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T he nitrogen cycle plays a highly important role in a closed environment like that of an aquarium. Due to its
presence, it is possible keep the fish and invertebrates alive, in a small viable space, therefore it is fundamental
to learn to know it, mainly in respect of the life forms that we nurture.
Until a few years ago, it was thought that the nitrogen cycle in its complexity, was a complete linear process.
However, most recent scientific discoveries have greatly revolutionized our well-established knowledge on the
nitrogen cycle and on the micro-organisms involved in such processes. As a matter of fact, the global cycle of
nitrogen in the environment, particularly in that of marine, has been integrated with at least three new links
which include:
In the first part of this article, I will try to review the essential and more predominant aspects of the nitrogen
cycle: the transformation processes of the main components (atmospheric nitrogen, ammonium ion, nitrite,
nitrate) and the role played by the bacterial species involved.
In the second part, new ways will be explored with particular reference on the role of bacteria, focusing on the
implications that these new discoveries have brought in the global cycle of nitrogen.
A complex network of reactions links these nitrogen forms in processes that as a whole, is called the nitrogen
cycle (Figure 1). The greatest source of nitrogen comes in the form of inert gas N2 (N N), representing 78% of
the atmosphere. A small part of the atmospheric N2 is fixed by particular bacteria called nitrogen-fixing
(nitrogen fixation) and is reduced to ammonium ion (NH4+) which can be easily usable for other organisms. In
a marine environment which inhabited by particular bacteria, ammonium is quickly oxidized to nitrate in aerobic
conditions (nitrification). Nitrate is then reduced again to an N2 gas in anaerobic conditions (denitrification),
thereby completing the cycle (Figure 1).
It is interesting to note that, ultimately, the ammonium ion in the water is in balance with ammonia (NH3) based
on the following stoichiometry:
The main source of nitrogen is obtained from the nourishment both of the fish and invertebrates, particularly in
the form of protein and single amino acids, assuming they are directly administered into the tank. Even minor
vitamins and other molecules like the DNA, contain nitrogen but the quantity is decisively less than that of
protein's. In proteins, nitrogen forms a part of the framework and of some single amino-acids' lateral chains, as
the tryptophan, asparagin, glutamine, lysin, arginine, histidine.
The oxidative degradation of amino acids leads to the release of ammonia nitrogen into the tank. In what way?
On one hand, the protein ingested by the fish or by the other organisms are broken down into single amino
acids. In turn, amino acids can be used to build new proteins within the organism or be oxidized to supply
energy. The degradation of amino acids by the animals leads to the elimination of varied by-products. For
example, the fish release nitrogen as ammonia, while the majority of organisms may release it in the form of
uric acid (fowls, reptiles), or urea (humans). On the other hand, in the presence of a strong organic charge,
protein and amino acids in waste products, in sediments and in organic decay are decomposed in a process
called ammonification, carried out by particular decomposer bacteria which release ammonium into the water
by degrading the aminoacidic nitrogen.
Nitrification
Nitrification occurs in two distinct stages:
1) Nitrosation: in the first stage, ammonium ion is oxidized to nitrite in two steps:
2) Nitration: the oxidation of nitrite to nitrate, which occurs through the activity of the nitrite oxidase enzyme,
completes the process of nitrification:
2NO2- + O2 2NO3-
The conventional view of nitrification occurs in the presence of oxygen and anticipates the oxidation of
ammonium to nitrate based on the following global synthetic formula (see Figure 1):
cies;
The nitrifying bacteria are generally obliged aerobes and obliged chemoautorophs because they directly use
CO2as a source of carbon, while organic substances can be toxic.
Denitrification
Here I describe the four stages of denitrification process in detail. The oxidation state of nitrogen is indicated by
the enclosing parentheses, after the names of chemical species.
Reduction of nitrate (+5) to nitrite (+3). This reaction is catalyzed by nitrate reductase (NAR) which exists in the
(internal) cytoplasmic part of bacterial membrane. Nitrate is carried within the bacterial cell by a specialized
carrier (AP in Figure 3), defined as antiport because it exchange ion nitrate upon entry with the nitrite which is
produced in the reaction and must be carried to the (external) periplasmatic space for the subsequent reaction.
2NO3- + 4H+ + 4e- 2NO2- + 2H2O
Reduction of nitrite (+3) to nitric oxide (+2). The nitrite which is now at the periplasmatic space is reduced
bynitrite reductase (NIR), releasing nitric oxide (NO). NO is a remarkably important molecule, from the bacteria
to humans (but this is another story).
2NO2- + 4H+ + 2e- 2NO + 2H2O
Reduction of nitric oxide (+2) to nitrous oxide (+1). NO is reduced by nitric oxide reductase (NOR) to nitrous
oxide (also called nitrogen protoxide otherwise known as the laughing gas). Both oxides represent a strong
stimulus to the reductase synthesis in the presence of nitrates and under anaerobic conditions.
2NO + 2H+ + 2e- N2O + H2O
Reduction of nitrous oxide (+1) to gaseous nitrogen (0). The last reaction in the denitrification process is the
reduction of nitrous oxide to molecular nitrogen in gaseous form by the nitrous oxide reductase. This reaction
should complete the denitrification process and conclude the nitrogen cycle.
N2O + 2H+ + 2e- N2 + H2O
Denitrification is one of the key processes within the nitrogen cycle and anticipates the reduction of nitrates to
gaseous nitrogen, passing through nitrite, nitric oxide (nitrogen monoxide) and nitrous oxide (nitrogen
protoxide).
The global reaction of denitrification (without considering the organic molecular degradation eventually
associated) can be synthesized with the following formula (for details see Figure 1):
Figure 4.Nitrogen cycle integrated with recently discovered reactions. Nitrogen oxidation states are pointed out.
DNRA
During the recent years, the anaerobic reduction of nitrates/nitrite or, DNRA (acronym
for DissimilatoryNitrate/nitrite Reduction to Ammonium) has stirred up a certain interest as a relevant reaction
both in the terrestrial and marine eco-systems. The reaction has been described in anoxic sediments and in the
presence of bacteria of the Thioploca and Thiomargarita species. Both types of bacteria are able to concentrate
nitrates within their own cells for the subsequent oxidation of sulphur-containing compounds in reduced form. In
this way, they are able to reduce nitrate to ammonium passing through nitrite as an intermediary compound
(blue line in Figure 4 and Figure 7). This reaction, although still needing clarification, would potentially supply
nitrite and ammonium to the ANAMMOX reaction (see subsequent paragraph) in anoxic sediments.
The question now is: under analogous conditions, can ANAMMOX occur in an aquarium? Obviously we are not
able to establish that but we can make some considerations. A driven DSB can reproduce optimum conditions
for this process. In fact, nitrogen bubbles are visible in the depths of the sediment. In line with this, it has been
experimentally observed (but not in an aquarium) that the higher the layer of sediment, the more pushed the
anoxic condition (oxygen inhibits the reaction) and the faster is the reaction. Therefore the high efficiency of a
DSB in removing nitrates can be also due to a non-canonical denitrification, besides the classic anaerobic
denitrification; obviously, the eventual presence of qualified bacterial strains has yet to be defined. Figure 7
illustrates the schematic diagram of integrated nitrogen cycle with the new reactions and the areas in which
they occur.
Figure 7. Nitrogen cycle as revised and integrated. Areas where reactions occur are made evident.
In conclusion, these constant discoveries clarify many obscure points of the nitrogen cycle but at the same time
they open up new horizons and paradigms that make these processes even more complex and fascinating. On
the other hand, I hope not to have further complicated the well-established ideas on nitrogen cycle, with this
article. I also wish that over time, these new discoveries can be applied and integrated with the aquarium, in
spite of the scarce scientific researches in this area. However, even if just theoretical, this knowledge allows us
to better understand that which can occur in our tanks and nurture our passion for aquarium.