Abstract: 9245 A. Introduction

1. General Discussion

Nitrifying bacteria and members of the domain Archaea convert ammonia to nitrite and then nitrate in a sequential process called nitrification.¹ Nitrification is crucial to the biogeochemical nitrogen cycle.

Two groups of nitrifiers are necessary to complete nitrification: ammonia-oxidizing bacteria (AOB), which convert ammonia to nitrite (NH₄⁺ to NO₂⁻), and nitrite-oxidizing bacteria (NOB), which convert nitrite to nitrate (NO₂⁻ to NO₃⁻). The most commonly isolated or identified nitrifiers from freshwater or wastewater systems are in the Nitrosospira and Nitrosomonas genera for AOB and in the Nitrobacter and Nitrospira genera for NOB.^2,3 Nitrosomonas europaea and Nitrobacter winogradskyi have been the most intensively studied species of AOB and NOB, respectively, and the genomes of these bacteria have been sequenced.^4,5 Nitrifying bacteria can occur in various habitats (e.g., soil and fresh, marine, brackish, waste, and treated drinking waters.) They are generally sensitive to visible and ultraviolet light,^6–9 so they are often found in light-free environments. The bacteria are found in a variety of shapes (e.g., rods, curved rods, spheres, spirals, and lobular forms, many with flagella) and range in size from approximately 0.3 to 11.7 µm. Many have intracellular membranes.^10,11

These bacteria are aerobic, Gram-negative, and generally chemolithotrophic (obtain energy via the oxidation of inorganic chemical compounds, such as ammonia or nitrite).^10,11 Nitrifiers are generally autotrophic, although mixotrophy has also been observed.^10,11 They use carbon dioxide and carbonates as carbon sources. Bacterial chemolithotrophic aerobic nitrifiers, once classified in the family Nitrobacteraceae, are now found in three classes (Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria) of the Proteobacteria phylum¹² and in the deeply branching Nitrospirae phylum.¹³

Two discoveries have significantly changed our understanding of nitrification.¹ One is anammox, the process of anaerobically oxidizing ammonia to N₂ gas using nitrite as the electron acceptor. The oxidizers are novel bacteria related to the Planctomycetales.¹⁴ This process was first characterized in wastewater treatment systems.¹⁵

The second discovery is that aerobic ammonia oxidation is mediated by organisms in the domain Archaea as well as by those in the domain Bacteria.^1,16 Although this section focuses on bacterial chemolithotrophic nitrifiers because of their known importance in drinking water^17,18 and wastewater systems,⁴ keep in mind that anammox and Archaea also probably affect nitrification in natural and managed systems.^1,19

To promote nitrification at a water or wastewater treatment utility, personnel allow nitrifying bacteria to grow in filter beds or aeration basins, where they convert ammonia to nitrate.¹⁷ The efficiency of this process depends on retention time, temperature, pH, dissolved oxygen, nitrogen, organic matter, toxic substances, and populations of heterotrophic and nitrifying bacteria and Archaea.

Nitrification can either help or hinder a utility’s overall effectiveness, depending on the application and the disinfectant the utility uses. In wastewater treatment, nitrification removes ammonia-nitrogen, which is toxic to some forms of aquatic wildlife.²⁰ In addition, it is often an indicator of the purification process’ overall efficiency. Ammonia also exerts an appreciable chlorine demand, whereas nitrate does not, so nitrification reduces the costs of chlorine disinfection. In water treatment, nitrification in filter beds can reduce source water ammonia, thereby reducing chloramine or breakpoint chlorination requirements (this occurs in some regions of the United States).

However, nitrification is a nuisance in drinking water distribution systems that use chloramines because it significantly reduces water quality.^7,18,21 Nitrification increases biomass in the distribution system and decreases chloramine residual (via abiotic chemical reactions between nitrite and chloramine).^18,21–23 Its biotic and abiotic reactions may substantially increase levels of heterotrophic plate count (HPC) bacteria, coliforms, and opportunistic pathogens.²⁴ Also, HPC bacteria use the organic compounds secreted by AOB as sources of energy,^21,25 so nitrification may impair a utility’s ability to meet the provisions of the U.S. Environmental Protection Agency Total Coliform Rule and Surface Water Treatment Rule²¹ (which requires utilities to maintain a disinfectant residual or keep the HPC level below 500 CFU/mL).

Although nitrification is an important water and wastewater treatment process, isolating and quantifying the related organisms is not a common practice. Because nitrification yields little energy, these bacteria grow at a much slower rate than most heterotrophic bacteria,²⁶ making them relatively difficult to grow in the laboratory. They also can be tedious to identify because of their slow growth rate and low isolation rates on agar plates.

Molecular methods for quantifying nitrifiers using real-time polymerase chain reaction to target group-specific 16S rRNA genes or functional genes (especially the ammonia monooxygenase gene, amoA) have been used to quantitate relative levels of nitrifiers in drinking water and wastewater treatment plants.^27–32 For example, recent investigations using molecular methods have identified the ubiquitous AOB Nitrosomonas oligotropha in full-scale drinking water distribution systems receiving chloraminated water.^22,23 However, these methods are still being refined; routine application in non-research laboratories is not yet feasible.