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THE NITROGEN CYCLE  |  IN DEPTH

by Leonard Ho

o   Nitrogen: The Essential Element

Nitrogen is an element vital to all life processes on Earth.  To appreciate the importance of nitrogen in our biosphere, simply realize nitrogen comprises 78% of the atmosphere, and is embedded in every living tissue!   It is a component of amino acids, proteins and nucleic acids.   With the exception of carbon, nitrogen is the most universal element of life  Put simply: Life could not exists without nitrogen.  Aside from organic development, nitrogenous compounds are also required by some organisms for metabolic functions and respiration.

Nitrogen exists in many states.  In its most common gaseous state of N₂, nitrogen forms very strong covalent bonds that can only be broken when great force or energy is applied (e.g. seismic event or lightening), or by particular bacteria species which use nitrogenous compounds for metabolism.  The fact is various bacteria are critical to every step of the nitrogen cycle.  It is these bacteria and their specific roles that are of particular interest to reefkeepers.

o   The Nitrogen Cycle: Defined

The nitrogen cycle is defined as the pathways for which nitrogen is recycled.  As with all elements, nitrogen is constantly combined and uncombined with other elements to form essential and nonessential compounds for life.  We term this progression from one compound to the next its pathway.  Tracking nitrogen’s pathway is critical to understanding the role nitrogen plays in the chemical functions of an ecosystem.  Consequently, we can apply this knowledge to the management of key processes in our captive reef ecosystems to ensure a balanced, healthy environment for our reef’s inhabitants.

To simplify the concept, we will trace the nitrogen cycle from its source of introduction to the eventual recycling of nitrogen within (and without) our reef aquariums.

Simplified Illustration of the Nitrogen Cycle 

The introduction of any organism, whether it

be coral or fish, constitutes an introduction of

nitrogen into the aquarium.

o   Introduction of Nitrogen

Nitrogen is introduced into the aquarium in a variety of ways.  Here are the three major contributors of nitrogen:

1. Introduced organisms.  Every living organisms, from fish to algae, all have great quantities of assimilated nitrogen in their tissues.  Remember that nitrogen is a fundamental ingredient for the formation of proteins and nucleic acids.  Every time you introduce fishes, corals, live rocks, shrimps, crabs, worms, macroalgae, etc., you introduce nitrogen into your system.

2. Food inputs.  Foods  are merely an extension of item #1.  Dead or alive, they are organic masses, and possess the same nitrogenous attributes as the decorative living organisms you introduce.

3. Inorganic inputs.   There are two major sources for inorganic nitrogen: the atmosphere and introduced water.  Atmospheric nitrogen (N₂) is incorporated into our aquarium water via nitrogen fixation by bacteria and cyanobacteria as ammonia (NH₃).  Inorganic nitrogenous compounds from our municipal water system enter our aquarium.  Even after employing extensive filtering systems such as reverse osmosis and deionization, trace quantities of nitrogen is still imported into our aquariums, and accumulate over time.  The most common compounds are the ammonias and its oxides: NO₂ & NO₃._.

o   Nitrogen Released Via Decomposition 

As mentioned, living organisms possess a large mass of assimilated nitrogen.  This localized organic concentration is immobilized (“locked up”) in the tissue cells until the organism dies, at which time the nitrogen is released back into the environment via aerobic decomposition.  This process is known as eutrophication.  Note that decomposition requires a lot of oxygen.  When large organisms die, decomposition demands large amounts of oxygen that  may be very taxing of the system.  This is the very reason why ORP (oxygen redox potential) levels drop significantly when decaying matter is present.

Organisms constantly reproduce, grow, and die in any ecosystem; our reef aquariums are no different.  Keep in mind that fish and corals are not the only organisms that inhabit our aquariums: phytoplankton, zooplankton, microalgae, macroalgae, worms, crustaceans, bivalves, and sponges are just a few of the other life forms found in our aquariums.  There exists a continual cycle of life and death.  When these organisms die and decompose, assimilated nitrogen is released back into your aquarium. 

Because food inputs are nothing more then introduced organic masses, they go through a similar process of releasing nitrogenous byproducts into the environment.  Foods are either consumed or unconsumed:

§         Foods consumed will pass through the organism.  The organism will process the food to assimilate the nutrients it needs, and discard the rest as excrement.  Although the organism will incorporate a considerable quantity of nitrogen, their excrements are still rich in nitrogenous compounds, which is quickly liberated into the water through advanced decomposition.

§         Foods that go unconsumed will eventually die (if not dead upon introduction) and decay, contributing previously assimilated nitrogen back into the environment via decomposition.

Decomposers include bacteria and fungi, with bacteria performing the greatest burden for decomposition in reef ecosystems.

Foods are either consumed and processed by

fish and other organisms, or go unconsumed.

Both will invariably add nitrogen to the system.

Fish and corals are not the only organisms

that inhabit our aquariums. Small organisms,

such as these fan worms, contribute

significantly to the nitrogen cycle.

o   Ammonification  & Nitrification 

The decomposition process produces large quantities of ammonia (NH₃) through the process of ammonification.   Heterotrophic microbes utilize the organic compounds of decomposing matter as their carbon source.  Ammonia (NH₃) is the byproduct of this consumption.  Ammonia, in its neutral state, exists as ammonium (NH₄⁺).

Ammonium has several divergent pathways from this point forth.  Plants and algae can assimilate ammonia and ammonium directly for the biosynthesis.  The remaining bulk of decomposed byproducts is utilized by bacteria in a process called nitrification. 

Nitrification is the oxidation (affixation of oxygen) of ammonium  by chemolithotrophic bacteria species.  During this process, specific species of nitrifying bacteria strip the ammonium of its hydrogen molecules as an energy source.   Oxygen molecules are then affixed to the stripped nitrogen, forming the oxide nitrite (NO₂).  Another group of bacteria utilize the enzyme nitrite oxidase that is then responsible for converting nitrite into nitrate (NO₃).   

In order for nitrification to occur, three constants must be present:

1. Nitrifying bacteria.  This is self-evident.  However, the species responsible for nitrification are not.  For the greater majority of reefkeeping’s history, Nitrosoma spp. and Nitrobacter spp. were thought to be responsible for nitrification (derived from freshwater studies).  However, recent studies have questioned this long-held supposition.

2. Ammonia/Ammonium.  NH₄⁺ is the energy source for these bacteria, and thus it is the motivation for nitrification.

3. Oxygen.  After all, nitrification is an oxidative process, in which oxygen is being affixed to nitrogen to form nitrite and nitrate.  While ammonium is the motivation for nitrification, oxygen is the mechanism used to achieve the desired results (ie energy generation).

The biochemicial processes of nitrification is described below:

1. NH₃(aq) + H₂O(l) à NH₄⁺(aq) + OH(aq)

2. NH₄⁺ + O₂ + 2e^- + 2H⁺à NH₂OH +H₂O

3. NH₂OH +H₂O + ½ O₂ à  NO₂^- +2H₂O + H⁺

4. NO₂^- + ½ O₂ à NO₃^-

In short, the processes described thus far progresses in the following manner:

decomposition à NH₃ à NH₄⁺  à  NO₂ à  NO₃

o   Recycling of Nitrogen 

First and foremost, let us dispel the misinformation that the nitrogen cycle “ends” at some point along the pathway.  The prevailing concept about the nitrogen cycle is that it ends with the formation of NO₃, or with the process of denitrification.  Neither is true.

As the term suggests, the nitrogen cycle is an unremitting succession of pathways for nitrogen.  It is never "completed."  There is no end to the nitrogen cycle;  Matter can neither be created or destroyed, and nitrogen is no different.  Instead, nitrogen is continuously recycled from one form to another.

So if the nitrogen cycle doesn’t end, what happens to the aforementioned byproducts of nitrification?  There are two primary routes for nitrate reduction: Assimilative and Dissimilative pathways.

"There is no end to the nitrogen cycle;

Matter can neither be created or destroyed,

 and nitrogen is no different.  Instead,

 nitrogen is continuously recycled from one

 form to another."

Assimilation

Assimilative reduction is  process whereby nitrate is reduced to organic nitrogen for the construction of organic matter.  Assimilation may occur in either the presence or absence of oxygen, and only enough nitrate is reduced to fulfill the organism's requirements.  Photosynthetic plants, algae, and zooxanthellae (the symbiotic dinoflaggellates found in photosynthetic corals, anemones, and some sponges) assimilate NH_3,  NO₂, and/or NO₃ for biosynthesis of proteins, amino acids, and nucleic acids.  Of these, nitrate is the most utilized compound because it is very low in toxicity, and is readily acceptable.  When utilizing nitrate, the organisms performs the following reduction:

 NO₃ à NO₂ à NH₂OH à NH₃ à R-NH₂ (organic N)

Nitrite is the least utilized compound for biosynthesis since it is the most toxic.  In the presence of acid, nitrite forms nitric acid, a known and dangerous mutagen.  In advanced organisms such as fish, nitrite will bind with red blood cells and hinder their capability of transporting O2, asphyxiating the organism.

Animals that  consume plants and algae subsequently assimilate nitrogen for biosynthesis of their own tissue (N.B. Plant matter is the ultimate source of nitrogen for most animals).  When both plants and animals die and decompose, the assimilated nitrogen is released back into the environment, and the whole cycle begins over again.  

Various organisms, like this macroalgae

(Halimeda sp.), can bioassilimate nutrients

directly from the water.

Dissimilation

Dissimilative nitrate reduction is the antithesis of assimilative nitrate reduction. Whereas the assimilative pathway is generally aerobic and only uses enough nitrogenous compounds to meet an organism's requirements (i.e. no excess is produced), the dissimilative pathway is generally anaerobic and produces copious amounts of excess byproducts.  Denitrification is the key dissimilative pathway for nitrate reduction.

Denitrification is the terminology used to describe the conversion of nitrogen oxides (NO₂ and NO₃) back into gaseous nitrogen (N₂, N₂O, or NO).  Denitrification results in nitrogen being lost from the local environment (e.g. water) to the atmosphere.  This process, as most processes are in the nitrogen cycle, is accomplished primarily by bacteria species.  However, unlike nitrification, denitrification is an anaerobic process, meaning it occurs in the absence of oxygen.  Denitrifying bacteria metabolize nitrogenous compounds (with the assistance of the molybdenum-containing enzyme, nitrate reductase) in the reverse way that nitrifying bacteria does: they turn oxides back into nitrogen gas or nitrous oxides for energy generation.  These gases then volatize, returning back into the atmosphere.   

Because the enzyme nitrate reductase is synthesized only when O2 is repressed, anoxic conditions are obligatory for most denitrifying bacteria.  This is why the denitrification process predominantly occurs in deeper substrates and in areas of stagnant flow where oxygen levels are depressed.  And this is why deep sand beds are effective as a nitrogen export mechanism.  As water slowly diffuses deeper, aerobic organisms strip all available oxygen for respiration.  In the deep, oxygen-deprived layers, denitrifying anaerobes are given the opportunity to convert nitrogen compounds into nitrogenous gases. 

The net loss of nitrogen to the atmosphere will be regained via  introduced foods and water.  Some nitrogen will also inevitably be fixated and incorporated back into the aquarium by bacteria and cyanobacteria.  

Deep sand beds are suited for the process

of denitrification because the fine substrate

media stagnates water flow,  creating an

ideal anaerobic condition for anaerobes.

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o   And so ....  

.... these processes repeat endlessly in our reef aquariums, unseen yet nevertheless of utmost significance.

The nitrogen cycle is the continuous recycling of nitrogen as an essential building block for life.  The understanding  of these mechanisms is critical to the application of methodologies that promote a balanced, healthy, captive ecosystem.