Nitrogen cycle

GFA nitrogen cycle

GFA nitrogen cycle


Ammonia, nitrite and nitrate are the main forms of inorganic nitrogen found in recirculating fish cultures.  The major source of organic nitrogen is the protein-rich supplementary feed that is given daily to the fish.  Ammonia is produced as the main end product of protein catabolism and is excreted by fish through their gills.  Ammonia is also released to the water by the decomposition of uneaten feed and feces by heterotrophic bacteria present in the system. Among the inorganic nitrogen species, ammonia and nitrite are toxic and most often associated with stress or even mortality of fish.  Nitrate is considered to be less toxic than ammonia but may damage fish at high concentrations.  Inorganic nitrogen transformation in closed intensive recirculating systems including G.F.A, is usually mediated microbial and includes two major processes-nitrification and denitrification.



Nitrification –


Nitrification is a two-step aerobic process that is carried out by two autotrophic bacterial species; ammonia is first oxidized to nitrite (reaction I), which is then followed by its oxidization to nitrate (reaction II):


I)   2NH3   +   3O2      ->             2HNO2   +   2H2O    (+ energy released)

II)  2HNO2   +   O2     ->             2HNO3                     (+ energy released)


The major groups of bacteria carrying out nitrification have been identified in both freshwater and marine environments.  Ammonia-oxidizing nitrifiyers have been found to belong to the b-subdivision of the Proteobacteria and are typified by Nitrosomonas europaea.  Nitrite-oxidizing nitrifiyers belong to the a-subdivision of the Proteobacteria of which Nitrobacter winogradskyi is a representative species.  Until recently it was assumed that species of ammonia- and nitrite-oxidizing bacteria are identical in marine and freshwater environments.  However, the use of 16S ribosomal RNA (rRNA)-targeted oligonucleotide probes have revealed the identification of a group of nitrifying bacteria responsible for ammonia oxidation in freshwater that are different from the bacteria responsible for ammonia oxidation in seawater.  Recent studies showed that the ammonia oxidizer Nitrosomonas europaea appears to be present at high levels in seawater aquaria and at very low levels in freshwater aquaria.  We have identified both Nitrosomonas and Nitrospira spp. in our warm-water mariculture system.

Several types of biofilter configurations have been used in recirculating aquaculture systems for nitrification.  These include submerged biofilters, trickling biofilters, rotating biological contractors (RBC), bead filters and fluidized-bed filters and each design has advantages and disadvantages.  An advantage to using RBC and trickling biofilters over the others, for example, is the ability of these filters to oxidize water during operation and provide some carbon dioxide stripping.  Submerged biofilters or fluidized-bed filters, on the other hand, require continuous oxidation of water during operation.  G.F.A systems employ trickling filters as the major ammonia removal platform. Its parallel benefits that include ammonia removal, carbon dioxide stripping, water oxygenation and its biological stability, make it the perfect choice for G.F.A systems.



Denitrification –


Biological reduction of nitrate to nitrogen gas occurs by denitrification, which is carried out by facultative anaerobic heterotrophic bacteria.  In place of oxygen, these bacteria are capable of using nitrate, nitrite, nitric oxide or nitrous oxide as terminal electron acceptors in the presence of an organic carbon source that serves as electron donor (reaction III):

III) HNO3   +   (CH3OH)X  ->   HNO2  ->   N2O(g)  ->   NO(g)  ->   N2(g)

Denitrification closes the nitrogen cycle and releases nitrogen to the atmosphere.  Denitrifiyers are found among many bacterial genera including Paracoccus, Pseudomonas, Alcaligenes, Flavobacterium, and Hyphomicrobium.

Since denitrification is inhibited in the presence of oxygen, filtration systems that are established for denitrification are generally preceded by a mechanism designed for decreasing oxygen concentrations.  In some cases, oxygen consumption is coupled to the oxidation of carbon sources through activity of heterotrophic bacteria such as Pseudomonas and Bacillus spp. present in nitrifying biofilters.  The oxygen concentration that will completely inhibit denitrification is dependent on the denitrifying bacterial species present in the system.  Oxygen acts via inhibition of both synthesis and activity of enzymes involved in denitrification resulting in the accumulation of denitrification intermediates, NO, N2O, and NO2, depending on oxygen concentration.

As most denitrifying bacteria are heterotrophic, they require organic carbon compounds as a source for electrons and protons.  Such compounds include carbohydrates, organic alcohols, amino acids and fatty acids.  For example, utilization of acetate as a carbon source for denitrification proceeds as follows:

IV) 5CH3COO-   +   8NO3-   +   3H+  ->  10HCO3-   +   4N2 (g)   +   4H2O

The C/N ratio required for complete nitrate reduction to nitrogen gas by denitrifying bacteria depends on the nature of the carbon source and the bacterial species.  As noted above, carbon source limitation will result in the accumulation of denitrification by-products such as NO2 and N2O.  In addition, denitrification rates will depend on the variety of available carbon source.  For example, in anaerobic reactors denitrification was shown to be faster in the presence of acetate compared to glucose and ethanol.  The limiting factor of the process in marine and land sediments is the availability of an organic carbon source.


G.F.A technology takes full advantage of the anaerobic water treatment and uses it both for nitrate removal and organic solids digesting. Water from the bottom of the fish tanks rich with organic particles are collected to a central basin were few process are taking place in parallel manner: solids settling and digesting, oxygen uptake and nitrate reduction (denitrification). Thus the organic solids that are usually removed out from recirculating systems as fast as possible, are used in the G.F.A. technology as a “fuel” to activate the nitrate reducing bacteria and allow water to go back to the culture tanks with no losses – zero discharge technology.

part of
Croatian Center of Renewable Energy Sources (CCRES)

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