Nitrogen cycle

Despite the Very Large Amount of Combined Nitrogen Compounds present in the biosphere, lithosphere, and hydrosphere, over 99.9% of all global nitrogen is in the atmosphere in the form of dinitrogen. A small amount of this nitrogen gas is converted to oxides during electrical storms in the atmosphere and is washed out in rainfall. However, most transformations of nitrogen are catalyzed by microorganisms, which are thus of critical importance in the control of nitrogen availability for the growth of crop plants, in the treatment of waste, and in the conversion to forms that are leachable and result in contamination of groundwaters. 

I. INTRODUCTION Some aspects of the biochemistry and microbiology of each nitrogen cycle process are presented here, followed by ecological and environmental implications. The most reduced forms of nitrogen are the organic nitrogen compounds, such as amino acids, and the first product of their decomposition, ammonia (NH_ 4 or NH3) (Fig. 59.1). The most oxidized form is nitrate (NO_ 3), and between ammonia and nitrate are compounds of different oxidation states: the gases dinitrogen (N2), nitrous oxide (N2O), nitrogen monoxide (NO or nitric oxide), and nitrogen dioxide (NO2); and the nonvolatile hydroxylamine (NH2OH) and nitrite (NO_ 2). Oxidative reactions (leading to the right in Fig. 59.1) tend to occur under aerobic (oxygenated) conditions, and reductive reactions leading to the left occur mostly under low-oxygen or anaerobic conditions. Environmental conditions dictate the type and abundance of microorganisms occurring in nature and they thus can critically control the kinds of nitrogen transformations that are likely to occur. 

II. DINITROGEN FIXATION A. Biochemistry and microbiology The ability to fix (assimilate) gaseous nitrogen is restricted to the archaea and the eubacteria (together, the prokaryotes) and is found in higher life forms (eukaryotes) only in association with N2-fixing (diazotrophic) bacteria. The fixation of dinitrogen is catalyzed by an enzyme complex, nitrogenase, consisting of two subunits of dinitrogenase reductase (an iron protein), two pairs of subunits of dinitrogenase (an iron–molybdenum enzyme), and an iron–molydbdenum cofactor (FeMoCo). Dinitrogenase reductase uses ATP as the energy source to transfer energized electrons to the dinitrogenase enzyme. The very strongly triplebonded molecule of dinitrogen is successively reduced on the surface of the FeMoCo cofactor, yielding two molecules of ammonia as the first detectable product. The ammonia is then used in the synthesis of amino acids and proteins (Section III). During dinitrogen reduction, some electrons and ATP are used to reduce protons to dihydrogen (H2), a wasteful process. The overall stoichiometry of these reactions is N2_8e__8H__16ATP → 2NH3 _16ADP_16Pi_H2 Some (mainly aerobic) bacteria have adapted by using a recycling (or uptake) hydrogenase to reoxidize the H2, returning electrons to the electron-transport chain and regaining valuable ATP. Nevertheless, nitrogen fixation remains a very energy-expensive process and the availability of energy and reducing power is frequently a major limiting factor. Another limiting factor is the presence of oxygen (O2). Because the nitrogenase complex is extremely sensitive to O2 inactivation, it is active only in cells or environments with very low O2 concentrations. One species of Azotobacter possesses a third protein in the nitrogenase complex, the FeS II or Shethna protein, which affords some protection from O2 inactivation. An interesting property of the nitrogenase complex is that in addition to being able to reduce dinitrogen to ammonia, it can also reduce a number of other small molecules that are somewhat similar in size and conformation to N2. 

For example, it was discovered in the late 1960s that nitrogenase could reduce acetylene to ethylene (H–C≡C–H to H2C_CH2) and this reaction provided a very simple, inexpensive, and very sensitive assay for nitrogenase activity that could be applied to systems varying in complexity from enzyme preparations to soil samples. The sample to be assayed is exposed to C2H2 and the product C2H4 is readily detected by gas chroma-tography. In addition, nitrogenase reduces hydrogen cyanide (H–C≡N), hydrogen azide (H•N_•N_•N), nitrous oxide (N•N_•O_), and other compounds. All of these reactions consume ATP, so the reduction of N2O by nitrogenase is different from the reaction catalyzed by the N2O reductase of denitrifying bacteria, which contributes to ATP synthesis (Section V.A), and which, interestingly, is inhibited by (but does not reduce) C2H2. About 20 catalytic proteins, cofactors, and other components are necessary for N2 fixation. They are coded for by genes (nif ) that may be tightly clustered, as in Klebsiella pneumoniae (Fig. 59.2), or scattered throughout the bacterial chromosome, or even present on very large megaplasmids (as in some of the rhizobia that infect the roots of legumes and cause the formation of N2-fixing root nodules). The nif system is tightly regulated to conserve resources for the cell, and it is repressed by both O2 and NH_ 4. Microorganisms possessing nif genes and able to fix N2 are taxonomically and phylogenetically very diverse, but because the nitrogenase structural genes are highly conserved, some believe that the process is very ancient in the evolutionary sense, and that it may have evolved during an early geological period of decreased lightning that resulted in a nitrogen limitation. Table 59.1 shows some selected N2-fixing genera in major phylogenetic groups, indicating the very varied ecological types represented. It should be noted that two other, rather rare, nitrogenases are known, one containing vanadium instead of molybdenum, and the other containing only iron. They are produced only in molybdenum-deficient environments and their ecological significance is not clear. B. Ecology of N2 fixation The very high ATP requirement and the high O2 sensitivity of the nitrogenase system means that N2-fixing organisms are active only in certain environments. 

1. Environments with high C and energy availability, such as readily decomposable high-C organic matter, high-C root exudates (e.g. N2 fixers associated with sugar cane) or photosynthate (e.g. cyanobacteria, and root nodules formed on legumes or other plants). 2. Cells or environments with low O2 concentrations. Anaerobes such as Clostridium spp. naturally must avoid O2 to grow, whether the environment is nitrogen-deficient (i.e. they need to fix N2) or not. Facultative anaerobes such as Klebsiella spp. fix N2 only when O2 is absent, but some obligate aerobes such as Azotobacter can fix N2 under aerobic conditions by using a type of “respiratory protection” in which they develop a special branch of their electron-transport chain that permits them to consume O2 (and therefore also organic carbon) at a high rate to maintain reducing conditions in the cytoplasm where the nitrogenase is located. Indeed, Azotobacter has the highest respiration rate per unit cell mass of any known organism. Many free-living diazotrophs have no specific mechanisms of O2 protection. When they must fix N2 they become microaerophilic and grow only in environments having O2 concentrations less than about onetwentieth of that in the atmosphere, that is, 2.25_M dissolved O2. Such bacteria are members of the genera Acetobacter, Azospirillum, Aquaspirillum, Gluconacetobacter, Magnetospirillum, Pseudomonas, and the diazotrophic methanotrophs. Some of these organisms are found inhabiting the rhizosphere of many plants, including rice. A very unusual diazotrophic Streptomyces actinomycete was reported to use CO as carbon and energy source, but did not reduce acetylene and its DNA did not hybridize with molecular probes for the highly conserved nifH and nifDK genes. Cyanobacteria such as Anabaena produce specialized cells called heterocysts about every 10 or 12 cells in a chain of photosynthetic cells. Such heterocysts lack the O2-producing photosystem II and support nitrogenase with carbohydrate translocated from the neighboring photosynthetic cells. 

Most Anabaena and related spp. are active in the free-living state in rice paddies and other aquatic systems. However, an Anabaena sp. lives symbiotically in specialized cavities in the fronds of the aquatic fern Azolla, which can contribute large amounts of fixed nitrogen in rice paddies. Other cyanobacteria do not develop heterocysts. Species of Trichodesmium are filamentous and fix N2 only in the center of colonial aggregates where O2-producing photosynthesis does not occur. They are significant in, for example, the N-deficient Caribbean Sea, where, however, wave action can disrupt aggregates and allow O2 to inhibit N2 fixation. The single-celled Gloeocapsa has evolved another strategy in which it photosynthesizes during the day and fixes N2 at night. Yet other cyanobacteria, with fungi, form symbioses called lichens that have a great capacity to colonize rocks and other inhospitable substrates. They are therefore important primary colonizers in nature, accumulating organic matter and allowing other forms of life (such as mosses, ferns, and higher plants) to become established. Rhizobia of the genera Rhizobium, Bradyrhizobium, and Sinorhizobium are soil bacteria that can infect the roots of plants of the leguminosae (alfalfa, peas, beans, vetches, and soybean). Others can lead to the formation of stem nodules on some tropical plants such as Sesbania. To initiate infection, they attach to root hairs, exchange signal molecules with the plant, pass into the root-hair cell by reorientating its growth and causing an invagination of the cell wall, and promote the synthesis of an infection thread that infects cells in the root cortex. 

Bacteria are released from this thread and differentiate into specialized cells called bacteroids, in which the nif and associated fix genes that support nodule N2 fixation are derepressed. The bacteroids use plant dicarboxylic acids to fuel the reduction of N2 to ammonia, which is converted to amino acids or ureides, mainly by host enzymes, for transportation to the rest of the plant. Oxygen is required by the bacteroids and is supplied at a very low free concentration but at a high flux by leghemoglobin, a carrier having a very high affinity for O2. The leghemoglobin comprises an apoprotein coded for by the plant and a heme moiety coded for and synthesized by the bacteroids. Nodules formed on the roots of nonlegumes are called actinorhiza. The causal agents are species or strains of the genus Frankia, filamentous actinomycetes that can infect often commercially important trees and shrubs of genera such as the temperate Alnus (alder) and the semi-tropical Casuarina (horsetail pine). Such genera are important ecologically as colonizers of nutrient-poor soils and moraines, and are used for soil stabilization and windbreak purposes. 

III. ASSIMILATION AND AMMONIFICATION Most microbes in nature can not fix N2 and must obtain their nitrogen supply in the form of ammonia (NH_ 4), nitrate (NO_ 3), or free amino acids. Ammonia is assimilated by the glutamate dehydrogenase or (in N-deficiency) the glutamine synthetase-glutamate synthase (GS-GOGAT) system. Nitrate is reduced by assimilatory nitrate and nitrite reductases to NH_ 4 (Fig. 59.1), a system that is repressed by NH_ 4, but is unaffected by O2. Growth may be limited by C or N availability, and in agricultural soils supplied with residues having a high ratio of C to N, such as straw, the N-starved microorganisms may be serious competitors with the plant roots for available nitrogen. This suggests the advisability of reducing the C content by composting plant material before its application to soils. Nitrogen tied up in microbial biomass components is released only on death or lysis of the cells. Other microbes then produce a variety of proteolytic enzymes and deaminases that degrade proteins, as well as other enzyme systems that attack nucleic acids and wall components. The major ultimate product of these reactions is NH_ 4 and the process is termed ammonification or mineralization (Fig. 59.1). The ecological implication of the assimilation and ammonification reactions is that the microbial biomass in terrestrial and aquatic systems is in a state of turnover, with the two processes often being in a steady state such that there is no marked change in the concentration of NH_ 4. Perturbations of the ecosystem can upset the steady state, high C inputs resulting in net assimilation and high N inputs resulting in net ammonification. A great variety of microbes carry out these two processes and activity is likely in both aerobic and anaerobic environments. IV. NITRIFICATION The oxidation of NH_ 4 through NO_ 2 to NO_ 3 is carried out mainly by two highly specialized groups of lithotrophic bacteria that use the oxidation reactions as their sole source of energy and reducing power to fix CO2 to the level of cellular organic C components. They can thus grow in completely inorganic environments, providing NH_ 4, CO2, and O2 are available. They are of great environmental importance because they convert the relatively immobile NH_ 4 to the anionic NO_ 3 which is mobile; can be leached into lakes, rivers, or groundwaters; and is a major substrate for denitrification. 

The nitrifying bacteria are also of significance in aerobic secondary sewage treatment, in which NH_ 4 from ammonification is converted to NO_ 3. NO_ 3 in drinking water obtained from contaminated lakes, rivers, or aquifers can be reduced to NO_ 2 in the human gut and cause the conversion of hemoglobin to methemoglobin, with a great loss in O2-carrying capacity. This condition is referred to as methemoglobinemia (in infants, called blue babies). The NO_ 2 can also react with amines forming carcinogenic nitrosamines. A. Biochemistry and microbiology 1. NH_ 4 oxidation The oxidation of ammonia (NH3 is the actual substrate) is catalyzed by an ammonia monooxygenase (AMO) in an inner membrane. O2 is an obligate requirement because one of the atoms is incorporated into the substrate, the other is reduced to water, and hydroxylamine (NH2OH) is produced. Reducing power is supplied from the ubiquinone pool (Fig. 59.3). The NH2OH is oxidized to NO_ 2 by means of a hydroxylamine oxidoreductase that donates the electrons released to a cytochrome 554, and then through a cytochrome 552 to the terminal oxidase that reduces O2 to water on the inner face of the inner membrane (Fig. 59.3). The overall reactions are NH3_O2_2H__2e_ → NH2OH_H2O NH2OH_H2O → NO_ 2 _5 H__4e_ The redox reactions in the membrane are associated with the extrusion of protons into the periplasm, thus creating a proton-motive force and supporting ATP synthesis. ATP and reducing power are needed for growth and fixation of CO2 by the Calvin cycle using ribulosebisphosphate carboxylase-oxygenase (RuBisCO). The ammonia monooxygenase genes (amo) show significant homology with those of methane monooxygenase (pMMO), providing some evidence that the genes may be evolutionarily related. During the oxidation of NH_ 4, small amounts (about 0.3% of the N oxidized) of NO and N2O are produced, the amounts increasing in O2 deficiency to 10% or more of the N oxidized. It is known that the NO is produced from NO2 _ by a copper-containing nitrite reductase (CuNir, Fig. 59.3), but the mechanism by which N2O and, in a few cases, small amounts of N2 are produced is not known. An NO reductase has not been isolated or characterized from these organisms. It was recently shown that NO (or NO2) is required for effective oxidation of ammonia. Thus, air sparging of a Nitrosomonas eutropha culture can reduce NO concentration and inhibit growth. An NO-binding agent also inhibits growth. The mechanism of action of NO and NO2 is not known. 

In Nitrosomonas sp., the genes for AMO, amoA and amoB, coding for the two subunits, are duplicated, there are three copies of hao, and some of the cytochrome genes are present in more than one copy. There is evidence that these genes are more scattered on the chromosome than are those of the N2 fixation and denitrification systems. Ammonia-oxidizing bacteria are limited to six genera that are members of the beta subdivision of the Proteobacteria, except for Nitrosococcus oceanus, which is in the gamma subdivision (Table 59.2). Some are found commonly in soils; others in aquatic environments. Many have arrays of cytomembranes arising from infoldings of the cytoplasmic membrane and these are believed to contain important components of the NH_ 4-oxidizing system. However, much remains to be discovered about the detailed biochemistry of this system. 2. NO_ 2 oxidation The oxidation of NO_ 2 appears to be a one-step process catalyzed by a nitrite oxidoreductase, an enzyme containing iron and molybdenum that may be located in the membrane. The reaction is NO_ 2 _H2O → NO_ 3 _2H__2e_ It is not yet clear how a proton-motive force is generated for the support of ATP synthesis. The nitrite oxidoreductase can operate in the reductive direction and some strains of NO_ 2 oxidizers are reported to be able to grow anaerobically. However, both the NO_ 2 oxidizers and the NH_ 4 oxidizers are essentially aerobic organisms, requiring O2 for growth and activity. The fixation of CO2 is by means of ribulose bisphosphate carboxylase-oxygenase.   Nitrite-oxidizing bacteria are confined to four genera (Table 59.2) in the alpha subdivision of the Proteobacteria. 

All are lithotrophic but some can grow mixotrophically (that is, they can grow using a combination of lithotrophic and heterotrophic pathways) and one, Nitrobacter hamburgensis, can grow heterotrophically on organic carbon alone. B. Ecology of nitrification The obligatory requirement of the nitrifiers for O2 means that nitrification occurs only in aerobic environments. Ammonia must come from ammonification, either in the same location or by diffusion from neighboring anaerobic environments where excess NH_ 4 accumulates (Fig. 59.4). Nitrification provides NO_ 3, the most important nitrogen source for plant growth, and occurs readily in most agricultural and other soils, especially at near-neutral pH values, and the production by nitrifying bacteria of NO and smaller amounts of N2O makes such environments important global sources of these trace gases. NO plays important roles in tropospheric chemistry and is a factor in acid precipitation and ozone turnover. It was recently recognized that NO from nitrification or other sources can be consumed under aerobic conditions by many heterotrophic bacteria that convert it to NO_ 3 as the major product. This process can greatly modify the net flux of NO to the atmosphere. N2O is radiatively active, absorbing infrared radiation from Earth and thus acting as a greenhouse gas. When N2O diffuses to the stratosphere, it is converted photochemically to NO, which then catalyzes the conversion of ozone to O2. Based on the behavior of nitrifying bacteria in pure culture, it was believed that nitrification would not occur in environments with pH values 2–6.5. However, it is now known that at least some of the nitrification that can occur in acidic forest soils is brought about by NH_ 4 oxidizers such as Nitrosospira spp. growing in colonial aggregates that are surrounded by less acid-sensitive NO_ 2 oxidizers. Such aggregates are observed in aquaculture and other aquatic systems in which nitrification is an important process reducing the potential problem of ammonia toxicity and providing the denitrification substrate NO_ 3. In agriculture, the potential losses of fertilizer nitrogen through leaching or denitrification of the nitrification product NO_ 3 is considered to be a serious problem. A number of “nitrification inhibitors,” such as nitrapyrin (sold as N-Serve by the Dow Chemical Company) are commercially available and usually specifically inhibit the ammonia monooxygenase enzyme, thus preventing the conversion of anhydrous ammonia (NH3) or cationic NH_ 4 fertilizers to the leachable and denitrifyable anion NO_ 3. Sewage treatment and certain other waste-treatment processes promote nitrification in a secondary activated sludge reactor in which biologically available organic carbon (often monitored as BOD, biochemical oxygen demand) is mineralized and converted to microbial biomass and CO2. The NO_ 3 that is produced, if fed into a river or lake as receiving water, can stimulate eutrophication and the growth of algae and macrophytes (aquatic higher plants), so it is preferably subject to tertiary or nitrogen-removal treatment in which denitrification is encouraged (Section V). 

C. Heterotrophic nitrification Although it is clear that most of the global nitrification is catalyzed by the lithotrophic nitrifiers, there are some heterotrophs that can produce NO_ 2 and NO_ 3 from reduced nitrogen compounds or from ammonia by mechanisms that are not yet clear. RNH2 → RNHOH → RNO → RNO2 → NO_ 3 NH_ 4 → NH2OH → NOH → NO_ 2 → NO_ 3 In the heterotrophic nitrifier–denitrifier Thiosphaera pantotropha (recently reclassified as a strain of Paracoccus denitrificans), there do not appear to be genes homologous to the amo or hao of lithotrophic nitrifiers, so the nitrification mechanism would seem to be different. Other heterotrophic nitrifiers include fungi such as Aspergillus flavus, relatively acid insensitive and perhaps responsible for much of the nitrification in acidic forest soils, or bacteria such as Arthrobacter sp. However, there is no evidence that any of these organisms can conserve energy from the oxidations that they catalyze, and so the evolutionary significance of this process is not known. It is difficult to distinguish between lithotrophic and heterotrophic nitrification activity, but in laboratory experiments, use can be made of the fact that acetylene (in low ppmv concentrations) inhibits the Amo of the former but not the oxidation system of the latter, and chlorate inhibits lithotrophic NO_ 2 oxidation but apparently not heterotrophic NO_ 3 production. V. DENITRIFICATION When oxygen becomes limiting, some microorganisms, mainly aerobic bacteria, have the ability to switch to the use of the nitrogen oxides NO_ 3, NO_ 2, NO, and N2O as terminal acceptors of electrons in their metabolism. This process is known as denitrification, and it permits organisms to continue what is essentially a form of aerobic respiration in which the end product is dinitrogen. However, intermediates sometimes accumulate. Denitrification is of major importance because it closes the global nitrogen cycle, maintaining a balance in atmospheric dinitrogen; it is responsible for significant losses of fertilizer nitrogen in agriculture; it is a critical process in the nitrogenremoval component of modern tertiary wastewatertreatment plants; leakage of the intermediate N2O adds to the greenhouse gas load of the troposphere and also indirectly causes catalytic destruction of stratospheric ozone; and it is possible that the longterm accumulation of nitrous oxide in closed systems may cause human or animal health problems. 

A. Biochemistry and microbiology Most of the denitrifying organisms of significance in nature are bacteria, and they have an aerobic type of electron-transport (cytochrome) chain leading to O2 as the terminal electron acceptor. However, when O2 is deficient or absent, a cellular regulator (Anr or Fnr) switches on the synthesis of a series of reductase enzymes that successively reduce NO_ 3, NO_ 2, NO, and N2O to N2. This type of respiration is less efficient than O2 respiration, and so growth of the microorganisms is slower under anaerobic conditions. The NO_ 3 reductase (Nar) is a membrane-bound molybdo-enzyme with the active site on the inner face of the membrane (Fig. 59.5). Also, a soluble NO_ 3 reductase enzyme (Nap) is located in the periplasm (between the inner and outer membranes) of some gram-negative bacteria, and probably enables the organism to adapt to rapid onset of anaerobiosis. NO_ 2 reductases (Nir) are of two types, copper-containing and cytochrome cd1-containing, and are periplasmic. NO reductase (Nor) is membrane-bound, and N2O reductases (Nos) are also mostly periplasmic Cu enzymes, except in the gliding bacterium Flexibacter canadensis, in which it is in the membrane. Nitrate is the major N oxide substrate for denitrification. It is taken up through the inner membrane by means of an antiport (at least in some bacteria) that is O2 sensitive, and thus NO_ 3 uptake is inhibited by O2. The NO_ 2 product from reduction of NO_ 3 exits via the NO_ 3 –NO_ 2 antiport and undergoes successive reduction by the periplasmic NO_ 2 reductase (Nir), the membrane-bound NO reductase (Nor), and the periplasmic N2O reductase (Nos) (Fig. 59.5). Electrons for these reductions are supplied via NADH dehydrogenase, the ubiquinone pool, a cytochrome bc1 complex in the inner membrane, and other cytochromes in the periplasm (not shown in Fig. 59.5). These membrane redox reactions result in the extrusion of protons (H_) into the periplasm and the generation of a proton motive force that allows the cell to make ATP. The genes encoding the denitrification system are generally chromosomal and are tightly linked, as shown in Fig. 59.6 for Pseudomonas stutzeri. The NO_ 2 and NO reductase genes, nir and nor, are coregulated, presumably to avoid the possible toxicity of NO, because NO could accumulate if Nor activity was deficient. Both low O2 and the presence of a nitrogen oxide are necessary for the transcription of the denitrification genes. 

Organisms exist that possess only certain subsets of the four reductases; thus the nitrogen oxide substrates used and the final products can vary with the species (Fig. 59.7). Phylogenetically, bacteria that are able to denitrify are found widely distributed in very diverse groups (Table 59.3), and this has led to much speculation about the evolutionary significance of this distribution.  Afew fungi (for example, Fusarium oxysporum strains) were recently shown to denitrify, and they produce N oxide reductases with some similarities to those in bacteria. Indeed, a dissimilatory NO_ 3 reductase (DNar) is found in the mitochondria of F. oxysporum. However, there is no evidence that such fungi are important denitrifiers in nature. Most denitrifiers are typical heterotrophs, such as Pseudomonas, Alcaligenes, and Ralstonia spp., using organic carbon as a source of reducing power. B. Ecology of denitrification 1. Primary controlling factors Denitrifiers are ubiquitous, growing best aerobically, so some potential for denitrification exists in many habitats. The actual activity depends on three primary factors—limitation of O2, availability of N oxides, and availability of reductant (mainly as organic carbon, but some bacteria can use H2 or sulfide). a. Oxygen Depletion of O2 occurs in environments in which its consumption by biological activity is greater than its rate of supply by mass transport or diffusion. Thus, aquatic sediments, waterlogged or irrigated soils, and the centers of large water-saturated soil aggregates are some of the habitats in which the genes for N oxide reductases of denitrifiers will be derepressed and the enzymes synthesized. If respiratory activity is high and O2 is supplied by molecular diffusion (as in biofilms), the gradients of O2 concentration can be very steep, conditions becoming nearly anaerobic within hundreds of micrometers of an air-saturated boundary layer. Although O2 represses the synthesis of the N oxide reductases and inhibits the activity of the enzymes, some denitrifying systems are reported to be relatively insensitive to O2. However, studies using microelectrodes have shown significant denitrification only in the anaerobic or nearly anaerobic zone. The N2O reductase is often reported to be the most sensitive to O2, so exposure of a denitrifying system to O2 may result in the release of N2O rather than N2. Indeed, the Nos enzyme seems to be generally more sensitive than the other reductases to unfavorable conditions. Thus, low pH values and inhibitory components in the environment can cause the release of N2O. 

The marked inhibition of N2O reduction by acetylene, coupled with the measurement of N2O accumulation by gas chromatography, is used as a sensitive assay for denitrification.   b. N oxides In environments in which O2 depletion occurs, the denitrifiers become energy limited unless reducible N oxides are present. Nitrification is ultimately the major source of these oxides, but they also come from precipitation and fertilizers. As mentioned in Section IV.B, nitrification, the oxidation of NH_ 4 to NO_ 2 and NO_ 3, is an obligately aerobic process that often occurs close to denitrification zones, on opposite sides of aerobic–anaerobic interfaces, and so these processes are frequently tightly coupled (Fig. 59.4). Such interfaces can occur in soils, but they are very important in aquatic sediments, where both the sediment and the water column may be sources of NO_ 3. The relative importance of sediment nitrification and the water column in providing NO_ 3 for denitrification may depend on the depth of O2 penetration, and thus on the steepness of the NO_ 3 diffusion gradient from the surface to the zone of denitrification. Sediment nitrification is likely to be the major source of NO_ 3 for denitrification in cases in which O2 penetrates deeply, the NO_ 3 gradient from the surface to the interface is less steep, and the nitrification potential occurs close to the aerobic–anaerobic interface. c. Reductants In environments with low organic-matter content, the supply of electron donors (and thus energy) may be a critical factor. Denitrification capacity may be highly correlated with water-soluble soil organic carbon, and denitrifiers are reported that degrade various aromatic compounds anaerobically. Such bacteria are of interest in the bioremediation of contaminated environments. Other bacteria can use dihydrogen, elemental sulfur, or oxidizable sulfur compounds as electron donors, but it is not known whether this is of significance in nature. 2. Accumulation of intermediates and effects of plants Transient nitrite accumulation occurs, at least in the cultures of some denitrifiers, when NO_ 3 concentrations above about 0.3 mM inhibit the reduction of NO_ 2. The NO_ 2 is reduced once the NO_ 3 concentration decreases sufficiently. As mentioned earlier, O2 exposure and low pH values can cause the escape of N2O from a denitrifying system, but the escape of NO appears to be minimal because of the very high apparent affinity of NO reductase for NO. Thus, denitrification is not as significant a contributor of NO to the atmosphere as nitrification. Active plant roots are strong sinks for O2 and NO_ 3 and are sources of oxidizable organic carbon released in exudates. 

The roots of aquatic macrophytes act as sources of O2. Therefore, in either case, they have the potential to both stimulate and inhibit denitrification in the rhizosphere. The actual outcome seems to depend on the availability of NO_ 3 and carbon in the O2-depleted part of the rhizosphere soil. 3. Dissimilatory reduction of nitrate to ammonia (nitrate ammonification) In highly reducing habitats, such as sludges and the rumen, where the ratio of available organic carbon to electron acceptor (NO_ 3) is high, the NO_ 3 is reduced not by denitrification to gaseous products but via specific NO_ 3 and NO_ 2 reductases to NH_ 4 (Fig. 59.1). Both enzymes are soluble and cytoplasmic, and the process acts mainly as an electron sink. It is found in some fermentative organisms such as E. coli and some species of Clostridium. VI. SUMMARY AND GLOBAL IMPLICATIONS The microorganisms involved in the many N-cycle processes respond to changes in their environment and in the availability of their substrates. Human influences, especially since the 1930s, have greatly changed both environments and substrates. Increases in biological N2 fixation in legume crops and in chemical production of inorganic fertilizers have roughly doubled the total N inputs to the terrestrial N cycle. The retention of N has not been sufficient to prevent losses of inorganic N and other nutrients to rivers, lakes, and oceans, and it is now known that riparian ecosystems such as wetlands or forests along shorelines are important in restricting (through assimilation and denitrification) such input of NO_ 3 to rivers and lakes. 

The conversion of N forms has increased volatile losses of NH3, NO, and N2O to the atmosphere, and the long-range transport and deposition of these molecules or their oxidation products. This has resulted in the “N-saturation” of many previously N-limited natural terrestrial and aquatic ecosystems, with consequent changes in flora, decreased diversity, and increased acidity both from rainfall of nitric acid (from the oxidation of NO) and from the nitrification of NH_ 4. Other effects on the atmosphere itself include the contribution of NO to photochemical smog and of N2O to both the greenhouse effect and the destruction of stratospheric ozone.