Aquarium Microbes: Part 1: Nitrification

By Robert T. Ricketts, a.k.a. RTR

Introduction
What do you keep in your tanks? A big school of Tetras? A pair of Cichlids? A few billion bacteria? Don’t gag. The "bugs" are the most numerous things in our tanks and are ultimately responsible for our success if we are to keep our fish alive and healthy. Every solid surface in our tanks has a biofilm on it, of bacteria, algae, etc., composed of whatever type(s) of microorganism that finds that surface and adjacent water conditions hospitable. These bugs are not the enemy or in any way unwelcome. They aid us in the upkeep and function of the micro-ecologies needed to make our tanks stable and safe for our wet pets.

Hopefully, most of us are aware that most life forms are sensitive to their own waste products. Waste would not be waste if the critter could still use it, it is waste because they cannot, and most often because it is toxic if retained. Also we are aware that fish produce ammonia (NH3) as a primary nitrogenous (nitrogen-containing) waste product. We know that ammonia can kill fish at what seems to us to be fairly low concentrations. This pair of articles is aimed not at details of bacterial growth and metabolism, but at giving just a glimpse at the massive populations of bacteria and other microscopic life forms that are so very important to our aquarium systems. I hope that I can clarify the some of the roles these "good guys" play in our tanks, and a few things that can go wrong when we intentionally or accidentally abuse our microscopic helpers.

The most familiar groups of bacteria to tank keepers are the two types needed to process nitrogenous waste to relatively harmless forms. For years just about everybody has said these are Nitrosomas and Nitrobacter species, and most books will refer to these two forms. Now it appears that those bacteria may play a role in saltwater, but have little or no function in freshwater systems. Research from Dr. Tim Hovanec and others pointed to Nitrospira and Nitrosomonas as the freshwater bacteria primary in oxidizing nitrogenous waste in captive FW systems.

But in the end, we do not care as much about what their names might be as we do care about what they do in our tanks. I said before that ammonia is a primary nitrogen waste product for fish, which happens to be excreted largely through the gills, but also in part in urine, less in solid waste although many ammonia precursors are available there. Ammonia has a great affinity for water, and diffuses readily and rapidly throughout any available volume of water. Land animals must arrange to get rid of nitrogen wastes from the blood through the kidneys as urea in urine. Fish can rid themselves of much ammonia just by the circulation of the blood through their gills, where it will readily and rapidly diffuse out into the surrounding water.

This is great for the fish in the wild, less favorable within the limited confines of out tanks. We have limited water volume, so ammonia builds up quickly. Ammonia tests used in the hobby read in milligrams per liter (mg/l), or as parts-per-million (PPM or ppm), 1 mg/l = 1 ppm for our purposes. Ammonia is toxic as low as 0.25 ppm as other than very short-term exposures. Between 0.5 and 1.0 ppm there can be long term or permanent damage to the gills by chemical burning. At levels much higher than 1.5 ppm death may occur at any time. There is a significant difference in the range of resistance of any particular genus or species, and by the age of the particular fish, to ammonia damage, or in exact levels of damaging or fatal concentrations. Ammonia it is never healthy at levels detectable by standard aquarium test kits. Even the lowest detectable levels, maintained for any length of time, can have negative effects. Ammonia is also added to the tank water as metabolic waste from the microbes and infusoria in our tanks, and from their action on fish solid waste and uneaten food, decaying plants, etc.

Mother Nature has provided bacteria that "eat" ammonia, oxidizing it to nitrite and gaining energy for use in their own metabolism. You can think of ammonia for these bugs as serving a similar role to carbohydrates for us. These ammonia-oxidizers will grow and multiply to populations sufficient to consume whatever levels of ammonia are present in their native water (our fish tank). Great, they have gotten rid of the toxic ammonia that would otherwise kill our fish, not as a public service, but by working for a living as we do. Wonderful, BUT their nitrogenous waste product is nitrite ion (the same nitrogen atom, oxidized by the addition of two oxygen atoms, NO2).

Nitrite is also highly toxic to fish. Again 0.25 ppm nitrite-N is not good other than very short term, above 0.5 ppm dangerous, and 1.0 ppm can be fatal. At nitrite-N concentrations, the numbers are even worse than for ammonia. If you wish to use true nitrite ion concentrations (the whole NO2 ion), the nitrite-N reading should be multiplied by 3.3 - on the boards you should specify which figure you are using. That is because 0.25 ppm of nitrite-nitrogen is >0.75 ppm of nitrite ion.

Again Mother Nature has provided for our nitrite issue. A second type of bacteria uses nitrite just as the first group used ammonia, as an energy source. It "eats" nitrite and oxidizes it to nitrate (the same nitrogen atom, but now with a third oxygen atom attached, NO3, nitrate ion). Guess what? Nitrate is essentially non-toxic short term at these concentrations, more than two orders of magnitude less toxic than the preceding two metabolites, ammonia and nitrite. Long-term toxicities are a slightly different matter. High levels of nitrates, above 40-50 ppm, (along with other less testable pollutants) in the tank water can slow growth, suppress breeding, depress the immune system - making the fish more susceptible to disease, etc.

Again there are apparently significant species variations in nitrate ion and other pollutant tolerance, and the whole area is poorly studied. Most laboratory studies use nitrate alone, the simplest case. That test platform does not, and cannot, reflect the real-world situation in out tanks. Nitrate to us is a symptom and indicator of general pollution. For general use, 20 ppm to maybe 40 or 50 ppm might be an acceptable range, but I try to keep my tanks at or below 10 ppm, or at least below 20 ppm, with 10 ppm as the post-partial water change goal. The "acceptable level" has to be set by each tank keeper, depending on their own level of comfort and their plans for the tank inhabitants by considering the importance of breeding, growth rates, and long life balanced against the upkeep required. Nitrate testing is for me a useful measure of the general pollution level in the tank. The other pollutants are much harder to measure, impractical at home, so nitrate serves as general pollution indicator in other than heavily planted tanks with light fish loads.

The bacteria responsible for oxidizing ammonia to nitrite, and nitrite to nitrate are called lithotrophic bacteria, or lithotrophs. This name means "rock-lover" or "rock-grower", because they are found firmly attached to solid, usually quite firm (to them if not necessarily so to us), substrates. To these bacteria, plastic foams (sponges) are hard, as are most synthetic fibers. These bacteria do not occur as functional metabolizing and multiplying forms in the water column, only when attached. They can and will occupy any solid surface in the tank - gravel, rocks, glass, filter sponges or floss of fiber mats, ceramic or glass beads, plastics, etc. They can occur any of these places, but the most favorable dwelling for them is in the filter.

That is the case whether it is gravel for a UG or RFUG, sponges, floss, beads, spheres, or whatever, in canisters or HOT/HOB power filters, or in fluidized bed filters. This is the most favorable location for them because the constant flow of water is providing the bugs with the two things they need most after an attachment site - oxygen and an energy source (ammonia or nitrite) along with an assortment of other nutrients. The bugs in such a home will have a competitive edge over bugs living dispersed on the glass or ornaments in the tank or even the gravel, who have to rely on diffusion or much lower water currents for their food and oxygen needs. Unfortunately, neither of the originally named two types, nor the most likely current candidates have a spore or other resting forms, so in practice both types must be obtained live and actively metabolizing.

Where do we get these wonderful tank helpers? Well, it is possible to get them with fish. As fish nibble on their environment a lot in the search for food, they tend to have ingested a few good guy bacteria as they grazed, and when we bring them home, some of these bugs may survive the fish’s gut to initiate a colony in our tanks. This is the old way to "cycle" a tank, using a few, hopefully relatively ammonia resistant, fish to start off. It does work, but it is anything from lightly to severely stressful for the fish (and keeper). It can lead to the development of numerous diseases in and on the fish (from the stress-reduced resistance of the fish).

Thus we are multiplying the chances for fish death not just from direct toxicity of the metabolites, but by disease, and also from inappropriate treatment with shotgun medications (used in attempts to combat and control the stress-induced diseases). Those same medications may also directly suppress or kill the good guy bacteria we have been trying to cultivate, kicking us back to square one and starting the whole cycle over, but with weakened fish. If you have existing tanks, you can prepare for additions by getting your filters in advance and running them for 3–6 weeks on existing, healthy, balanced, mature tanks along with those tanks’ regular filters. This will inoculate the new filter quite well. Then it can be moved to the new unit and it will be ready for at least starter fish. Mature colonies of these good-guy bacteria are not static - if they are eating well they multiply by division.

Old biofilms (large or tiny sheets of attached bacteria, joined to one another and the surface they grow on) can shed or "shale" off. Some of these loose fragments of film or individual bacteria may be in the water column temporarily. But they cannot metabolize there. If they cannot attach to a vacant surface soon they will die. Or you can use Dr Chris Cow’s fishless cycling techniques (3), which I strongly support. There have been many earlier attempts to cycle tanks without fish, and some even worked- but many required access to and experience in a laboratory, or were poor and uncontrolled processes. Dr Cow’s technique is not cut-and-dried. It may need a bit of minor juggling depending on local conditions {Insert URL - Fishless considerations}, but it is far better, safer, and surer than any other I have seen and tried. The most difficult part of the technique is learning patience while the process is establishing the needed bacteria in the tank.

If you do use fish to cycle the tank, monitor the metabolites regularly, at least once a day - and twice is better. If or when they move to detectable levels, reduce your feeding markedly. It is hard to starve a fish (other than Loricariids, sucker-mouth catfish, or other specialized feeders, and even that is not short term). Drop back to every other day or even every third day feeding. This will reduce the waste load. I suggest water changes whenever the metabolites reach any potentially toxic levels, to bring the concentrations back down to merely undesirable ranges, or even better, to undetectable levels. The nitrification bacteria seem to "saturate" (grow and divide at maximum rate) at ammonia or nitrite levels well below hobby test kit detection, so are growing at full speed before we can read their "food" by our kits.

The filters should not be disturbed (unless they are clogged) with this water change, nor the gravel vacuumed (unless you have overfed and have visible decaying/molding food on the substrate - then vacuum lightly - the lesser of two evils). The preferred site is the filter, but new colonies may appear anywhere. Over time the colonies in the filter will carry almost the whole load, but this cannot be guaranteed initially. Add stock to the tank very slowly. The water changes done to reduce the concentrations of toxic metabolites will not slow the development of the good-guy bacterial colonies. That is due to the already mentioned fact that these bacteria are growing and dividing as fast as they can below the lowest detectable level of our test kits. If we reduce the concentration of ammonia in the tank from say 1.0 ppm to 0.25 ppm or lower, we are reducing the damage to the fish markedly. We will be protecting the fish against damage or death, without slowing the development of the bacteria.

So long as there is detectable or just undetectable ammonia (or nitrite) in the tank, the bacteria are multiplying as fast as they are can to meet the available food supply. These bacteria are not fast growing. Their division and multiplication is very slow in relation to most other bacteria. If you start cycling with no inoculum, or with a very small inoculum, the total cycle is going to be longer. Slow growth means a longer time spans to a mature colony. This is one advantage to fishless cycling. The higher levels or titers used there do not give faster growth, but do in the end give much larger colonies of each type of bacteria. When you measure ammonia or nitrite in a tank with fish, you are taking a snapshot of the ammonia present at that moment, not the rate of accumulation of one or several days, unless you have been testing and recording data in the past.

Fishless cycling uses single daily doses of more ammonia as needed to maintain levels greater than the maximum produced in an overstocked tank in 24 hours. If that can be cleared within 24 hours, and the resulting nitrite cleared in the same 24 hours, then your filter will handle a full bioload from day one. With fish cycling, the colony will handle the daily production of the fish present now, period. Adding more fish means a mini-cycle that may or may not be detectable by standard hobby test kits, depending on the number of fish added in relation to the original stock. High titers cannot be allowed during fish cycling, it will injure and can kill the fish (both counter-productive and cruel). I hope that makes sense and is clear. The take-home lesson here is that the level or titer of ammonia or nitrite shown by hobby teat kits does not affect the growth rate for the bugs in the ranges we can detect, but does affect total colony population size. By the same token, excessive titers of either metabolite (ammonia or nitrite) can inhibit the bacteria we want to support. Some to enough food is good, too much can be a problem.

One other point is that if the initial inoculation is small, as it is by adding fish, the ammonia-oxidizers will build numbers so slowly that the nitrite-oxidizers may starve before there is enough nitrite to "feed" them, so they may die off in part or completely. This can give an even longer nitrite "spike" (detectable accumulation of nitrite) than the ammonia spike was. This can be defeated by re-inoculation of the filter/tank after any extended (10 days to 2 weeks or more) ammonia spike.

By the way, the first time I used the term "cycle" above, I put it in quotation marks. This was intentional. The nitrogen cycle is well known and recognized in biology and ecology, and we borrow the term for the process of conditioning our tanks by development of the two needed bacterial types for the oxidation of ammonia through nitrite to nitrate. In reality, we do not have a complete nitrogen cycle in our systems. If we did, we would need to have cyanobacteria or other life forms capable of "fixing" atmospheric nitrogen (taking nitrogen gas from the air or dissolved in water and incorporating it into body mass). Then we would need the use of that fixed nitrogen by other creatures in the food chain until it got to the level of our fish and was used by them until excreted as ammonia waste, oxidized to nitrite and then nitrate.

That nitrate would then need to be reduced (via anaerobic bacteria such as in a plenum or denitrator) to nitrogen gas, N2, and returned to the atmosphere to complete the loop of the cycle. So we really have only a very small slice of the nitrogen cycle in our tanks, but we use the broader term routinely. It is okay; it is that part of the full nitrogen cycle that we have in our tanks. We do lots of things that are worse.

The next section of this article will talk about some of the other good guy bacteria and other life forms that inhabit our tanks, and which are needed to bring the tank to "maturity" (balance, with no upsets in nitrification or water clarity causing cloudiness) and keep it at that level indefinitely. Plus a few things that can go wrong, and some thing that can help the tank come into balance, or at least help us understand why and how it is out of balance.