Water Tech: Care and Feeding of Nitrifying Bacteria

Article submitted by CassCo Products, Inc. www.cassco-bio.com

Aquarium hobbyists use the term “New Tank Syndrome” to describe fish that are poisoned by high levels of ammonia (NH3). This phenomenon also occurs when starting up new ponds.

Ammonia is produced by the bacterial mineralization of fish waste, excess food, and the decomposition of animal and plant tissues. Additional ammonia is excreted directly into the water by the fish themselves. Ammonia poisoning causes damaged tissue, especially to the gills and kidney. It also causes physiological imbalances such as impaired growth, decreased resistance to disease and death. High levels of nitrite are also a problem. 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.

The successful pond keeper realizes the importance of establishing the nitrogen cycle quickly and with minimal stress on the fish and aquatic life. Nitrifying bacteria oxidize toxic ammonia and nitrite. Nitrosomonas sp. breaks down the ammonia into less toxic nitrite (NO2). Nitrobacter sp. breaks down nitrite (NO2) into nitrate (NO3). This nitrate can then either be used by plants, as a nutrient source, or can be further broken down into dinitrogen gas (N2) through the activity of other species of bacteria.

Nitrifying bacteria are classified as obligate chemo-lithotrophs. 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, and fix inorganic carbon dioxide (CO2) to fulfill their carbon requirements. They are largely non-motile and must colonize surfaces such as gravel, sand or synthetic bio-media, for optimum growth. They secrete a sticky, slime matrix, which they use to attach themselves.

The two most common species of Nitrifying bacteria, mentioned above, are Nitrosomonas and Nitrobacter. They are gram negative bacteria, ranging between 0.6 and 4.0 microns in length. They are obligate aerobes and cannot reproduce or convert ammonia or nitrites without oxygen.

Nitrosomonas utilize 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. Nitrobacter use nitrites for their energy source during its conversion to nitrate (NO3).

Most of the energy produced by Nitrosomonas (up to 80%), is devoted to fixing CO2 and little energy remains for their growth and reproduction. As a consequence, they have a very slow reproductive rate. Because little energy is produced from these reactions, Nitrosomonas have evolved to become extremely efficient at converting ammonia and nitrite. Scientific studies have shown that Nitrosomonas are so efficient, that a single Nitrosomonas cell can convert the same amount of ammonia as approximate 1,000,000 heterotrophic bacteria (standard pond bacteria).

Nitrifying bacteria reproduce by binary division. Under optimal conditions, Nitrosomonas may double in number every seven hours. Nitrobacter may double every 13 hours. This is an extremely long time considering that standard heterotrophic bacteria can double in as little as every 20 minutes. For example, in the time it takes a single Nitrosomonas cell to double in population, a heterotrophic culture could have reached a population of many trillions of cells.

Nitrifying bacteria do not 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 water temperature, pH, dissolved oxygen levels, salt, micronutrients, light, and inhibitory chemicals. Below we will touch briefly on each of these.

**Water Temperature**

The water temperature for optimum growth of Nitrifying bacteria is approximately 75 – 85°F. Growth rate is cut to 50% at approximately 65°F, and cut by 75% at approximately 50°F. Growth is zero at approximately 40°F or below. Nitrifying bacteria will die if frozen, or if water temperature reaches 120°F.

**pH**

The pH for optimum growth of Nitrosomonas is approximately 7.8 – 8.0. At pH levels below 7.0, Nitrosomonaswill grow more slowly, and at a pH of 6.5, Nitrosomonas growth is inhibited. The pH for optimum growth of Nitrobacter is approximately 7.3 – 7.5. Nitrobacter will grow more slowly at the higher pH levels, (typical of marine aquaria). It is important to note that all Nitrification is inhibited if the pH drops to 6.0 or less. At a pH of 6.5, most 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 drops to 2.0 mg/L ­(2 ppm) or less. Nitrobacter is more strongly affected by low DO than Nitrosomonas.

**Salinity**

Some nitrifying bacteria will grow in salinities ranging between 0 – 6 ppt (parts per thousand). Other nitrifying bacteria will grow in salinities ranging from 6 – 44 ppt. Adaptation to different salinities may involve a lag time of 1-3 days before the Nitrifiers will experience exponential growth.

**Micronutrients**

All species of nitrifying bacteria require a number of micronutrients. Most important 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 in the absence of phosphates. Sufficient phosphates are normally present in regular drinking water. Other essential micronutrients are available in our drinking water as well. The increasing popularity of high-tech water filters for de-ionizing, distilling, and reverse osmosis (hyper-filtration), produces water that is stripped of these essential nutrients. While these filters are generally excellent for producing high purity water, this water is inhibitory to nitrifying bacteria.

**Light**

Nitrifying bacteria are photo-sensitive, especially to blue and ultraviolet light. After they have colonized a surface, this light poses no problem. But during the first 3 to 4 days, many of the cells may be suspended in the water column. Any bulbs that emit UV or near UV light, should remain “off” during this initial 3 to 4 day time frame.

Chlorine & Chloramines

Before adding bacteria to a pond or aquarium, all chlorine must be completely neutralized. Most US cities treat their drinking water with chloramines. Chloramines are more stable than chlorine. The type of chloramines formed is dependent on pH. Most chloramines exist as either monochloramine (NH2Cl) or dichloramine (NHCl2). These com-pounds are created by adding ammonia to chlorinated water. Commercial chlorine reducing chemicals, such as sodium thiosulfate, will break the chlorine: ammonia bond. Chlorine (Cl) is reduced to harmless chloride (Cl-) ion. Each molecule of chloramine that is reduced, will produce one molecule of ammonia. When treating, first neutralize all chlorine. Then eliminate ammonia.

The cells of Nitrifying bacteria are reddish (Nitrosomonas) to brownish (Nitrobacter) in color. The liquid solution found in commercially available products is normally a light red color, primarily due to the natural pigment of the bacterial cells. Commercial products normally have a musty smell. Sometimes the solution may turn dark brown or black and smell like rotten eggs. This is rare but not unusual. This is due to the presence of residual sulfates that have been reduced to sulfides. This has no relationship to the viability of the bacterial cultures. The concentration of sulfides is only a few parts per billion and is not toxic when diluted in the aquarium or pond. If desired, these sulfides can easily be “de-gassed” before use, by removing the bottle cap and allowing oxygen to penetrate into solution.

Article submitted by CassCo Products, Inc.
www.cassco-bio.com