One of the most important, and least understood, aspects of successful aquarium keeping is biological filtration and its function in the nitrogen cycle. Traditionally, novice aquarists become disillusioned at the frequently experienced high death rates of their aquatic pets after setting up a new aquarium. Statistically, as much as 60% of the fish sold for a new aquarium will die within the first 30 days. Two out of every three new aquarists abandon the hobby within the first year.
Known as "New Tank Syndrome" these fish are poisoned by high levels of ammonia (NH3) that is produced by the bacterial mineralization of fish wastes, excess food, and the decomposition of animal and plant tissues. Additional ammonia is excreted directly into the water by the fish themselves. The effects of ammonia poisoning in fish are well documented. These effects include: extensive damage to tissues, especially the gills and kidney; physiological imbalances; impaired growth; decreased resistance to disease, and; death.
Nitrite poisoning inhibits the uptake of oxygen by red blood cells. Known as brown blood disease, or methemoglobinemia, the hemoglobin in red blood cells is converted to methemoglobin. This problem is much more severe in fresh water fish than in marine organisms. The presence of chloride ions (CL-) appears to inhibit the accumulation of nitrite in the blood stream.
The successful aquarist realizes the importance of establishing the nitrogen cycle quickly and with minimal stress on the aquarium’s inhabitants. Aquarium filtration has advanced from the old box filters filled with charcoal and glass wool to under gravel filters, then trickle filters, and most recently - fluidized bed filters. Every advance has been to improve upon the effectiveness of biological filtration which in turn increases the efficiency of the nitrogen cycle. The availability of advanced high-tech filtration systems has lent added importance to the understanding of basic aquatic chemistry.
Nitrifying bacteria are classified as obligate chemolithotrophs. This simply means that they must use inorganic salts as an energy source and generally cannot utilize organic materials. They must oxidize ammonia and nitrites for their energy needs and fix inorganic carbon dioxide (CO2) to fulfill their carbon requirements. They are largely non-motile and must colonize a surface (gravel, sand, synthetic biomedia, etc.) for optimum growth. They secrete a sticky slime matrix which they use to attach themselves.
Species of Nitrosomonas and Nitrobacter are gram negative, mostly rod-shaped, microbes ranging between 0.6-4.0 microns in length. They are obligate aerobes and cannot multiply or convert ammonia or nitrites in the absence of oxygen.
Nitrifying bacteria have long generation times due to the low energy yield from their oxidation reactions. Since little energy is produced from these reactions they have evolved to become extremely efficient at converting ammonia and nitrite. Scientific studies have shown that Nitrosomonas bacterium are so efficient that a single cell can convert ammonia at a rate that would require up to one million heterotrophs to accomplish. Most of their energy production (80%) is devoted to fixing CO2 via the Calvin cycle and little energy remains for growth and reproduction. As a consequence, they have a very slow reproductive rate.
Nitrifying bacteria reproduce by binary division. Under optimal conditions, Nitrosomonas may double every 7 hours and Nitrobacter every 13 hours. More realistically, they will double every 15-20 hours. This is an extremely long time considering that heterotrophic bacteria can double in as short a time as 20 minutes. In the time that it takes a single Nitrosomonas cell to double in population, a single E. Coli bacterium would have produced a population exceeding 35 trillion cells.
None of the Nitrobacteraceae are able to form spores. They have a complex cytomembrane (cell wall) that is surrounded by a slime matrix. All species have limited tolerance ranges and are individually sensitive to pH, dissolved oxygen levels, salt, temperature, and inhibitory chemicals. Unlike species of heterotrophic bacteria, they cannot survive any drying process without killing the organism. In water, they can survive short periods of adverse conditions by utilizing stored materials within the cell. When these materials are depleted, the bacteria die.
Biological Data
There are several species of Nitrosomonas and Nitrobacter bacteria and many strains among those species. Most of this information can be applied to species of Nitrosomonas and Nitrobacter in general, however, each strain may have specific tolerances to environmental factors and nutriment preferences not shared by other, very closely related, strains. The information presented here applies specifically to Nitrosomonas and Nitrobacter strains.
Temperature
The temperature for optimum growth of nitrifying bacteria is between 77-86° F (25-30° C).
Growth rate is decreased by 50% at 64° F (18° C).
Growth rate is decreased by 75% at 46-50° F.
No activity will occur at 39° F (4° C)
Nitrifying bacteria will die at 32° F (0° C).
Nitrifying bacteria will die at 120° F (49° C)
Nitrobacter is less tolerant of low temperatures than Nitrosomonas. In cold water systems, care must be taken to monitor the accumulation of nitrites.
pH
The optimum pH range for Nitrosomonas is between 7.8-8.0.
The optimum pH range for Nitrobacter is between 7.3-7.5
Nitrobacter will grow more slowly at the high pH levels typical of marine aquaria and preferred by African Rift Lake Cichlids. Initial high nitrite concentrations may exist. At pH levels below 7.0, Nitrosomonas will grow more slowly and increases in ammonia may become evident. Nitrosomonas growth is inhibited at a pH of 6.5. All nitrification is inhibited if the pH drops to 6.0 or less. Care must be taken to monitor ammonia if the pH begins to drop close to 6.5. At this pH almost all of the ammonia present in the water will be in the mildly toxic, ionized NH3+ state.
Dissolved Oxygen
Maximum nitrification rates will exist if dissolved oxygen (DO) levels exceed 80% saturation. Nitrification will not occur if DO concentrations drop to 2.0 mg/l (ppm) or less. Nitrobacter is more strongly affected by low DO than NITROSOMONAS.
Salinity
Freshwater nitrifying bacteria will grow in salinities ranging between 0 to 6 ppt (parts per thousand) (specific gravity between 1.0000-1.0038).
Saltwater nitrifying bacteria will grow in salinities ranging from 6 up to 44 ppt. (specific gravity between 1.0038-1.0329).
Adaptation to different salinities may involve a lag time of 1-3 days before exponential growth begins.
Micronutrients
All species of nitrifying bacteria require a number of micronutrients. Most important among these is the need for phosphorus for ATP (Adenosine Tri-Phosphate) production. The conversion of ATP provides energy for cellular functions. Phosphorus is normally available to cells in the form of phosphates (PO4). Nitrobacter, especially, is unable to oxidize nitrite to nitrate in the absence of phosphates.
Sufficient phosphates are normally present in regular drinking water. During certain periods of the year, the amount of phosphates may be very low. A phenomenon known as "Phosphate Block" may occur. If all the above described parameters are within the optimum ranges for the bacteria and nitrite levels continue to escalate without production of nitrate, then phosphate block may be occurring. In recent years, with the advent of phosphate-free synthetic sea salt mixes, this problem has become prevalent among marine aquarists when establishing a new tank.
Fortunately, phosphate block is easy to remedy. A source of phosphate needs to be added to the aquarium. Phosphoric Acid is recommended as being simplest to use and dose, however, either mono-sodium phosphate or di-sodium phosphate may be substituted. When using a 31% phosphoric acid mixture, apply a one time application of 1 drop per 4 gallons of water to activate the Nitrobacter. This small dosage of phosphoric acid will not affect the pH or alkalinity of marine aquaria.
Minimal levels of other essential micronutrients is often not a problem as they are available in our drinking water supplies. The increasing popularity of high-tech water filters for deionizing, distilling, and reverse osmosis (hyper-filtration) produce water that is stripped of these nutrients. While these filters are generally excellent for producing high purity water, this water will also be inhibitory to nitrifying bacteria. The serious aquarist must replenish the basic salts necessary to the survival of the aquarium’s inhabitants. These salts, however, usually lack these critical micronutrients.
Nutriment
All species of Nitrosomonas use ammonia (NH3) as an energy source during its conversion to nitrite (NO2). Ammonia is first converted (hydrolyzed) to an amine (NH2) compound then oxidized to nitrite. This conversion process allows Nitrosomonas to utilize a few simple amine compounds such as those formed by the conversion of ammonia by chemical ammonia removers.
Nitrosomonas is capable of utilizing urea as an energy source.
All species of Nitrobacter use nitrites for their energy source in oxidizing them to nitrate (NO3).
Color and Smell
The cells of nitrifying bacteria are opaque to brownish in color. What you see are actually clumps of bacteria stuck together by their own slime matrix.
Most nitrifying bacteria solutions have an "earthy" smell.
Caution: solutions that contain dark brown or black liquids and/or product that smell of sulfur or rotten eggs can contain spoiled or even contaminated bacteria. If you suspect that the product is spoiled or contaminated, do not apply to closed aquatic system.
Light
Nitrifying bacteria are photosensitive, especially to blue and ultraviolet light. After they have colonized a surface this light poses no problem. During the first 3 or 4 days many of the cells may be suspended in the water column. Specialized bulbs in reef aquaria that emit UV or near UV light should remain off during this time. Regular aquarium lighting has no appreciable negative effect.
Chlorine and Chloramines
Before adding bacteria or fish to any aquarium or system, all chlorine must be completely neutralized. Residual chlorine or chloramines will kill all nitrifying bacteria and fish.
Most US cities now treat their drinking water with chloramines. Chloramines are more stable than chlorine. It is advisable to test for chlorine with an inexpensive test kit. If you are unsure whether your water has been treated with chloramine, test for ammonia after neutralizing the chlorine. You can also call your local water treatment facility.
The type of chloramines formed is dependent on pH. Most of it exists as either monochloramine (NH2Cl) or dichloramine (NHCl2). They are made by adding ammonia to chlorinated water. Commercial chlorine reducing chemicals, such as sodium thiosulfate (Na2S2O2) break the chlorine:ammonia bond. Chlorine (Cl) is reduced to harmless chloride (Cl- ) ion. Since dichloramine has two chlorine molecules, a double dose of a chlorine remover, such as sodium thiosulfate, is recommended.
Each molecule of chloramine that is reduced will produce one molecule of ammonia. If the chloramine concentration is 2 ppm then your aquarium or system will start out with 2 ppm of ammonia. Chlorine Remover will reduce up to 2 ppm of chlorine at recommended dosages. During the warmer months chlorine levels may exceed 2 ppm. A double dose would be required to effectively eliminate the excess chlorine.
Adding Bacteria
After all the chlorine has been safely neutralized, nitrifying bacteria should be added to rid the aquarium of ammonia. Depending on the aquarium pH, 3-4 days may be advisable before adding your fish in order to minimize stress. If the water supply does not contain chloramines, and there is no ammonia, nitrifying bacteria should be added at the same time as the fish. Nitrosomonas and Nitrobacter species of bacteria belong to the family NITROBACTERACEAE - the true nitrifiers. Five genera are generally accepted as ammonia-oxidizers and four genera as nitrite-oxidizers. Of these, Nitrosomonas (ammonia-oxidizers) and Nitrobacter (nitrite-oxidizers) are the most important. Marine species are different from those that prefer fresh water, and yet, are very closely related. Each species has a limited optimum range for survival. They are the most efficient, and most important, group of nitrifying bacteria and are ubiquitous (world-wide) in their distribution.
Aquatic Solutions Nitrifying Bacteria is autotrophic instead of heterotrophic sludge (organic) consuming bacteria. Heterotrophic bacteria are not nitrifying bacteria and the use of heterotrophic bacteria will provide little to no benefit at establishing a sound or "cycled" ammonia and nitrite.