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The Nitrogen Cycle: A Nano Reefer's Perspective
I. INTRODUCTION
The purpose of this article is to familiarize the reader with the nitrogen cycle and related various issues that are of interest to aquarists (nano reefers in particular). Nitrogen is a very important element to all living things and it is often a limiting nutrient in both terrestrial and aquatic ecosystems. Nitrogen is very abundant, in fact nitrogen gas (N2) makes up about 78% of the Earth's atmosphere. However, most organisms can't use nitrogen in its gaseous form because it is largely inert. Instead, they rely upon other, usually photosynthetic, organisms to "fix" nitrogen from N2 gas into more readily usable (labile) forms. Nitrogen, like most other elements on Earth, goes through a cycle during which it is swapped between ion pairs and various compounds. This is where the term "nitrogen cycle" originated. We will begin our discussion of the nitrogen cycle with nitrogen fixation.
II. NITROGEN CYCLE: NITROGEN FIXATION (a minor feature in nano reefs)
N-fixation is carried out by relatively few organisms, many of which are photosynthetic microorganisms. Some of the more well known aquatic microorganisms that perform N-fixation form a subset of the cyanobacteria. Cyanobacteria are also known as "blue-green algae". These microorganisms are commonly found in both fresh and saltwater aquaria. Cyanobacteria can form mats or films that cover solid surfaces. These mats/films come in a variety of color shades (despite their name) such as red, orange, blue-green, and black. Cyanobacteria are not the only N-fixers you might find in an aquarium, but they are certainly one of the most common.
N-fixation is an energy intensive process, largely because of the energy needed to break the very strong triple covalent bond between the nitrogen atoms in N2 gas. N-fixation can occur in the absence of biology but only during high energy events such as lighting strikes, combustion or the Haber–Bosch process which is used industrially to make fertilizer and explosives. These abiotic processes don't fix much nitrogen globally, certainly much less than is needed by all the life on earth. Most N-fixation on earth is done by biological microorganisms.
Terrestrially, biological N-fixation occurs in soils and specialized plant structures but regardless of where it is done, it is done not by "higher" organisms. N-fixation is done solely by "simple" microorganisms. Some other sources (such as Wikipedia) state that some terrestrial plants are capable of N-fixation, but it has been conclusively shown that it is microorganismal endosymbionts within the plant that do the N-fixation, not the plant itself. A review of the ecology and mechanisms of Rhizobium and legume nodules can be found in the references section.
Biological N-fixers can't consume enough matter (matter consumption is called heterotorphy) to produce the energy needed to sustain N-fixation. They can't even provide for their own nitrogen needs in this way and instead use energy from the sun (photoautotrophy). When N-fixers fix nitrogen they reduce N2 gas to NH3 (ammonia). Once nitrogen has been fixed from N2 to NH3, it can be made available to other, non-fixing organisms. NH3 can be incorporated into amino acids (which form proteins), nucleic acids (which form RNA & DNA), and other useful biological compounds. In aquatic environments N-fixing photoautotrophes are exclusively microorganisms, most of which are bacteria (prokaryotes).
In a nano reef, unless it is completely overrun by cyanobacteria, N-fixers directly contribute very little to the pool of labile nitrogen.
III. NITROGEN PROLIFERATION: (minor in nano reefs compared to food addition)
There are four basic ways that fixed nitrogen can be made available to non-fixers: 1) leaks, 2) excretion, 3) natural death, or 4) sloppy feeding.
- All aquatic organisms have, to some degree, "leaky" cells. This means that some of what is inside will leak out despite the organism's best efforts to keep it in.
- All living things (aquatic or not) produce waste and they all need to excrete it. In many cases, the waste of one organism contains a food (and nitrogen) for another.
- All organisms, if they are not killed, die eventually anyway. When they die, their contents can either diffuse out (because there is no cellular machinery to keep it in) or are actively broken down by other organisms.
- Many are made a meal by other organisms before they die naturally. This is your classic predator/prey relationship. I think my microbial ecology professor put it best when he said that a microorganism that has been killed by a predator it is like "a can of dog food that has been smashed with an ax." When they are killed and consumed, they spill a lot of their cellular contents into the surrounding water.
Regardless of out labile nitrogen gets out of the organism that fixed it, it is quickly consumed and altered by other living things. In your nano reef, N-fixers directly contribute very little to nitrogen proliferation, especially when you consider the addition of food or the death of a larger tank inhabitant.
IV. NITROGEN CYCLE: NITRIFICATION (the bulk of the nano reef nitrogen cycle)
The nitrogen cycle consists of more than just the conversion of N2 into NH3. In fact, many organisms don't use NH3 as their primary nitrogen source. Two other labile forms you may find in the water of your nano reef are nitrite (NO2-) and nitrate (NO3). The rest of the nitrogen cycle consists of the transformation of NH3 → NO2- and NO2- → NO3- and the reverse reactions.
In the presence of oxygen (water column): N2 + energy → NH3 → NO2- → NO3-
In the absence of oxygen (sediment): NO3- → NO2- + N2O → NH3 → N2
There is also a third process, that (at the time of this writing) is not well understood. This process is called ANAMMOX. This is an acronym for Anaerobic AMMonium OXidation. The chemical equation for this process can be found below.
This process is done in the absence of oxygen (hence, anaerobic) and is therefore likely to occur in the same areas as denitrification (see Section VII). If this process is part of the nitrogen cycle in a nano reef, it is likely to be a very small contributor. Because it is likely to be a minor feature and because it is not well understood, it is a concept that goes beyond the scope of this article. I will therefore make no further mention of it.
In nano reefs (and freshwater aquaria), the oxic stages of the nitrogen cycle are generally completed by bacteria of the prolific genera Nitrosomonas and Nitrobacter. The individuals of genus Nitrosomonas take in NH3 and oxidize it to NO2-. The individuals of Nitrobacter take in NO2- and oxidize it to NO3-. These conversions require oxygen and hey take place in the water and live rock areas that are rich in dissolved oxygen. Both Nitrosomonas and Nitrobacter genera are known as "nitrifiers" and the steps of the nitrogen cycle they complete are known as "nitrification." There are other bacteria and archaea that are known to be nitrifiers, but they are not as abundant as the two genera mentioned above. When you "cycle" your tank, you are waiting for the populations of the various nitrifiers to grow to a number large enough to process the nitrogenous wastes present in the tank.
This is a great place to reiterate that the NH3 in your tank doesn't come only from leaky, dead, and dying nitrogen fixers. It is also released as a byproduct of both decomposition and heterotrophic metabolism. When something dies in an aquarium, be it a fish, coral, macro algae, or cyanobacterial mat, the nitrogen that was a part of that organism either leaches out or is released as waste by decomposers. This is is why you have an "ammonia spike" when you add live rock to your aquarium.
As you may have learned in your other research, live rock is usually out of the water for many hours or even several days before it arrives at your door or your local fish shop. When it arrives many of organisms on and in it are dead or dying from their prolonged exposure to the environment. Sponges, macro algae, micro algae, and your precious nitrifying bacteria are the among the first organisms to succumb after being removed from the water. When you place your new live rock in your tank, all those organisms continue to decompose, releasing "wastes" and "nutrients" into your water. It can take some time for this widespread decomposition to run its course and as any experienced aquarist can tell you, the end result often doesn't smell pretty.
NH3 is also released by the healthy, metabolizing organisms in your tank because no organism is 100% efficient at processing its food. When your fish, snails, crabs and (to a lesser degree) corals feed, some of the nutrient nitrogen in their food is excreted as solid and liquid waste. In the healthy and stable nano reef, this excreted nitrogen (some in amino acids and some in NH3) is quickly and efficiently snapped up by the various organisms in the tank and much of it is quickly shunted through nitrification and into NO3. This is why NO3 builds up over time. The attentive nano reefer will perform water changes or enact other methods to remove it from their tank.
This means something that may not be obvious: there is always NH3, NO2- and NO3- in your tank whether your test kit says so or not. This goes for even the most rock solid, stable, and vibrant reef tanks. Your test kit has a detection limit below which it cannot distinguish zero from "very low." This constant, low level presence of labile nitrogen compounds isn't a bad thing. In fact, you couldn't stop it if you tried. However, it bears mentioning here so that the reader can more fully understand the dynamic nature of their little slice of ocean and the nature of their test kits as well.
NH3, NO2-, and NO3- can be toxic to many organisms, especially the fish and invertebrates that we wish to keep in our nano reefs. NH3 and NO2- are both toxic at very low concentrations and NO3- becomes toxic only at very high concentrations or after prolonged exposure to elevated concentrations. This is why you need to wait until your cycle is "complete" to add any livestock. I say "complete" with quotation marks because the definition of when your cycle is complete depends on who is defining it. Like all hobbies, reefing has its "experts" and almost all hobbyists have an opinion on almost every aspect of their hobby. That is why I am going to present parts the next section as my opinion and not as fact.
V. “CYCLING” A NANO: THE BEGINNING OF WASTE MANAGEMENT
I call a cycle complete when NH3 and NO2- fall below the detection limit of my tests irrespective of my NO3 readings, others wait until even NO3- has reached undetectable levels. I prefer not to wait for NO3- to naturally become undetectable because there are only two ways that it can happen: 1) organisms take the NO3- in and use the nitrogen in build their cells/tissues or 2) it is used in denitrification.
Let's talk about the first way NO3- can naturally disappear from your water. Micro algae are present in all aquariums and they are very efficient at taking in NO3-. Some can also use NO2- and NH3. Even the most novice aquarist can tell you that it is a pain and a constant struggle to control the nuisance micro algae in their tank. A more experienced aquarist will be able to give you pointers on how to control algae but such advice will come in two flavors: top-town and bottom-up control.
Top-down control is the control exerted by organisms that graze on the algae (snails, crabs, etc...) and bottom-up control is when the aquarist limits the amount of nutrients that are added to the tank. The successful reefer will employ a mixture of both methods and the specific mix must be tuned to each tank. I believe that allowing the micro algae to feast upon the nitrogen (and other nutrients) that are floating around a newly cycled tank is directly in opposition to the notion of bottom-up control. The aquarist will spend the rest of their tank's life battling and controlling nuisance algae and I think it is a bad idea to give them free-reign during the initial stages of the tank's life. I believe that this is an especially bad way to start a smaller tank such as a nano.
In smaller tanks, our top-down control methods are very limited because of our small water volumes. Tangs just don't do well in 20 gallon tanks and a little bit of green hair algae goes a long way in a 5.5g. I believe that a reefer may make his/her life easier by removing the NO3- as their cycle nears completion and before the micro algae really comes out to play. I always recommend that people do a water change amounting to 50% or more of their tank's volume as soon as the NH3 and NO2- are no longer detectable. The idea of changing such a large water volume usually freaks people out, but unless you have oodles of sponges or other air-sensitive organisms on your rock you have nothing to worry about.
VI. NITROGEN CYCLE: DENITRIFICATION (the reefing holy grail or Texas chainsaw massacre?)
Denitrification is the other natural way that NO3- can be removed from aquariums. It is a process that may be slow to get going and not because the bacteria that perform it grow slowly. It may be slow because it takes a time to develop an organic carbon food base. Where your nitrification cycle may be fully operational in as little as one week, denitrification genrally takes 3-6 months at a minimum to get going. Denitrification is essentially the reverse of nitrification; during denitrification, NO3- is converted back into N2 gas. This process is done by two classes of bacteria: the “obligate” and the “facultative” anerobes. Obligate anaerobes are organisms that cannot survive in the presence of oxygen and facultative anaerobes are those that can tolerate some oxygen. One genus of bacteria that may perform denitrification in marine aquaria is Pseudomonas. There are many, many other bacteria that can perform denitrification; too many to list here. As a rule, denitrification cannnot proceed in the presence of atmospheric oxygen concentrations, therefore the organisms that do it must be able to create areas of very low dissolved oxygen. This requirement means that there are only a few places in a nano reef that it may occur.
The first place it may occur is in tank substrate. In my opinion, microzones are the single most important factor that allow for the tremendous bacterial diversity that we see in nature. In aquaria, anaerobic microzones can form anywhere water flows very slowly or not at all. Marine aquarium substrate (typically aragonite sand) is the perfect marterial to support such microzones because it generally has a small grain size. Small grain size translates to large surface area to volume ratio, which means you have a lot of bacterial habitat for a given volume of substrate. Small grain size also means that the grains can pack together closer, which drastically reduces water flow velocities and inter-grain pore volume. Small pore volume makes it easy for aerobic bacteria to drag O2 concentration down, because there is less water to hold the oxygen. Several different aquarium designs to provide more anaerobic area have been proposed but they were all developed for larger (55+g) reef systems. Some examples: the plenum and deep sand bed (DSB). I have read about some nano reefers that have attempted to adapt these methods for use in smaller tanks, but I haven't yet seen anyone report on whether they worked.
The second place it may occur is under the surface and in the pores of your live rock (LR). There will be areas in or on your LR where water will be come hypoxic (very low in O2) or anoxic. It was previously thought that denitrification required complete anoxia to proceed, but there many species have been identified in the last 10-20 years that can accomplish denitrification in the presence of a little oxygen. Another method that some hobbiests have proposed to create more anoxic area is by constructing a denitrification coil. Again, this is a piece equipment developed for larger systems and is known to be hard to tune. One reason they might be used on larger systems is because they are safer there than on a nano. An accidental release of toxic N2O, NO2--. NH3, or HS (hydrogen sulfide) is less destructive in a larger water volume.
Before you decide that denitrification is the best thing you have ever heard of and you decide to build a system with a plenum and a coil, you need to be aware of some of the risks inherent to systems with large anoxic areas. Anoxia fosters not only denitrifying bacteria but many others, most of which create compounds that can kill all your prized livestock if even a little bit makes it into the main area of your tank. Denitrification itself creates substances that are toxic in low doses, such as NO2--, NH3, and N2O. If some of your rockwork falls and disturbs your 6-month-old DSB, you may experience an NH3 or HS spike which can wipe out your fish and corals in a matter of hours. If your spouse accidentally bumps the flow-control on your denitrifcation coil, you can expect to have the same problem as my DSB example. Any of these problems are magnified in a nano because bad things happen very quickly in small water volumes.
Of course, much of this talk of denitrification methods can be rendered moot by one important point: denitrifying bacteria are heterotrophic. They “eat” organic carbon and “breath” NO3-. Specifically they consume dissolved organic carbon (DOC). The size and activity level of your denitrifier population is limited by how much DOC and NO3- you have floating around in your water. In the real world, DOC will be the limiting factor in aquaria because there is always plenty of NO3- around. This is why some people have tried dosing tanks with sugar, ethanol, or other soluble organic substances. This was a big thing in marine aquaria about 12-15 years ago and some people who tried it reported a virtual stripping of NO3- (and sometimes PO43+) from the water. Once again, this was done in larger systems because it is easier not to screw things up in a big system. It worked so well in some systems that the corals were shocked by the rapid shift in water chemistry. Of course, NO3- isn’t the only water chem parameter that you’re toying with when you dose DOC, you’re also monkeying with your dissolved oxygen and dissolved inorganic carbon. Don’t forget that the denitrifiers aren’t the only heterotrophes in your tank; there are plenty of aerobic ones as well. When you add DOC, you give a bump to all the heterotrophic bacteria not just the denitrifiers. This means that you can expect to see a drop in dissolved oxygen and a corresponding increase in dissolved inorganic carbon (CO2 + H2O <=> H2CO3, HCO3-, CO32-) which will be evident as a pH drop. Abruptly lowered oxygen and a perturbed pH in a nano? Recipe for dead livestock.
When a draft of this article was presented to the Nano-Reef.com community for review, questions were raised that required a review and revision of this section (VI). While researching for the revision of this section I had conversations with microbial ecologists and microbiologists regarding the ecology and behavior of denitrifiers. These converstions provided information that is in direct opposition to hobby conventional wisdom. Specifically, that denitrification is likely a feature in every marine aquarium large and small. Our hobby “experts” have always assumed that denitrification requires profound, or at least solid anoxia and to be a significant feature in aquaria. Professional microbial experts disagree. It is more likely that the limitation of denitrification in aquaria is the amount of dissolved organic carbon present in the system.
VII. THE END (or is it the beginning?)
I hope that you have had as much fun reading this article as I had writing it. Long though it is, what I have written here barely scratches surface of the topics discussed. I also hope that this article has helped to increase your curiosity regarding the aquatic nitrogen cycle, especially as it relates to nano reefs. There is a wealth of knowledge about these topics both on the web and at your local library. I have provided a few easy reading references for those readers who wish to delve deeper than I have here. Within these references, the savvy reader will find the citations of professional science papers that can provide all the gritty details regarding these topics.
Happy Reefing!
Isaac M. Hagenbuch,
Estuarine Ecology Lab
University of South Carolina
Department Biological Science
Date: 12.04.07
VIII. REFERENCES
• Sverdrup, Keith A., Duxbury, Alyn C., Duxbury, Alison
B. (2003) An Introduction to the World's Oceans, 7th Edition.
McGraw-Hill, NY. pp. 376
• Campbell, Neil A. & Reece, Jane B. (2005) Biology, 7th Edition. Pearson Education, Inc. pp. 539, 542, 763, 1188, 1197
• Miller, Charles B. (2004) Biological Oceanography. Blackwell Publishing, USA pp. 61
• Neera G (2007). Symbiotic nitrogen fixation in legume nodules: process and signaling. A review. Agrononomy for Sustainable Development 27, pp. 59-68
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