20 Biological Nitrification and De-nitrification

Babita Khosla

21.1 INTRODUCTION

Nitrogen is one of the primary nutrients critical for the survival of all living organisms. It is a necessary component of many biomolecules, including proteins, DNA, and chlorophyll. Although nitrogen is very abundant in the atmosphere as dinitrogen gas (N2), it is largely inaccessible in this form to most organisms, making nitrogen a scarce resource and often limiting primary productivity in many ecosystems. Only when nitrogen is converted from dinitrogen gas into ammonia (NH3) does it become available to primary producers, such as plants.

In addition to N2 and NH3, nitrogen exists in many different forms, including both inorganic (e.g., ammonia, nitrite, nitrate) and organic (e.g., amino and nucleic acids) forms. Thus, nitrogen undergoes many different transformations in the ecosystem, changing from one form to another as organisms use it for growth and, in some cases, energy. The major transformations of nitrogen are nitrogen fixation, nitrification, denitrification, anammox, and ammonification (Figure 1). The transformation of nitrogen into its many oxidation states is key to productivity in the biosphere and is highly dependent on the activities of a diverse assemblage of microorganisms, such as bacteria, archaea, and fungi.

Figure: 1 Nitrogen Cycle in nature

(Image: https://www.dreamstime.com/royalty-free-stock-photos-nitrogen-cycle -cycle-processes-transform-one- form-to-another-illustration-flow-image69353365)

Since the mid-1900s, humans have been exerting an ever-increasing impact on the global nitrogen cycle. Human activities, such as making fertilizers, industrialization and burning fossil fuels, have significantly altered the amount of fixed nitrogen in the Earth’s ecosystems. Now, instead of a primary nutrient, nitrogen is one of the major pollution sources that contribute to environmental quality problems. Industries like tannery effluents, textile, landfill leachate, fertilizer industries etc., produce ammonia rich effluents. Ammonia when present in water exists in two forms ammonium ion (NH4+) and free ammonia (NH3) depending on the pH of water. At higher pH ammonia is toxic to aquatic organisms and also for terrestrial organisms. Though many technologies are available for the removal of ammonia from industrial effluents but most of them are expensive and some are facing operational difficulties. Generally, removal of ammonia using traditional nitrification and denitrification is inexpensive and easy to maintain method of choice in waste water treatment. Bacteria remove nitrogen from wastewater by a two step biological processes: nitrification followed by denitrification. Technically, it is a three step process: ammonification precedes nitrification and denitrification.

Ammonification: In fresh wastewater the nitrogen present is primarily combined in proteinaceous matter and urea as organic nitrogen. The majority of the organic nitrogen contained in raw sewage is converted to ammonia through a process of decomposition by heterotrophic bacteria, known as ammoniafication. Ammonia nitrogen may exist in aqueous solution as either ammonium ion or unionized ammonia. The relationship between the two forms is pH and temperature dependent and may be expressed in accordance with the following equation:

NH3 + H2O → NH4+ + OH-

At a pH greater than 7.0, the reaction is displaced to the left and ammonium ions are predominant at pH less than 7.0.

Free ammonia in concentrations above 0.2 mg/l, has been shown to be fatal to several species of fish. Ammonia toxicity should not be a problem in receiving waters with pH below 8.0 and ammonia nitrogen concentrations less than about 1 mg/l.

Nitrification: Nitrification is an essential process in the nitrogen cycle of soils, natural waters, and wastewater treatment systems. Nitrification is the biological process and actually the net result of two distinct processes: the oxidation of ammonia (NH3) or ammonium

(NH4+) to nitrite (NO2−) by ammonia-oxidizing bacteria and the oxidation of nitrite (NO2-) to nitrate (NO3-) by the nitrite-oxidizing bacteria.

The stoichiometry of nitrification is

NH4+ + 1.5O2 → NO2- + H2O +2H+

2NO2- + O2 → 2NO3-

The overall reaction is as follows:

NH4+ + 2O2 → NO3- + 2H+ + H2O 21.2 Nitrifying microorganisms Ammonia oxidising bacteria:

Two groups of bacteria– Nitrosomonas sp. and Nitrobacter sp. collectively known as nitrifying bacteria are known as the principal organisms involved in nitrification processes. The first group of nitrifiers is the ammonia oxidizers, which oxidize ammonia to nitrite. In most natural waters, ammonium is present predominantly as the positively charged ion, ammonium (NH4+), but the enzyme responsible for the first step of the reaction uses the gaseous form, NH3, which is usually a minor component at equilibrium. There are two very different groups of ammonia-oxidizing microbes. One is the well-known bacterial group (ammonia oxidizing bacteria), which includes a few different kinds of bacteria that all make a living by generating reducing power (ATP) from the oxidation of ammonia and using that energy to fix carbon dioxide (Bock and Wagner, 2006). These organisms are considered to be autotrophs, since they derive energy for growth and synthesis from the oxidation of inorganic carbon (CO2) compounds, using the Calvin cycle rather than from organic compounds. Ammonia is their only energy source, and their main metabolic product is nitrite. In addition to the net production of nitrite they are also capable of producing nitrous oxide (N2O).

Both of these groups have rather specific environmental requirements in terms of pH, temperature, and dissolved oxygen and reproduce at much slower rates than heterotrophic bacteria. They are strict “aerobes,” meaning they must have free dissolved oxygen to perform their work. Various heavy metals and organic compounds have been found to suppress or inhibit the growth of nitrifiers.

Ammonia oxidising archea:

A second distinct archaea group of soil bacteria widespread in most soils has recently been recognized as ammonia-oxidizing microbes. The ammonia-oxidizing archaea are abundant in  many environments and in the ocean and many terrestrial systems, they far outnumber the ammonia oxidising bacteria. These ammonia-oxidizing archaea oxidize ammonia to nitrite and produce nitrous oxide and nitrite from ammonia, but the enzymatic pathways are quite different. They are also thought to be predominantly autotrophic, but they fix CO2 using the 3-hydroxypropionate/4-hydroxybutyrate pathway, rather than the Calvin Cycle. Although the enzymes and pathways differ for the ammonia oxidising bacteria and archea, aerobic ammonia oxidation in both groups apparently proceeds by the same stoichiometry.

NH3 + 1.5O2 → NO2- + H2O +H+

Anammox Organisms: A third group of bacteria, members of the Planctomycetes phylum, are capable of oxidizing ammonium using nitrite instead of oxygen and producing N2 instead of nitrite. This metabolism is strictly anoxic and the process is known as anaerobic ammonia oxidation, or anammox. Anammox organisms are strict autotrophs, and apparently use the acetyl-CoA pathway for CO2 fixation. Their growth is extremely slow, with generation times on the order of 2 weeks. The cells contain an internal membrane-bound ‘organelle’ called the anammoxosome, in which the anammox reaction is localized. The cell membranes contain unique lipids called ladderanes, after their diagrammatic appearance as a ladder, which form the very dense membrane (van Niftrik and Jetten, 2008). The net reaction for anammox involves a 1:1 combination of ammonium and nitrite in the production of

N2. NO2- + NH4+ → N2 + 2H2O

Thus, unlike conventional nitrification, anammox results in the loss of fixed nitrogen from the system, and is ecologically equivalent to denitrification, rather than to nitrification. Anammox results in the anaerobic removal of ammonium using nitrite, derived from either aerobic ammonium oxidation or partial denitrification, as the oxidant.

Nitrite-Oxidizing Bacteria The second functionally defined group of nitrifying microbes is the nitrite-oxidizing bacteria (NOB), which include several genera. The best-known cultivated members, in the genus Nitrobacter, are chemolithoautotrophic, like the ammonia oxidising bacteria, using nitrite as an energy source and CO2 as a carbon source via the Calvin cycle (Bock and Wagner, 2006). However, the lesser known genus, Nitrospina, is apparently most abundant in the ocean, and uses the reductive tricarboxylic acid pathway for CO2 fixation. Many strains are known to possess heterotrophic capabilities and are considered mixotrophic or facultative autotrophs. Although they have limited metabolic capabilities for uptake and degradation of organic molecules, they can supplement their growth with organic carbon and, in some cases, grow slowly in the absence of nitrite when certain organic substrates are present. The oxidation of nitrite is even less energy yielding than ammonia oxidation, so perhaps this ability for heterotrophic growth is not surprising. Aerobic nitrite oxidation proceeds by the following stoichiometry:

2NO2- + O2 → 2NO3-

There are no other pathways, nor any different kinds of bacteria or archaea known to be capable of or involved in nitrite oxidation in the environment.

Heterotrophic Nitrifiers: The ability to nitrify, via pathways involving the inorganic transformations normally associated with the autotrophic nitrifiers described above, or via

pathways involving organic intermediates but resulting in the net oxidation of ammonium, has been attributed to some heterotrophic bacteria and fungi. Heterotrophic nitrification does not conserve energy (i.e., is not linked to ATP production) and the rates observed are much slower than rates found in cultivated conventional nitrifiers. Autotrophic nitrifiers are susceptible to inhibition by a number of naturally occurring substances, including secondary metabolites of some trees, for example. AOB are inhibited by acidic conditions, which pertain in some soils. These observations led to the suggestions that heterotrophic nitrification might be particularly important under conditions in some soils that are very unfavorable for known autotrophic nitrifiers.

21.3 Factors affecting Nitrification:

Several environmental factors that might control nitrification in various ecosystems have already been mentioned. They include the kinds of things that affect biological processes in general, as well as those particular to the metabolism of nitrifiers: temperature, salinity, light, organic matter concentrations, substrate (ammonium and nitrite) concentrations, pH, and oxygen concentration. A few of the interesting and unique interactions of nitrifiers with their environment are explored below.

Oxygen: Nitrifiers are obligate aerobes, i.e. they require free molecular oxygen and are killed off by anaerobic conditions. Maximum nitrification occurs at a D.O. (Dissolved Oxygen) level of 3.0 mg/l. Significant nitrification occurs at a D.O. level of 2.0 to 2.9 mg/l. Nitrification ceases at D.O. levels of <0.5 mg/l. Approximately 4.6 kg of oxygen are required for every kg of ammonium ions oxidized to nitrate (This compares with a requirement of 1 kg of oxygen to oxidize 1 kg of carbonaceous B.O.D.). An absence of oxygen for <4 hours does not adversely affect nitrifiers when oxygen is restored. To ensure effective nitrification always maintain a D.O. level of ≥1.5 mg/l.

Temperature: Nitrification is temperature sensitive. The optimum temperature for nitrification is generally considered to be 30°C. The nitrification ceases below 5°C and above 45°C. The nitrification rate is approximately just half at 16°C as compared to 30°C.

Alkalinity and pH: Nitrifiers use alkalinity as a carbon source, i.e., they use an inorganic form of carbon. In an activated sludge process during nitrification the oxidation of ammonium ions to nitrous acid (HNO2) destroys alkalinity. Any transformation that involves the production or consumption of hydrogen ions is pH sensitive, and ammonia oxidation is no exception. Hydrogen ions (H+) are produced when ammonium ions are oxidized to nitrite:

2NH4+

Low pH eventually inhibits both adjustment.

+ O2 → 4H+ + 2NO2- + 4H2O

H+ + NO2- → HNO2

groups in culture, and activity can be restored by pH

Denitrification

Denitrification is a natural soil microbial process by which certain species of bacteria under anoxic conditions reduce nitrate nitrogen to the gaseous end-products of N2, NO2 or N2O which can then escape from solution to the atmosphere. Denitrification occurs when soil bacteria use nitrate for their respiration in the place of oxygen in the air. Denitrification most commonly occurs in wet or waterlogged soils with an abundance of nitrate where the oxygen supply for respiration is restricted.

Denitrification is a two-step process and, using methanol as the electron donor, may be represented by the following equations:

2CH3OH + 6NO3- →, 6NO2- + 2CO2 + 4H2O

3CH3OH + 6NO2- → 3O2 + 3CO2 + 3N2 + 3 H2O + 6OH-

The overall reaction using methanol may be expressed as:

5CH3OH + 6NO3- + 6O2 → 5 CO2 + 3N2 + 7H20 + 6OH-

The optimum pH values for denitrification are between 7.0 and 8.5 and denitrification is an alkalinity producing process

The most common denitrifying bacteria are Bacillus denitrificans, Micrococcus denitrfjicans, Pseudomonas stutzeri, and Achrornobacter sp. Unlike the autotrophic nitrifying bacteria responsible for nitrification, denitrifying bacteria are facultative organisms, they can use either dissolved oxygen or nitrate as an oxygen source for metabolism and oxidation of organic matter. If dissolved oxygen and nitrate are present, bacteria will use the dissolved oxygen first. That is, the bacteria will not lower the nitrate concentration. Denitrification occurs only under anaerobic or anoxic conditions.

21.4 Factors affecting Denitrification:

Presence of Nitrate: Denitrification only occurs when nitrate is present. One method for minimizing denitrification is to maintain a minimum concentration of nitrate needed to support healthy plant growth. This can be accomplished through techniques such as split fertilizer applications, fertigation, or the use of controlled-release fertilizers. Another  approach is the use of a nitrification inhibitor added to N fertilizer. Nitrification inhibitors temporarily restrict Nitrosomonas bacteria from converting ammonium to nitrite. Slowing this process reduces the rapid appearance of nitrate that commonly follows fertilization with ammonium based fertilizer, or manure.

Temperature: Temperature affects the growth rate of denitrifying organisms, with greater growth rate at higher temperatures. Denitrification can occur between 5 and 30oC and these rates increase with temperature.

Soil wetness: The presence or absence of oxygen is one of the largest factors determining the extent and duration of denitrification. Denitrification can occur in aerobic (adequate oxygen) conditions, but to a relatively insignificant degree. Wet soils are generally the trigger for denitrification to occur. Nitrogen gases can begin to appear as soon as 15 minutes after saturation if conditions are favorable. At higher soil moisture, N2 tends to become the major product of denitrification.

Presence of dissolved carbon: Denitrifying bacteria obtain their energy from soluble organic carbon. Therefore denitrification is enhanced in soils with a ready supply of organic carbon, such as manure, compost, cover crops, or crop residues. Soluble carbon also influences the end product of denitrification. Production of N2 commonly dominates with an adequate supply of soluble carbon, while N2O and NO production is more likely if soluble carbon limits microbial growth. There have been experimental attempts to stimulate denitrification in the field to remove nitrate from water by adding soluble carbon (such as edible oil, molasses, and other rapidly degraded carbon sources) as energy sources for microbes. Similarly, passing nitrate-rich surface water through a reactor or through a constructed wetland to stimulate denitrification is a common technique for water treatment. Soils that go through a prolonged dry period followed by rainfall or irrigation typically have a burst of soluble carbon that can support a spike in denitrification. Waterlogging also stimulates the release of soluble carbon into the soil that may support rapid denitrification.

21.5 Roles of Nitrification- Denitrification

Agricultural and Terrestrial Systems: Nitrogen is the main component of fertilizers applied in many agricultural systems. Addition of N as ammonium is advantageous because it is easily assimilated by plants and, due to its positive charge, it binds to soil particles and is somewhat resistant to loss in runoff. Nitrifying bacteria in the soil can  convert the ammonium to nitrate, which is more easily lost in the soil solution, thus reducing the efficiency and increasing the cost of fertilizer application. Nitrification inhibitors are therefore often applied along with fertilizers, to slow down this conversion and increase the amount of N available to the plants. Not only is the nitrate more susceptible to physical loss from the system, but it is also the substrate for denitrification. If soils become waterlogged to the extent that interstitial spaces become anoxic, denitrifying bacteria present in the soils will switch to anaerobic metabolism, in which they respire oxides of nitrogen, beginning with nitrate, instead of oxygen. Nitrate respiration leads to the removal of nitrate by its conversion to N2 gas, which is not biologically available to most plants and is lost from the system by evasion.

Not only is this N lost from the bioavailable pool, but it plays a very important role in the atmosphere as a greenhouse gas. N2O has a radiative forcing that is on the order of 200 times more potent per molecule than CO2, the most abundant greenhouse gas. Thus, N2O fluxes from agricultural systems to the atmosphere are potentially of concern.

Wastewater Treatment:  Nitrate nitrogen is the highly oxidized form of nitrogen and regarded as an undesirable substance in public water. Although it occurs naturally in water, elevated levels of nitrate in groundwater usually result from human activities, such as over use of chemical fertilizers in agriculture and improper disposal of human and animal wastes High nitrate concentration in drinking water may cause serious problems in humans and animals (Nugent, et al., 1988). The high concentrations (90-104 units mg/l) have been shown to cause methemoglobinemia in infants under four months old. In order to protect against this effort, the United States Environmental Protection Agency (USEPA) has established the maximum contamination level of nitrate in drinking water at 10 mg NO3-N /L, which corresponds to the maximum allowance recommended by the World Health Organization (WHO).

The discharge of nitrate nitrogen into receiving waters from wastewater treatment plants will not result in any oxygen demand in terms of NOD. Nitrate is, however, an important nutrient for algae growth and, when present in excessive quantities, may be responsible for promoting eutrophication in streams and lakes. Thus, in certain cases, its discharge might have to be limited or prohibited to prevent excessive algae growth.

Tertiary wastewater treatment is designed to remove inorganic nitrogen from the stream by nitrification. When carried out by nitrifying bacteria, this is an obligatory aerobic process, and requires aeration to allow their growth and activity to produce nitrate. Water containing the nitrate thus produced is then subjected to an anoxic treatment in which denitrification reduces the nitrate to N2. In this form, the effluent does not increase the bioavailable N in the receiving waters.

The Marine Environment: The nitrogen cycle of the ocean is interesting because of the role of N as a limiting nutrient for primary production in the sea, and because the ocean is the ultimate repository for waste from land, in the form of wastewater effluent and natural drainage from rivers. Nitrogen loading in natural waters, from excess fertilizer applications as well as wastewater effluent, has increased in recent decades, such that the impact in coastal waters is now detectable. Nitrification plays a part in both of these processes as described above.

Together with ammonification, nitrification and denitrification forms a mineralization process that refers to the complete decomposition of organic material, with the release of available nitrogen compounds. This replenishes the nitrogen cycle.

References:

• Princic, I. Mahne, F. Megasus, E. A. Paul, and J. M. Tidje, “Effects of pH and oxygen and Ammonium concentrations on the community structure of nitrifying bacteria from wastewater,” Applied and Environmental Microbiology, vol. 64, no. 10, pp. 3584-3590, 1998.
• Bock E and Wagner M (2006) Oxidation of inorganic nitrogen compounds as an energy source. The Prokaryotes 2: 457–495.
• Kuenen JG (2008) Anammox bacteria: from discovery to application. Nature Reviews Microbiology 6: 320–326.
• Van Niftrik L and Jetten MSM (2008) Anaerobic ammonium-oxidizing bacteria: Unique microorganisms with exceptional properties. Microbiology and Molecular Biology Reviews 76: 585–596.
• Walker CB, De La Torre JR, Klotz MG, et al. (2010) Nitrosopumilus maritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine crenarchaea. Proceedings of the National Academy of Sciences of the United States of America 107: 8818–8823.