microscope and take some photos. It's a light microscope, so I couldn't see enough detail to know what species it was. But I could see enough to take a stab at which genus of cyano I had.  A genus is a group of species that all share some common characteristics. I found a website called phycokey (amazing website based at the University of New Hampshire) that helps you identify which genus of cyano you're dealing with based on some pretty obvious features. Here is the link.

This is a low mag view and as you can see my cyano is filamentous (chains of cells not single cells). The filaments are not branched.

This is x400 magnification. You can see that the bluey green pigment isn't confined to sub-compartments of the cells like it would be in an algae (algae can grow in chains as well). That's a good start, I don't have to re-name this blog. The next question you have to answer on phycokey is 'are the filaments tapered?'. From these photos you'd have to say most are not, but maybe I have a mixed cyano population because that filament coming in from right-field is definitely tapered. Anyway, next question is 'do the filaments have heterocysts?'. Heterocysts are specialized cells that enable cyano to fix inorganic nitrogen from the atmosphere. I looked at a load of photos of filamentous cyanobacteria that have heterocysts, and the heterocysts always looked different from the other cells in the chain. These cells all look the same so I'm saying no. Next question is 'do the filaments have a sheath?'. For some cyano that have sheaths, multiple filaments are contained within the sheath and the photos of cyanos where single filaments had sheaves looked nothing like these so I'm saying no. That leaves two possibilities, a group of species where the filaments form spirals or Oscillatoria.

This is a photo from the web of Oscillatoria posted by Paul G. Davison Professor of Biology at The University of North Alabama. I see some of the filament ends are tapered as well. Well i'm pretty happy to call my cyano an unknown species from the genus Oscillatoria. But what does that tell me? In Appendix II I'll post what I have discovered about Oscillatoria species.

Chapter VI. Questions

So, the cyano was not 'self sustaining' after all, nothing is. That was some woolly thinking by me. No matter how much I removed of the slime, how much I diluted it in the water, how low I knocked it down with antibiotics, it would grow back. The conditions in my tank were obviously ideal for its growth. I'm not saying that controlling cyano by phosphate limitation (achieved by stimulating plant and algal growth) doesn't work. I'm just saying it didn't work for me. Maybe I didn't have enough plants/the right types of plants or add enough nutrients. Maybe it works if you have more lights. I note from re-reading the cyano section of The Krib archive that great success has apparently been had using phosphate limitation while adding CO2. But surely it should be possible to have a cyano free tank without injecting CO2! From what I have read the strategy may be flawed anyway. It seems to me to be based on the assumption that plants and algae, the so called 'higher' lifeforms (eukaryotes), are more efficient at phosphate uptake than cyano (cyanobacteria are prokaryotes). I can find no evidence to support this idea, but I can find people who say otherwise.

"Phosphate uptake in cyanobacteria follows saturation-type kinetics with kinetics parameters (K1/2 and Vmax) comparable to those of eukaryotic algae"
Maria del Carmen Avendano and Eduardo Fernandez Valiente. Effect of Sodium on Phosphate Uptake in Unicellular and Filamentous Cyanobacteria. Plant Cell Physiol. 35(7): 1097-1101 (1994)

These scientists have measured rates of phosphate uptake in the lab and basically found cyano and algae take it up at the same rate and with the same efficiency so when phosphate levels are low, neither should have any particular advantage.

"Phosphorus storage in cyanobacteria appears to be much larger than in other species and this capacity was reported to give them a competitive advantage over diatoms and chlorophytes when P was supplied in pulses"
Santosh Kumar Singh, Vandana Pandey, and Kapil Deo Pandey. Phosphate uptake kinetics and its regulation in N2-fixing cyanobacterium Anabaena oryzae Fritsch under salt stress. African Journal of Biotechnology. 6 (20):2363-2368 (2007)

This work suggests that when phosphate is low cyano may even out-compete the algae (diatoms and chlorophytes are algae) because they can store it better. Anyway, this type of speculation is pointless because cyanobacteria are a very diverse group. If you're interested read The Ecology of Cyanobacteria: Their Diversity in Time and Space  it's on google books. They're so diverse that even if you knew which species you had in your tank, you might not be able to predict how they would behave because they vary so much within species. There is a suggestion on The Krib that cyano plagues in fish tanks are "probably Oscillatoria or Lyngbya". I haven't found out if anything is known about the relative efficiency of phosphate uptake in these species, but not knowing what species my cyano is makes it a moot point. It was time for a change of tactic.