H

Dinitrogenase

Dinitrogen dihydride N2H2 -1

Dinitrogen tetrahydride (N2HJ -2

Dinitrogenase Ammonia NH3 -3

2H i

Dinitrogenase a

Nitrogen Species

Figure 13.19 Nitrogen fixation pathway.

strategies include producing a physical barrier, forming a protective complex with another protein, and/or maintaining high rates of respiration to deplete oxygen near and within the cell. Thus, another advantage of being within a root nodule is the more restricted flux of oxygen. Klebsiella and some other free-living bacteria can also produce a thick slime layer. Being in the rhizosphere, where root exudates produce a higher concentration of respirable organic substrates, helps limit oxygen concentration.

Several of the filamentous cyanobacteria fix nitrogen only in a few differentiated cells (unusual in prokaryotes) with thick walls, called heterocysts (see Section 10.5.4 and Figure 10.20). The nitrogen is then distributed to the other cells in the filament, which in return provide organic substrates to the nonphotosynthetic heterocyst. Many cyanobac-teria may also prefer reduced levels of oxygen (perhaps 10% of saturation).

Most nitrogenases will also cometabolize other triple bonds, such as those of acetylene (HC CH) and hydrogen cyanide (HC N). In fact, acetylene reduction to ethylene (H2C=CH2) is often used as an indirect means of measuring nitrogen-fixing activity.

Dissimilatory Nitrate Reduction, Including Denitrification A wide variety of aerobic prokaryotes are also able to utilize nitrate (NO=) as a terminal electron acceptor in the absence of oxygen. In many cases, including with strains of Escherichia coli, Bacillus, Staphylococcus, Spirillum, some actinomycetes, Aquifex, and some archaea, the product of this dissimilatory nitrate reduction is nitrite (NO=). A few of these organisms are able to further reduce the nitrite to ammonia, but this type of ammonification (as opposed to the mineralization of organic nitrogen) appears to be of only minor importance in most environments.

However, under similar conditions, anaerobic respiration by a fairly broad array of otherwise aerobic prokaryotes, such as some Pseudomonas, Thiobacillus denitrificans, Paracoccus, and a few archaea, results in a sequential conversion (Figure 13.20) of nitrate

Nitrogen Oxidation State

Nitrate reductase Nitrite(NO2J +3 |

Nitrite reductase ri th

Nitric oxide +2

Nitrite reductase ri th

Nitric oxide reductase Nitrous oxide +1

Nitrous oxide reductase

Dinitrogen N2 0

Nitrogen Species

Figure 13.20 Denitrification pathway.

to nitrite to elemental nitrogen gas (N2). The term denitrification is applied to this process because N2 is unusable by most organisms, so that these reactions represent a loss of available (fixed) nitrogen to an aquatic or soil environment. (Similarly, the release of small amounts of the gaseous intermediates NO and N2O represent losses of nitrogen from the system.)

A general reaction for oxidation of a carbohydrate through denitrification can be written as

If the organic material oxidized is methanol, the reaction can be written instead as

Example 13.5 An anaerobic groundwater contains 100 mg/L of carbohydrate with an empirical formula of CH2O. How much nitrate would have to be added to meet the stoi-chiometric requirement for complete oxidation of this contaminant?

Answer From equation (13.10), 1.0 mol of nitrate would be needed per 1.25 mol of CH2O, which has an apparent molecular weight of 1(12) + 2(1) + 1(16) = 30:

• , xT~ xr , ™ ^ ,T mol CH2O\ / 1 mol NO3- \ / 14 g NO3- \

Under anaerobic conditions we often expect unpleasant odors to develop. When nitrate is present, however, this typically does not happen. Further, the active organisms are mainly aerobes that have the additional ability to respire using nitrate as an alternative terminal electron acceptor. Thus, sanitary engineers, and subsequently, environmental engineers and scientists, have traditionally referred to conditions in which oxygen is absent but nitrate is present as anoxic rather than anaerobic. However, please be aware that most biologists and others outside our field do not make this distinction, using anaerobic and anoxic as synonyms to refer to any system in which oxygen is absent.

Although most denitrifiers are organotrophs, some are lithotrophs utilizing hydrogen or sulfide. Recently, autotrophic nitrification (Section 13.2.2) also has been linked to denitrification, in a process dubbed anammox (anoxic ammonia oxidation). In oxidizing ammonia as an energy source, the organisms involved can reduce nitrate to nitrite and then reduce the nitrite to nitrogen gas:

This process does not seem to play a major environmental role in nitrogen cycling. However, it may be important to the organisms involved in some circumstances, particularly when aerobic conditions (under which the nitrate is produced) are followed by anoxic ones. This occurs in soils that are periodically flooded, as well as in some wastewater treatment plants.

In general, denitrification does not occur in the presence of oxygen. Since approximately 20% less energy is available to an organism when it is utilizing nitrate, there

Figure 13.21 Clustered nitrifying cocci within activated sludge floc.

has been strong selective pressure for microbes to develop control mechanisms to suppress nitrate reduction in the presence of O2. However, denitrification can occur in anoxic microenvironments (microscopically localized environments having different physical and chemical conditions) within heterogeneous aerobic systems, such as moist soils and microbial colonies.

Nitrate Assimilation A wide variety of bacteria, archaea, fungi, algae, and most plants can utilize nitrate as their source of nitrogen. However, to do this, they must reduce it to an oxidation state of —3 (the level of ammonia and organic nitrogen). This is referred to as assimilatory nitrate reduction. While dissimilatory reduction can be linked to energy-yielding respiration, assimilatory reduction requires energy. As a result, the biomass yield of organisms growing on nitrate as their nitrogen source will be slightly lower than when ammonia is used. Hence, an organism that is able to use both ammonia and nitrate will utilize ammonia preferentially when it is available. On the other hand, the presence of oxygen does not inhibit assimilatory reduction.

13.2.2 Nitrogen Oxidation

The oxidation of ammonia to nitrite, and nitrite to nitrate, is known as nitrification. There is no known biochemical oxidation of nitrogen gas, although there are abiotic mechanisms (electrochemical, photochemical, thermal). Similarly, there is no known pathway for direct conversion of ammonia to nitrogen gas (the reverse of nitrogen fixation).

Autotrophic Nitrification Most nitrification results from the activity of aerobic, autotrophic chemolithotrophs, referred to as nitrifying bacteria (Figure 13.21). They utilize reduced nitrogen (ammonia or nitrite) as their energy source, carbon dioxide as their carbon source, and oxygen as their electron acceptor.

The overall oxidation really consists of two separate steps, carried out by different bacteria (Figure 13.22). Ammonia oxidation involves the transformation of ammonia to nitrite, a six-electron transfer (—3 to +3), and is sometimes called nitrite formation or

Nitrogen Oxidation State

Ammonia Monooxygenase (AMO)

Hydroxylamine |nh2Oh|Q-1 Nitrogen

Oxidation

J3L ^Statej

Hydroxylamine Nitrite NO- +3 Nitroxyl noh +1 oxidoreductase 2

(HAO) Nitrite

Oxidase

Nitrite NO2 +3 Nitrate NO

Nitrogen Nitrogen

Species Species

Figure 13.22 Steps in autotrophic nitrification: (a) ammonia oxidation; (b) nitrite oxidation.

nitritification. In the second step, nitrite oxidation or nitratification, nitrite is converted to nitrate, a two-electron change (+3 to +5). Similarly, the bacteria can be referred to as ammonia oxidizers (or nitritifiers) and nitrite oxidizers (nitratifiers). No known nitrifier can oxidize ammonia all the way to nitrate.

Balanced equations for the individual reactions and their sum can be written as

Note that the equations are written with ammonium and nitrite as the reactants, since it is these ionic forms that are expected to be predominant at neutral pH values. However, there is some evidence that the forms actually used by the bacteria are nonionized ammonia (NH3) and nitrous acid (HNO2). Also note that 2 mol of oxygen is used and that 2 mol of strong acid is produced from 1 mol of weak acid.

Known organisms carrying out the first (nitritification) step are the proteobacteria Nitrosomonas (p), Nitrosospira (p), and Nitrosococcus (g). The second (nitratification) step is performed by the proteobacteria Nitrobacter (a), Nitrococcus (g), and Nitrospina (8), and by Nitrospira, a member of the Xenobacteria. Thus, although all of the nitrifiers were once included in the same family because of their activities, it is now recognized that they are phylogenetically diverse. Also, at one time most nitrifiers were assumed to be either Nitrosomonas or Nitrobacter. However, it is now recognized, based on genetic techniques, that although these are the most often cultured species, they are not necessarily the most common or most active in the environment. Hence, nitrifying activity should not be assigned to these genera unless they are actually identified.

As shown in Figure 13.22, ammonia monooxygenase (AMO) is the enzyme responsible for catalyzing the first reaction of nitrification, in which ammonia is oxidized to hydro-xylamine. Hydroxylamine oxidoreductase then produces a transient intermediate

(nitroxyl) while forming nitrite. Luckily, hydroxylamine itself rarely accumulates, as it is a potential mutagen. Nitrite oxidation is catalyzed by nitrite oxidase.

AMO shows some similarity to methane monooxygenase (MMO), the enzyme used by methanotrophs (Section 10.5.6) to oxidize methane. In fact, many ammonium oxidizers and methanotrophs can aerobically cometabolize each other's substrate as well as a number of other compounds, including trichloroethylene.

Compared to oxidation of organic compounds, relatively little energy is available to nitrifiers. It takes about 35 mol of NH4+ for ammonia oxidizers to fix 1 mol of CO2. Nitrite oxidizers require even more substrate, about 100 mol of NO2~ per mole of CO2 fixed. Since the nitrite typically comes from the ammonia (100 mol of ammonia produces 100 mol of nitrite), this means that ammonia oxidizers are usually more abundant than nitrite oxidizers. Also, cell yields based on their energy source are much lower for nitri-fiers than for most heterotrophs, often in the range 5 to 20% rather than 50 to 60%.

The fact that nitrifiers appear to belong to only a few genera suggests that there may be more limitations on their activity than would be true if they were more diverse. In fact, compared to many heterotrophs, nitrifiers are slow growing. Under ideal conditions, minimum doubling times are around 8 hours. Also, there are no known thermophilic nitrifiers, so that autotrophic nitrification does not occur in systems with temperatures above ^42°C. Furthermore, since there are no known sporeformers, elevated temperatures actually kill the nitrifiers; this means that activity is slow to return to a system (being dependent on reinvasion or reinoculation) even once elevated temperatures decrease. Optimum temperature is usually around 28 to 30° C, and activity is usually minimal at temperatures below 10°C.

Similarly, pH can be limiting. Optimum pH values are around 7.5 to 8, with almost no activity below pH 6. This may in part be because of the unavailability of nonionized ammonia at low pH values. Also, nitrite is more toxic at low pH, where it is present as nonionized nitrous acid. At high pH, toxicity from ammonia becomes a problem. On the other hand, although they are aerobic [with the exception of the anammox process, equation (13.12)], nitrifiers can survive for prolonged periods under anaerobic conditions and are effective at utilizing low concentrations of oxygen. In other words, they have a low Ks (half-saturation coefficient, Section 11.7.2) value for dissolved oxygen, typically below 0.5 mg/L. Similarly, they require only small amounts of their energy sources to approach maximum activity rates (Ks values for ammonia- or nitrite-N of 1 mg/L or less).

A close relationship between ammonia and nitrite oxidizers can be expected, since the product of the first group is the substrate for the second. Thus, the two groups are typically located in close physical association. Ammonia oxidizers are usually more abundant, since about three times as much energy is available from ammonia oxidation as nitrite oxidation. Typically, only traces of nitrite are seen in the environment. Thus, nitrification is often treated as though it was a single step, involving one group of bacteria. However, accumulations of nitrite can occur under transient conditions, particularly since the nitrite oxidizers appear to be a little more sensitive to low pH and high concentrations of ammonia and nitrite.

Heterotrophic Nitrification Some heterotrophic bacteria and fungi are able to oxidize nitrite to nitrate, and/or occasionally, ammonia to nitrite. This does not appear to provide any benefit to the organism and hence is considered a type of cometabolism. Perhaps in some cases this represents assimilatory nitrate reduction enzymes working in reverse.

It is generally believed that heterotrophic nitrification plays only a small role in nitrogen cycling. However, in some environments, such as acid soils, in which autotrophic nitrification is severely inhibited, it may have a local effect.

13.2.3 Nitrogen in Environmental Engineering and Science

If nitrogen is second only to carbon in terms of the complexity of its cycling, it probably also is second only to carbon in terms of its importance in environmental engineering and science. The range of concerns includes wastewater and potable water treatment, surface and groundwater contamination, agriculture, sludge and solid waste management, human and environmental toxicity, and bioremediation. What is more, these problems may be interdependent. For example, in agriculture, nitrogen is often the limiting nutrient for many important crops (e.g., wheat, corn, cotton), so that loss of fixed nitrogen is a major concern. However, overapplication of chemical fertilizers, manures, or wastewater treatment sludges, which typically are high in nitrogen, can lead to groundwater (from leaching) and/or surface water (from runoff) contamination. In fact, nitrate is the leading groundwater contaminant in the United States, mostly as a result of agricultural practices.

Oxygen Demand As noted in equation (13.15), complete stoichiometric oxidation of ammonium requires 2 mol of O2 per mole of ammonium-N. This translates into 64/14 = 4.57 mg O2/mg NH4+-N oxidized to nitrate. Because the organisms also reduce CO2 and assimilate some N for cell constituents, the actual ratio is a little lower, typically 4.33 mgO2/mg NH4+-N (3.22 for ammonium oxidation to nitrite and 1.11 for converting nitrite to nitrate). This represents the N-BOD (Section 13.1.3).

Consider typical sewage (Table 13.4), with a BOD of 200 mg/L and a reduced nitrogen content of 30 mg/L (half organic-N and half ammonium-N). During secondary treatment (biological treatment required under federal law; see Chapter 16), the oxygen required to reduce the BOD to 20 mg/L (90% removal) is 180 mg/L. For the nitrogen, typically the organic-N is converted to ammonium, and about 5 mg/L is assimilated, leaving 25 mg/L. If this remaining nitrogen is oxidized, it would require 25 x 4.33 = 108 mg/L of oxygen, an increase of 60% of the original carbonaceous demand. Some industrial wastewaters may have much higher ammonium concentrations (even 1000s of mg/L) and thus represent even greater N-BODs.

TABLE 13.4 Typical Carbonaceous and Nitrogenous Biochemical Oxygen Demand of Treated3 and Untreated Sewage

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