Part 4: The Nitrogen Cycle

The Stuff That Water’s Made Of
Part 4: The Nitrogen Cycle

by Lenny Llambi
First published in Fincinnati, the official newsletter of the Greater Cincinnati Aquarium Society
Aquarticles.com

So far you could say that this series of articles has been leaning toward the "chemistry side of things." We’ve discussed water’s ability to dissolve a variety of different chemicals and the pressure that these chemicals produce called osmotic pressure. Conductivity measures the total amount of dissolved chemicals, while general hardness specifically measures anions such as calcium and magnesium. Finally, last edition; we covered how pH measures the amount of acids vs. bases that are dissolved in water, and how alkalinity maintains a high pH. This edition we’ll discuss the nitrogen cycle, which is the biological process of reducing ammonia to nitrate. Bacteria that use nitrogenous molecules to receive their energy drive this cycle.

Where Proteins Go When They’re Used
Many explanations of the nitrogen cycle begin with ammonia and end with nitrate, but the whole process actually begins before ammonia, and ends after nitrate. The nitrogen cycle begins when our fish ingest and begin to breakdown the proteins found in their diet, called: mineralization. Proteins are long chains of individual molecules called amino acids. These amino acids are very similar to the simple sugars that make up carbs; however, amino acids contain nitrogen in addition to hydrogen, oxygen, and carbon. This nitrogen allows amino acids to bond to each other (called the peptide bond), thus forming proteins. There is a real complicated explanation behind the physics of this bond, but for our purposes, let’s just keep in mind that the peptide bond offers extra rigidity to the protein molecule. This is why proteins are used to build muscles, enzymes, fingernails, etc. These are all parts, which need to retain a solid frame or shape. Once proteins are separated into amino acids, each amino acid is split into an ammonium (NH4+) molecule and an organic acid molecule. This brings up an important point about fish waste. Due to the organic acid and ammonium byproducts of protein metabolization, fish waste is very acidic by nature, and will cause pH to drop. This is very problematic when keeping African cichlids or marine species, which enjoy a very high pH. As I mentioned in the previous installment, alkalinity neutralizes acids such as those found in fish waste so that pH is not affected.

Fish are not the only creatures that are contributing to the nitrogen cycle. Aside from any invertebrates you may have in your aquarium, you probably also have a plethora of bacteria that are breaking down the proteins that your fish “miss”. Bacteria from the genera Bacillus, Clostridium, and Pseudomonas break down any proteins found in excess food, fish waste, plant mulm, or any other source of organic matter you may have “laying around” your tank. On the marine side of the hobby, more and more hobbyists are using rock, which is impregnated with a variety of different bacteria and invertebrates, generically called “live-rock” as a natural source of filtration. One drawback to the use of this rock is that often times, after moving the rock, many of these creatures die in transit. This provides a huge amount of organic matter that is constantly being broken down, causing massive algal blooms and an extended cycling time (curing). I have had good success speeding up the curing process by using Hagen’s Waste Controlä, which is a concentrated culture of bacteria (mostly Pseudomonas) responsible for mineralization. By using this product, frequent water changes, and a lot of circulation, I have been able to cure live rock in two weeks as opposed to the usual month.

Ammonia or Ammonium?
As we discussed in the third article, pH determines whether ammonia (NH3) or ammonium (NH4+) is present in our tanks. Our fish actually excrete ammonium, which remains ammonium in acidic water. However, if ammonium is excreted into water with a pH above seven, it begins reacting with the bases in the water to become ammonia. Now it’s really a misnomer to label ammonia as toxic and ammonium as non-toxic. Both chemicals are lethally toxic to cells when ingested. What differentiates the two is how readily they are ingested. I mentioned in the first article that osmosis allows for water and other small chemicals to pass through the cell’s membrane. Well it just so happens that ammonia is small enough to be transported across the cell membrane almost as readily as water. On the other hand, ammonium, due to its charge, has to be actively pumped into the cell using pumps and channels in the cell’s membrane. Therefore, fish have a little more control keeping ammonium out of their cells. Now don’t take this the wrong way. Whatever the pH in your tank, you should make sure your ammonia/um is under control at all times.

Dirt-Eating Bacteria
At this point in the nitrogen cycle, we begin the nitrification process. Basically this process takes ammonium and converts it into nitrite, which is then transformed into the final nitrification product: nitrate. All of these reactions are performed by lithotrophic (roughly translating as: dirt-eating) bacteria of the genera Nitrosococcus, Nitrobacter, Nitrospira, Nitrosolobus, amongst others. One characteristic of these bacteria is that, in bacterial terms, they are incredibly slow growing. This is why it is essential that you exercise restraint while stocking a brand new aquarium. Your aquarium inhabitants are constantly excreting ammonium into the water, while the nitrifying bacteria are only reproducing every 8 hours (every 24 hours in saltwater). Moreover, the lithotrophic bacteria that feed on nitrites, producing nitrates, are actually inhibited by the presence of ammonia. So once you establish a population of bacteria that can handle the bioload in your aquarium, you have only begun the process of tackling your aquarium's capability of dealing with nitrites. One time-tested and approved way tracking the cycling process is to test for nitrites regularly until the nitrite concentration spikes and returns to zero. Dosing products such as Hagen’s CycleÔ, which is a concentrated formula of lithotrophic bacteria, will help establish populations of ammonia-reducing and nitrite-reducing bacteria in much shorter order. The second important trait about these lithotrophic bacteria is that they must anchor themselves to some sort of substrate. Make absolute certain that when you clean whatever substrate these bacteria have colonized, use aged aquarium water, as this water is devoid of any harmful chemicals (bacteria that is) present in tap water.

One last note about nitrites and nitrates, concerning their toxicity. These two molecules actually have the same exact effect. When nitrate is ingested it is not toxic, but it can actually be converted to nitrite, which is toxic. Once nitrite makes its way into the bloodstream it can react with a blood cell’s hemoglobin, which is the site where oxygen bonds to blood. Once nitrite turns the blood’s hemoglobin into methoglobin, blood is unable to carry out its most important task: supply oxygen to the body. This makes nitrite probably the most lethal molecule in the nitrogen cycle, because 1) it is not inhibited by pH like ammonia 2) nitrate must first be converted to nitrite before it becomes toxic. So, again, make sure you measure a nitrite spike and drop-off before you consider your aquarium cycled.

Making Nitrogen Gas
Bacteria in the genera Pseudomonas, Bacillus, and Alcaligines drive the final step in the nitrogen cycle. These bacteria convert nitrate into nitrogen gas, which then escapes into the atmosphere. However, these bacteria only perform this reaction in certain conditions. When these bacteria grow in an area where oxygen is readily available (aerobic), they utilize the available oxygen to break down sugars. However, when these bacteria find themselves in an area of low or no oxygen (anaerobic or anoxic); they actually utilize nitrates (notice the oxygen molecules in NO3) to break down sugars. Those of us who have ventured into maintaining coral reef aquariums can provide such an anaerobic environment by using a deep sand-bed of at least three inches where the bacteria colonizing the bottom layer are starved of oxygen. You can actually see bubbles of nitrogen gas rising from the bottom layers of sand. Those of us who stay on the fresh side of things can duplicate this phenomenon by not cleaning our sponges (as in sponge filter sponges). What? Not clean our sponge filters? That’s right! Now notice I didn’t say stop cleaning your aquarium altogether. However, in theory, if you allow your sponge filter, ceramic beads, biowheel, etc. to become clogged with bacterial growth, so that the inner layers are starved of oxygen; you will then be able to complete the nitrogen cycle in your very own aquarium. Although the deep sandbed in reef aquariums allows for substantially more nitrification to occur than in a clogged sponge filter, we “reefers” still have to take extra measures, including good ole water changes, to keep nitrates to a minimum, so use this tip as an insurance policy not a magic snake-oil.

Although I think that most every hobbyist is at least somewhat familiar with the nitrogen cycle, I hope that the in-depth discussion presented in this article will give everyone more insights. Well after this installment, there is only one more to go. In the final part of this article, I will go over gases in the aquarium. The GCAS HAP program has been growing by leaps and bounds, so I can’t very well skip over the gas of interest for all HAP participants: carbon dioxide. However, one gas that affects every kind of aquarist, but we rarely hear discussed, is oxygen. Next installment I will discuss oxygen’s importance and why I think more of us should take this important gas much more into account when we plan our aquariums, or when we deal with a problem in our aquariums. In the meantime let’s keep learning about and caring for our fish.