Nitrification Microbiology in RAS Biofilters

Which microbes oxidize ammonia and nitrite in aquaculture biofilters, how much oxygen and alkalinity they consume, how pH and temperature affect them, and how a new biofilter is started.

Fish excrete ammonia across the gills as a product of protein metabolism. In a recirculating aquaculture system (RAS) it is removed mainly by nitrification: the biological oxidation of ammonia to nitrite and then to nitrate inside the biofilm of the biofilter. This guide covers the organisms involved, their chemical demands and the start-up of a new filter; filter types and sizing are treated separately.

Two steps, several groups of microbes

Nitrifiers are chemoautotrophs: they take carbon from carbon dioxide and gain energy by oxidizing nitrogen compounds. In the first step, which is the rate-limiting one, the enzyme ammonia monooxygenase converts ammonia to hydroxylamine and hydroxylamine oxidoreductase converts it to nitrite; in the second step nitrite oxidoreductase oxidizes nitrite to nitrate (Wikipedia).

Ammonia-oxidizing bacteria (AOB): Nitrosomonas and Nitrosococcus are the classic genera; a UNU Fisheries Training Programme review also lists Nitrosospira, Nitrosolobus and Nitrosovibrio.
Ammonia-oxidizing archaea (AOA): members of Nitrososphaerota (formerly Thaumarchaeota), such as Nitrososphaera viennensis and Nitrosopumilus maritimus, dominate ammonia oxidation in soils and the sea (Wikipedia).
Nitrite-oxidizing bacteria (NOB): genera include Nitrobacter, Nitrospira, Nitrospina and Nitrococcus.
Comammox: some Nitrospira of sublineage II carry out complete ammonia oxidation to nitrate in a single organism. The process was predicted in 2006 and discovered in 2015; Nitrospira inopinata, the first isolate, has a high affinity for ammonia and grows slowly. Comammox has been found in aquaculture biofiltration units, drinking water and wastewater systems (Wikipedia).

Who actually nitrifies in RAS biofilters

Older RAS design literature, including SRAC Publication 452, attributes nitrification to Nitrosomonas and Nitrobacter. A 2017 study by Bartelme, McLellan and Newton (PMC open access) of a fluidized sand biofilter in a commercial freshwater yellow perch (Perca flavescens) RAS, in operation for more than 15 years at about 21.7 °C, found a different community. Archaea in the filter were almost entirely Nitrososphaera-affiliated AOA, which outnumbered Nitrosomonas by more than three orders of magnitude. Comammox Nitrospira and strictly nitrite-oxidizing Nitrospira coexisted at stable abundances, and comammox ammonia monooxygenase genes were on average 1.9 times as abundant as those of AOA. Nitrobacter was not detected.

The authors relate this pattern to the low ammonia concentration entering the filter (mean 9 µM), since AOA are known to outcompete AOB at low concentrations, and state that Nitrosomonas and Nitrobacter are typically absent or scarce in freshwater nitrifying biofilters. The wider bacterial community shifted with operating conditions such as feed size and conductivity, while the nitrifying consortium remained stable.

Oxygen, alkalinity and by-products

The overall reaction given in the FAO guide is NH₄⁺ + 2 O₂ → NO₃⁻ + H₂O + 2 H⁺, so nitrification both consumes oxygen and releases acid. Stoichiometry from Ebeling and co-workers (2006), as tabulated in the UNU-FTP review, per gram of ammonium nitrogen converted:

Oxygen consumed: 4.18 g.
Alkalinity consumed: 7.05 g as CaCO₃.
Carbon dioxide produced: 5.85 g.
Bacterial biomass produced: 0.20 g volatile suspended solids.
Nitrate nitrogen produced: 0.976 g.

In practice SRAC 452 recommends keeping alkalinity at 50 to 100 mg/L as CaCO₃ or higher and notes that sodium bicarbonate added at 17 to 20 percent of the daily feed weight usually keeps pH and alkalinity in range; calcium carbonate may dissolve too slowly, and caustic bases should not be added directly to the rearing tank. Nitrifying bacteria become inefficient below 2 mg/L of dissolved oxygen (SRAC 452), and the UNU-FTP review recommends at least 2 mg/L in the biofilter effluent because low oxygen favours heterotrophic bacteria.

pH, temperature and competing heterotrophs

pH: SRAC 452 gives an optimum of 7 to 8 for biofilter bacteria and inhibition below pH 6. The FAO guide recommends adjusting to pH 7.0 to 7.5, because the rate falls at lower pH while the share of toxic free ammonia rises at higher pH. The UNU-FTP review cites optima of 7.2 to 7.8 for Nitrosomonas and 7.2 to 8.2 for Nitrobacter and warns against rapid shifts of more than 0.5 to 1.0 units.
Temperature: nitrifiers work over roughly 7 to 35 °C and their rate rises with temperature (UNU-FTP review); the FAO guide gives 10 to 35 °C with an optimum near 30 °C. In the UNU-FTP experiment, cooling conditioned biofilters from about 19 °C to 8.3 °C reduced nitrification.
Organic load: heterotrophic bacteria grow faster than nitrifiers and compete with them for space when organic loading is high, which is why mechanical filtration ahead of the biofilter matters (UNU-FTP review).
Free ammonia and nitrous acid at high concentrations can inhibit the nitrifiers themselves (UNU-FTP review).

Starting a new biofilter

SRAC 452 states that activating a new biofilter to handle normal feeding rates takes at least one month, during which stocking and feeding should be greatly reduced. Pre-activation before stocking is optional: the filter is seeded with nitrifying bacteria and fed for two weeks with a synthetic medium containing 10 to 20 mg/L of ammonia nitrogen, trace elements and sodium bicarbonate to hold pH at 7.5, after which the solution is discarded. The FAO guide shows the typical sequence during start-up: an ammonia peak, followed by a nitrite peak, before nitrate accumulates.

During activation, overfeeding is the main cause of losses. SRAC 452 describes ammonia rising sharply first; about two weeks in, ammonia is converted to nitrite, and nitrite falls only when nitrite oxidizers become established. At the first sign of high ammonia, feeding should be stopped. Nitrite toxicity can be reduced with chloride, usually 6 to 10 parts chloride per part nitrite nitrogen (SRAC 452); the FAO guide reports that salt at about 0.3 ‰ inhibits nitrite uptake. A biofilter killed by chemical treatment or over-vigorous cleaning needs about 3 to 4 weeks to re-activate.