One aspect of seawater chemistry that every new aquarist becomes acquainted with is the nitrogen cycle. In particular, the conversion of ammonia to nitrite and then on to nitrate is mentioned in nearly every beginning text because of concerns about ammonia toxicity to fish. But what of the other parts of the cycle? Where does the ammonia come from, exactly? Where does it go? What role do organics play? What happens to the nitrate? Hopefully, this article will help shed some light on these less commonly discussed aspects of the nitrogen cycle in seawater.
The sources of nitrogen in a reef aquarium
There are essentially three sources of nitrogen in most reef aquariums. The first two are obvious to most reefkeepers: foods fed to the aquarium, and top-off water. The third is nitrogen from the air. The importance of the last source is likely small in most reef aquariums getting fish food, but it may be the only source in unfed aquariums.
Nitrogen in fish food
Nitrogen that enters the aquarium in fish food is almost entirely present as protein (or individual amino acids if they have been added to the food). Some vitamins and other biochemicals, such as DNA, contain nitrogen, but the quantity of nitrogen in these sources is typically far less than that supplied by protein. Figure 1 shows the structure of a portion of a protein. Nitrogen comes both from the backbone of the protein, and from some of the R groups for certain amino acids (tryptophan, asparagine, glutamine, lysine, arginine and histidine).
When these proteins are consumed by fish or other organisms, they are broken down first into individual amino acids. These amino acids can then be used to build more proteins for the organism, or they can be oxidized to provide energy. It is the oxidation of the amino acids that leads to release of nitrogen to the water column. The biochemical pathways involved can be very complex, but most vertebrates ultimately release the nitrogen coming from protein as ammonia, uric acid or urea depending on the species, although other nitrogen-containing organics are known to be excreted by certain species (e.g., trimethylamine oxide).
Most aquatic organisms in reef aquariums normally release nitrogen as ammonia, and this is likely to be the primary source of inorganic nitrogen in fed reef aquariums. A typical daily feeding for a 100-gallon aquarium might include a few grams of protein, which equates to about 500 mg of nitrogen. Not all of this nitrogen is released to the water column, but if it were, the resulting nitrate levels might increase by about 6 ppm per day.
Nitrogen in top off water
Occasionally, top off water may contain nitrate. Nitrate is especially prevalent in natural waters that receive agricultural runoff because it is used as a fertilizer. The U.S. EPA has established limits for nitrate in drinking water of 10 ppm, while permitted levels for ammonia and nitrite are substantially lower. Depending upon the source of the water used for the aquarium, substantial nitrate can be added to a reef aquarium getting a 2 percent daily replacement of evaporated water containing 10 ppm of nitrate, although this value (0.2 ppm per day) is small compared to that potentially coming from food.
Atmospheric nitrogen in reef aquariums
All reef aquariums have a substantial amount of nitrogen present in the water in the form of N2. Typical aquariums would have 10 to 11 ppm, depending upon temperature (higher temperatures would result in lower N2). This nitrogen, however, is not bioavailable to most organisms. One of the few nitrogen fixers potentially present in reef aquariums is cyanobacteria. These organisms can convert N2 into useful forms (e.g., proteins). When the organisms die or are eaten, these organic nitrogen compounds are released to the water column and can be used by other organisms.
In many reefs aquariums, the nitrate levels are high enough that the contribution from cyanobacteria may be negligible. In aquariums with no feedings, however, this source may be significant. Steve Tyree has told me that he has unfed aquariums that do well, and he attributes the nitrogen source to N2 fixation.
In addition to the fundamental inputs of nitrogen to the reef aquarium, there is a tremendous cycling of nitrogen through the system that would take place even in the absence of any new additions of nitrogen. Every time an organism dies it releases inorganic and organic nitrogen compounds to the water. Most of these are rapidly converted to ammonia, and then on to nitrite and nitrate. In a new reef aquarium these inputs can exceed that coming from fish food, and it can spike in older aquariums any time that a sizeable organism dies.
The release of organic compounds to the water column through the normal activities of living organisms is an incredibly poorly understood field, largely because of the infinite variety of possible organic compounds, the difficulty in identifying and quantifying them, and because many are released in low concentrations. These can range from metabolic waste products for which that the organism has no need (e.g., uric acid and urea) to those released for specific purposes. Intentionally released organics include molecules intended to signal other organisms of the same or different species (e.g., I want to have sex, get off my turf, here’s a nice place to make a home, don’t eat me). They also include functional molecules that go out into the water column and serve a specific purpose, such as sequestering iron to be returned to the original organism. Most aquarists are also aware that many reef organisms use chemical warfare to thwart nearby competitors, and these are frequently organic molecules of some type.
In the ocean, these released organic molecules become diluted in a far greater volume of water than is present in any reef aquarium. Consequently, studies of organics in the ocean give limited information about the nature and concentrations of organics in aquaria (Organics in the nitrogen cycle in seagrass beds). For reef aquariums in particular, there have been few useful studies of the organic species present. Consequently, their contribution to the overall nitrogen cycle is unknown despite the fact that many of them contain nitrogen. Nevertheless, aquarists should be aware that organics are part of the chemical cycles in their aquariums, and that the importance of any particular species will vary from aquarium to aquarium as the nature of the system changes (e.g., skimmed or not).
Where does the ammonia go?
Because many of the sources of nitrogen in a reef aquarium are rapidly converted into ammonia, tracking the ammonia through the cycle would be an interesting endeavor. Most beginning texts suggest that it is a done deal: ammonia becomes nitrite, which in turn becomes nitrate, and this may be largely true in saltwater fish only aquariums with little algae. In Captive Seawater Fishes, Stephen Spotte does a good job of detailing these aspects of the nitrogen cycle.
Unfortunately, it isn’t so simple in reef aquariums. Many organisms, including many species of algae and clams, will directly remove ammonia from the water column as their preferred source of nitrogen. The percentage of ammonia that becomes nitrite will certainly depend upon what’s in the aquarium, so one cannot give any general figure over the portion going down any particular pathway. As a rule, however, the more algae and clams that you have, the more likely that the ammonia is being used directly instead of following the nitrite/nitrate pathway.
One practical result of this ammonia usage is the reported lowering of the ammonia, nitrite and nitrate concentrations reached in cycling live rock in the presence of actively growing macroalgae. While the reduction of the nitrate peak could be due to usage of nitrate by the algae, the reduction of ammonia and nitrite as well suggests that it is diversion of ammonia by marine macroalgae that is the primary cause.
Where does the nitrate go?
In the old days of fish-only aquaria, nitrate had nowhere to go until it was removed by water changes, and often exceeded 100 ppm. In reef aquariums, however, there are many sinks for nitrate, including denitrators and commercial nitrate-binding products that I won’t discuss here. The first sink, of course, is the uptake by growing aerobic organisms: bacteria, algae and higher plants and animals. The nitrogen taken up is used for any of its many chemical purposes, but is mostly incorporated into tissues. In many aquariums this is the primary sink.
In other aquariums, aquarists have incorporated low oxygen regions that are suited for conversions of nitrate to N2. In these processes, organisms use the nitrate as other organisms use oxygen: to oxidize organic compounds and thereby gain energy. The equation below shows two of the basic chemical processes taking place for a hypothetical organic material being consumed. In the first, all of the nitrate is converted to N2. In the second, a portion of the nitrate ends up as ammonia.
C106H263O110N16P + 91.4 H+ + 94.4 NO3– > 106 CO2 + 55.2 N2 + 177.2 H2O + PO4—
C106H263O110N16P + 81.8 H+ + 84.8 NO3– > 106 CO2 + 42.4 N2 + 148.4 H2O + 16 NH3 + PO4—
How important either of these pathways is depends upon the setup. In my aquarium, the sand bed is not typically then endpoint of the nitrogen cycle. With all of the emphasis on sand beds these days, I’ll probably need to explain that a bit more…
In my basement I have several things that support my main aquarium. In addition to a large skimmer, I have two well-lit refugia that have a combined surface area similar to the main aquarium, the bottoms of which are covered with 4 to 8 inches of live sand. These refugia are the home to everything from corals to sea stars to marine macroalgae. When these refugia had little in the way of growing macroalgae, I was easily able to poke into the sand bed and release copious bubbles, presumably of N2. This indicated to me that the above process was functioning well.
On the other hand, when the refugia became choked with macroalgae, I found much less N2 in the sand. On this basis, I conclude that much of the nitrogen entering the aquarium as fish food is ending up in the macroalgae and is not being processed by the sand bed. Admittedly, the sand bed may still be the primary sink for nitrate, with most of the nitrogen being taken out of the system as ammonia by macroalgae. Without doing specific nitrate dosing experiments, I’m not able to distinguish the primary sink for nitrate itself.
Other inorganic nitrogen compounds
In addition to ammonia, nitrite and nitrate, there are other inorganic nitrogen compounds that are present in natural seawater and may make their appearance in our aquariums. Small amounts of the colorless gas nitrous oxide (N2O) are formed by bacteria in the denitrification of nitrate in low oxygen environments such as sand beds. This is the same nitrous oxide that is sometimes used as an anesthetic and has been used as a propellant in whipped cream. Just as with N2, this gas can be lost from the aquarium to the air.
Two other inorganic nitrogen species that can occur are hydroxylamine (NH2OH) and hyponitrite (HON=NO–). Hyponitrite is synthesized by organisms that are using nitrate to make amino acids. This reaction proceeds through hyponitrite and then ammonia as intermediates.
NO3– + 2H+ + 2e– > NO2– + H2O
2NO2– + 5H+ + 4e– > HON=NO– + 2H2O
HON=NO– + H+ + 2e– > NH3 + H2O
NH3 > amino acids
This may, in fact, be why many organisms prefer ammonia as a nitrogen source to nitrate: they can eliminate the need to resynthesize ammonia from the nitrate.
I hope this article suggests to reef keepers that there is more to the nitrogen cycle in reef aquariums than ammonia, nitrite and nitrate. Unfortunately, the picture is far from complete, as a full understanding of the organic portions of the nitrogen cycle will not likely happen for years to come.