Nitrogen cycle

Nitrogen cycle. All nutrients are recycled, but the nitrogen cycle is the first one you think of. It's the cycle you're referring to when you say that a new tank has "cycled." In truth, this ammonia-to-nitrite-to-nitrate "arc" of the nitrogen cycle, referred to as "nitrification," is only one section of the complete nitrogen cycle. In the less-familiar arc of this cycle, de-nitrification, nitrate is converted to nitric oxide (NO) and nitrous oxide (N2O) — some may be returned to ammonium that can be used by algae and plants— but eventually it's completely reduced (stripped of its oxygen) and returned to the passive store of atmospheric nitrogen (N2). In the complete cycle, atmospheric dinitrogen gas (N2) is tapped by cyanobacteria in the first place. That's the completed nitrogen cycle.
 
Nitrification requires copious amounts of oxygen. In denitrification, by contrast, nitrogen is resorted to as an electron receptor only when oxygen supplies are vanishingly low. A wide range of autotrophic and heterotrophic bacteria are involved in denitrification. In natural ecotopes, these processes are tightly coupled; in the incomplete ecotopes of our planted aquaria, the processes may become uncoupled, leading to build-up of nitrates, as an unnatural end-product.
 
A simplified, non-technical introduction to this process is a team-written article, "The Nitrogen Cycle" at Age of Aquariums. The writers take for their epigraph a quote from Antoine Lavoisier, the French chemist of the Enlightenment, "In Nature, nothing is created, nothing is lost, all is transformed." Another, more detailed and specific, is Robert T. Ricketts' article on microbes "Aquarium microbes: part 1: nitrification" archived at The Aquarium Wiki.
 
Identifying precisely which species of bacteria are responsible might not seem urgent — except that there are products on the hobby market that claim to have packaged the bacteria in question. Use of these canned bacteria is claimed to "jump-start" the nitrifying cycle in a new aquarium. Even if this is possible, it appears that sometimes the wrong strains of bacteria have been chosen! In March 1998, an Aquarium Frontiers article by Tim Hovanec compellingly reported for hobbyists the relevant published scientific research, a lot of it done by Hovanec himself. Hovanec and his co-workers have established that the bacteria traditionally identified as responsible for nitrification are not present in significant numbers in freshwater aquaria. The freshwater bacteria converting nitrite to nitrate, for example, appear now to be Nitrospira; a version of his article, "Bacteria revealed", is archived at  All Tropical Fish.  "It is important to know what bacteria are really responsible for nitrification because, in order to mimic nature, we have to understand what we are trying to copy," Hovanec wrote. "There are many brands of bacterial mixtures for jump starting or accelerating the break-in period of newly set-up aquariums on the market. On what basis were they formulated? From research that has been done thus far we know that just putting some N. europæa and Nitrobacter winogradskyi in a bottle does not work. Of course, using this type of mixture is appealing to a lot of people because most of us don't like to wait. However, there is a lot of evidence accumulating from various research efforts that shows that the nitrifying bacteria are not dominated by just two species of bacteria — one for ammonia oxidation and the other for nitrite oxidation. Further, the species that we had previously thought to be important may not be the critical ones." In 2002 Hovanec's new employer, Marineland Labs, introduced a nitrifying bacteria product based on this research, which claims to ensure virtually overnight nitrification "cycling" for freshwater aquaria. Some alternative and less expensive techniques for establishing the nitrification cycle are here in the "Fishless Cycling" page.
 
 
Nitrification. Each time I've read another account of the "nitrification" of ammonia to nitrate, it has made the process clearer. 
 
Briefly, I'd explain nitrification this way: Nitrogen makes up almost 80% of the atmosphere and yet there is a scrambling competition among living organisms for the use of it. The paradox is explained by the fact that dinitrogen (N2), the form the gas takes in the atmosphere, is inert. Dinitrogen gas consists of two nitrogen atoms so strongly bonded that very few creatures have the metabolic technique of breaking them apart and using them. "Fixing" nitrogen this trick is called. So, though nitrogen is dissolved in the water, just as all the atmospheric gases are, atmospheric dinitrogen plays no part in the nutrient cycle. Instead, almost all the effective nitrogen, that's been drawn into the biological cycle originally and perhaps ages ago by cyanobacteria, enters the cycle as ammonia, NH3. (A little may be fixed  even in the aquarium by certain strains of cyanobacteria.) Ammonia is a by-product of all aerobic metabolisms, including the metabolism of microscopic organisms. It is produced by the fishes and excreted through their gills. But it is also excreted by every snail, every copepod, and by fungi and bacteria. Ammonia is also produced in the de-composing part of the nutrient cycle. Talk about plant and animal residues being "broken down" by fungi and decomposing bacteria is quite literal. All organic matter, including animal feces and tissues, plant detritus and other organic sediments, even peat, is used by a variety of decomposers that utilize the organic carbon as a source of energy. These organisms' enzymes reduce the larger polymers to monomers such as amino acids, purines and pyrimidines. Some microorganisms can directly use these, but generally they are further reduced to ammonia (NH3) in a process called "nitrate assimilation."
 
Ammonia's less toxic form, ammonium (NH4). Your ammonia test kit may measure free ammonia (not bound to another molecule, such as chlorine) or it may measure the Total Ammonia Nitrogen (TAN) dissolved in the water sample. But not all ammonia is equally toxic. In acidic water, that is with pH levels below pH7.0, ammonia tends to collect an extra hydrogen ion. The positively-charged, or ionized, form of ammonia is NH4+, called ammonium. In this ionized form it is much less toxic to fish.
 
As pH drops, more and more ammonia is ionized to non-toxic ammonium. Here's a useful rule-of-thumb for the ammonia/ammonium ratio: for every one unit decrease in the pH measurement there's about a ten-fold decrease in the percentage of toxic ammonia. At pH 7.0 ammonia is about 0.33% of TAN, at pH 6.0 toxic ammonia represents only 0.03% of the total. The temperature comes into play also: at higher temperatures, more of the TAN is toxic ammonia. At 82°F there's almost twice as much non-ionized ammonia as there is at 68°, if the pH remains the same.
 
A table correlating non-ionized NH3 with temperature and pH is part of Neil Frank's article "Ammonia toxicity to freshwater fish: the effects of pH and temperature," archived at The Krib. It might be good to recall that toxic NH3 incurs chronic gill damage at levels as low as 0.05 mg/l. With time, gill damage becomes irreversible.
 
The sensitive nitrifiers. Two cohorts of bacteria act upon the ammonia/ammonium. The first oxidise the nitrogen component  to nitrite (NO2), still toxic to fish, and then a second cohort further oxidise the nitrite to only mildly toxic nitrate (NO3). These "chemolithotrophic" bacterial metabolisms that "eat" nitrogen operate at reduced efficiency when conditions aren't to their liking. The nitrifiers are slow to multiply, compared to most bacteria, even given ideal circumstances. Nitrifying bacteria are affected by several factors, including oxygen, warmth and pH. Nitrifiers require more oxygen than the "ordinary" metabolism of aerobic bacteria — the kind that metabolize organic carbon. So organic matter decomposing in their vicinity prevents them from multiplying and growing, in part by taking up the oxygen that they require. 
 
Researchers performing lab experiments on silt from Danish estuaries found that photosynthesizing diatoms in the sunlit surface layers of the substrate aided the nitrifying bacteria coexisting with them in the biofilm by the enriched levels of oxygen they produced as a metabolic bye-product: rates of nitrification pulsed gently in time with light-dark cycles. The researchers also found that the diatoms could outcompete the bacteria by reducing nitrogen to vanishingly small levels — an unlikely event in your aquarium, with all its ammonia-respiring fish!
 
In the aquarium, the efficiency of this nitrifying bacterial metabolism depends on large water surfaces exposed to oxygen. A bio-wheel unit in the filtration is so effective in nitrification because it brings the bacteria constantly into contact with vast reserves of oxygen in the atmosphere.
 
The nitrifiers compete poorly for oxygen with the community of bacteria that are breaking down organics, the ones responsible for much of the "biological oxygen demand" (BOD). Though quantifying BOD in terms of mg/l is a task for pros, if BOD is reduced, nitrogen conversion is enhanced. Simply stated, a heavy load of organic materials being degraded in your system inhibits the nitrifiers by competing with them for oxygen. Dissolved oxygen concentrations above 1 mg/l are essential for nitrification to occur. If DO levels drop below this level, nitrifications slows or ceases altogether. Nitrification also slows as temperature drops: no surprise there. In koi ponds, as water temperature drops to 50°F, nitrification slows to a halt. The surprise for me was to read that the rate of nitrification keeps increasing as temperatures increased, right up to 95°F, where you might expect some inhibition to set in. Temperatures higher than 35°C/95°F weren't tested.
 
The pH is also a vital factor in nitrification. Maximum rates of nitrification occur at pH values above 7.2, peaking at 8.3 (a common pH for marine tanks) then falling at higher values. What surprised me was the rate at which the effectiveness of nitrification dropped in acidic pH values: to less than 50% optimal efficiency at pH 7.0, to just under 30% at pH 6.5, and to just over 10% of maximal efficiency at pH 6.0. At these low pH values, nitrifying bacteria don't die, they just stop metabolizing and reproducing. Of course in these acidic conditions, most of the toxic NH3 is ionized to non-toxic NH4. But I had been under the impression (and had mentioned here) that the pH needed to drop quite low, below pH 4.8, more like the acidity of a peat bog rather than conditions in a home aquarium, to repress nitrification. Not so.
 
De-toxifying ammonia with sodium hydroxymethanesulfonate (AmQuel) or sodium hydroxymethane sulfinic acid (Bio-Safe). In an aquarium with a higher pH, ammonia can be a serious problem. AmQuel reacts with free ammonia to form a non-toxic stable water-soluble compound, which is scavengable by the nitrifying bacterial population. AmQuel's patented active ingredient is simply sodium hydroxymethanesulfonate — in case you were planning to make your own AmQuel at home! Even at pH under 7.0 AmQuel will scavenge any free ammonia. The recommended dosage is 5 ml. (about a teaspoonful) per 10 gallons. Unreacted AmQuel is also stable  — but you won't keep adding teaspoon after teaspoon, will you? Fresh activated carbon will remove un-reacted AmQuel, so it makes sense to remove carbon from the filtration while you're working with AmQuel. The pH does affect the speed at which it work — lower pH slows the reaction.
 
But be cautious about two possible effects in using AmQuel. In lightly-buffered, "soft" waters, overdosing AmQuel can cause a sudden pH drop. This is especially a problem if you are used to water with a higher pH, but you have recently started infusing CO2, which reduces the buffering and drops the pH. And, an important note, unreacted AmQuel in the water will give false positive readings for ammonia, if you're using the usual tests that are based on Nessler's Reagent. Use a salycilic test instead.
 
The Kordon Company posts a clear description of the workings of AmQuel. See also "Water Chemistry" material archived atThe Krib.
 
Marineland Labs offer a rival chloramine neutralizer/ammonia binder, Bio-Safe, with a similar active ingredient. I should think that the same cautions apply. Now, with chloramine ever more widely used, one of these two products should be your chlorine/chloramine neutralizer.
 
Competition for ammonium. In a well-planted aquarium, ammonium is also quite likely to be scavenged by plants before the nitrifying bacteria even get a chance at it.  Diana Walstad has taught us that aquatic plants prefer ammonium (NH4) and its toxic (to animals) counterpart ammonia (NH3) to nitrate, if they can get it, so in planted tanks like mine, plants and nitrifying bacteria are direct competitors in scavenging any available ammonium. Diana Walstad points out, "Nitrifying bacteria are helpful, if not essential, in tanks without plants. However, in planted tanks they compete with plants for ammonia. The energy nitrifying bacteria gain from oxidising ammonium to nitrate is an equivalent energy loss to plants." (The Ecology of the Planted Aquarium, p 63) The "if not" construction is always ambiguous, but I think she meant "helpful, even essential" rather than "helpful, though not essential."
 
I had always assumed that aquarium plants were assimilating nitrate, as I "knew" garden plants do. But no. It seems that when both ammonium and nitrate are available, tests show that aquatic plants don't take up appreciable quantities of nitrate until the ammonium is gone. The presence of some ammonium actually inhibits the uptake of bacterially-produced nitrate, not just in plants but in a range of other nitrate-users — algae and fungi too. Algae it now appears don't assimilate nitrate if the NH4 concentration is higher that about 0.02 mg/L. I don't have a hobbyist test kit that could even register that level of ammonium.
 
Diana Walstad avers that extensive bio-filtration may slow plant growth. At first I was skeptical, reading this. But soon I recalled a phenomenon I've noticed in a densely-planted 10-gallon tank I use intermittently for quarantine. It's a sturdy, balanced environment, but the bioload represented by fishes varies: it may have a single small Siamese Algae-Eater for a month, lie empty for a week, then receive a dozen tetras. A new load of fishes can trigger a noticable spurt of plant growth. Not unexpectedly. But then plant growth levels off again, I've noticed. Bacteria, hard to kill,  tend to live at the brink of starvation. When they are presented with new resources, they characteristically increase their population, until they are once again living on the edge, in true Malthusian fashion. Now, in my intermittently-used Q-tank, during the brief period while bacteria are catching up to take advantage of new sources of ammonia, there is a temporary supply of it to fertilize plants, resulting in the spurt of new growth. Soon, however, the system restabilizes in a new dynamic balance, available ammonium drops to zero, and plant growth returns to its previous maintenance level of leaf replacement. So, now I'm convinced that extensive bio-filtration may indeed slow plant growth. Many knowledgeable aquarists would remain more skeptical than I am.
 
Ways of exporting nitrate. The usual way of exporting nitrate end-products that build up in the aquarium is through partial water changes. Lowering nitrate levels is a major motivation for water changes.
 
Adsorbing nitrate. There are some chemical products that promise to reduce nitrate levels. Aquarium Pharmaceuticals, for one, makes Nitra-Sorb, a rechargeable ion-exchange resin medium that adsorbs nitrate. Nitrate is very much more objectionable to marine organisms, so resin media like Nitra-Sorb are indispensible in marine tanks.
 
The resin is designed to be rechargeable in brine, which should alert you that it exchanges the nitrate for sodium ions. I felt that potassium chloride would make a good substitute as the recharging brine, since the released potassium would be taken up by plants. I e-mailed Aquarium Pharmaceuticals and got this brief but encouraging e-mail response from Dave Schaeffer, 14 Nov 2002, "As you are removing anions, the cation choice is irrelevant." Well, that was good to hear! Your own decision whether to substitute a KCl brine will be based on your own assessment of how much less stressful potassium ions are than sodium ions in your water. In a planted tank, I think there's no question.
 
When nitrite reappears. Sometimes nitrite can reappear in detectable concentrations, even in a well-established aquarium. Incompleted processes of bacterial de-nitrification can be a cause of nitrite in the aquarium. But it also seems that the bacteria metabolizing nitrite to nitrate are more sensitive than their various partners. Cold may repress them, or lower pH values, or they may possibly be sensitive to a renewed spike of ammonia.
 
De-nitrification, you remember, is the other "arc" that completes the nitrogen cycle.

Comments

Yes, mini-cycle can happen in even well established tank for one reason or another. That's why I prefer to use Prime as my water conditioner. It can detoxify ammonia and nitrite up to 48 hours.

I had a tank setup over 2 & half years ago and for the last year it has sat with no lighting and no fish with only one long surving hardy plant. (exact species unknown but most probably Anubias barteri var.)

I decided to replace the lighting unit and get things back up and running but for several weeks now have had massive Nitrate NO3 readings. After many large water changes, gravel hoovering and filter cleaning unfortunately I still had readings >100 to 250 mg/l. Scratching my head I replaced the entire volume of water (over 180l) with fresh rain water! but my readings were still too high so I decided to go to my LFS and purchase 6 bunches of fast growing hardy plants 3 Egeria densa and 3 Limnophila aquatica. I also lowered my filtration and almost 24hrs later the nitrates were down to a manageable level between 25 to 50mg/l.

I also continue to replace 4g of water per day. I am really encouraged by the results of adding just a few plants.

For these reasons I found your article very interesting indeed.

By reducing the fitration the new plants have less competition from nitrifying bacteria and since there is no ammonium to speak of the plants appear to be taking onboard most of the nitrates.

Good result I think.