Part 2, Section 2

Microbial Transformations of Nitrogen

I. Overview

II. Biological Nitrogen Fixation (BNF)

III. Organic N dynamics (N ammonification and immobilization)

IV. Nitrification

V. Nitrate reduction

I. Overview Top

The two largest N pools are contained in the lithosphere and atmosphere (Table 1). The lithosphere N is primarily N₂ entrapped in igneous rocks and is essentially inactive as far as biogeochemical cycling is concerned. Thus, the largest biogeochemically active N pool is the atmosphere, which is 79% N₂ by volume.
In soils, most of the N is contained in organic forms within the soil organic matter; the mineral N (NH₄⁺, NO₃^-) pool is relatively small (Tables 1, 2).
Practically all of the N in the mineral and organic pools originates from atmospheric N₂ and is formed through "nitrogen fixation", which is any process in which elemental N (N₂) reacts to form a compound (FIG. 1).
N fixation is naturally mediated by biotic and abiotic mechanisms. The primary abiotic process occurs in the atmosphere when lightning catalyzes a chain reaction between N₂ and O₂ that results in the formation of N oxides (FIG. 1). The amounts of N fixed by this process, however, are small compared to that produced biologically by microorganisms. Thus, microbes play the main role in initiating the N cycle by transforming atmospheric N₂ into a form accessible to plants and all other organisms.
The N fixed by microbes is immediately converted to an organic form and assimilated into biomass. In the next step of the N cycle ("mineralization") the biomass dies, the organic compounds are degraded, and the N is released in a mineral form (NH₃). Ammonia ("ammonium") can then be assimilated back into biomass, or utilized as an energy source by bacteria that sequentially oxidize it to NO₂^- and NO₃^- ("nitrification"). To complete the cycle, microbes may use NO₃^- as an electron acceptor and reducing back to N₂ (denitrification).

II. Biological Nitrogen Fixation (BNF)

Diazotrophy is an ancient biological activity, and is found among members of the Bacteria and Archaea (FIG. 2A,B). In the latter group diazotrophy has been most extensively documented in the methanogens. BNF is not an obligatory function needed to sustain organisms' growth.

Absent from the list are Ecarya that in and of themselves can mediate BNF. Some Ecarya may still participate in the process through symbioses with diazotrophs. Examples are lichens (fungi with cyanobacteria), plants (legumes with rhizobia; "actinorhizal" plants with certain actinomycetes).

The diverse collection of diazotrophs includes aerobic and anaerobic organisms growing as heterotrophs and phototrophs. Use of inorganic electron donors (Fe⁺², H₂) has also been demonstrated in aerobic laboratory cultures. However, the environmental significance of free-living, aerobic diazo-litho-trophs is unknown.

B. Biochemistry of BNF

The triple bonded N₂ molecule is extremely stable and exceedingly difficult to break under ambient conditions.
For industrial production, NH₃ is formed from N₂ by using high pressures 200 atm (3,000 pounds per square inch) and high temperatures >400 °C (750 °F). To accomplish the same feat at ambient conditions, microbes utilize the enzyme nitrogenase.
Nitrogenase ("Nase") is a multicomponent enzyme, the two main constituents of which are dinitrogenase (aka, Nase I) and dinitrogenase reductase (aka, Nase II).
The biochemical feat that Nase achieves is the generation of the strong reductant (high energy electrons) needed to overcome the energetically unfavorable first step of breaking the triple bond and reducing N₂ to N₂H₂ (FIG. 3)
To accomplish this, Nase II transfers electrons one at a time to Nase I, each electron is energized during this transfer by coupling to hydrolysis of 2 ATPs. Nase I holds these high energy electrons, and after two are accumulated they are transferred to N₂.
Nase is also capable of binding substrates other than N₂ at other sites on the enzyme and reducing these as well (Table 3). One of the most important is H⁺. This is because the enzyme functions in an aqueous environment and so is always exposed to H⁺ (from water). Thus, H⁺ reduction to H₂ is a consistent side reaction in N₂.
Proton reduction represents a loss of energy (ca. 25% of ATP consumed). Some of the wasted energy may be recaptured by organisms that posses an "uptake hydrogenase" (aka, "Hup"). The Hup system catalyzes the release of electrons (H₂ -> 2 H⁺ + 2e-) that may then be recycled to generate ATP or reduce N₂.
The overall reaction for N₂ reduction is:

N₂ + 8H⁺ + 8e- +16 ATP -- Nase--> 2NH₃ + H₂ + 16ADP

₂

₃

Nase is highly susceptible to inactivation by O₂. Consequently, there must be mechanisms for protecting the enzyme in aerobic diazotrophs.
In one approach, called "conformational protection", a "redox" protein binds to Nase and causes it to change shape (conformation) so that O₂^-sensitive areas are protected. The protected form of Nase is inactive, but if O₂ levels drop the redox protein is released and Nase regains activity. Another strategy is "respiratory protection" wherein additional electron transport systems are developed to support high rates of oxygen reduction. Bacteria may also form barriers of cell clumps and/or extracellular polymers that serve to restrict oxygen diffusion ("diffusion protection").

C. Microbiology of diazotrophs

1. Free-living heterotrophs

Many bacteria (and probably archaeons) are capable of BNF in a free-living state and are widespread in aerobic and anaerobic environments. Although common constituents of microbial communities they are generally not very numerous (10² to 10³ g^-1) under typical conditions. Numbers may increase substantially (e.g., >10⁷ g^-1) when fresh organic residues are supplied to the soil.
The high cost of BNF and limited energy supplies available to free-living organisms results in relatively low amounts of N fixation compared to associative and symbiotic N fixers (TABLE 4).
Factors affecting N fixation by free-living diazotrophs are:

Energy supply: This is the single greatest factor affecting the level of BNF considering the high energy demand coupled with the characteristically energy-poor condition of soils.

Energy generation mechanism: Aerobic/anaerobic respiration generate greater amounts of energy than fermentation. The latter thus have less energy to power BNF.

O₂ level: Exposure to high levels of O₂ requires aerobic diazotrophs to protect Nase from inactivation and/or reduce Nase expression; both result in decreased efficiency of BNF. While, obligate and facultative anaerobes are not burdened with this problem any potential benefit for BNF is counterbalanced by the lower amount of energy generated (TABLE 5).

Availability of fixed N: NH₄⁺ levels in excess of available carbon may suppress BNF as the organisms will not expend energy reserves needlessly.

a.) Plant-associated (associative) forms

Associative diazotrophs preferentially colonize plant root surfaces (rhizoplane), soil surrounding plant roots (rhizosphere), leaf surfaces (phyllosphere), or even internal vascular regions.
Associative colonizing root surfaces are distinguished from symbiotic forms in that there is no structural differentiation associated with BNF by either the plant or bacteria (i.e., neither forms special structures that are needed for or enhance BNF).
Types of bacteria commonly identified as associative diazotrophs are the genus Azospirillim and a variety of genera in the Enterobacteria group (i.e., Klebsiella, Enterobacter).
The limitations on BNF for associative diazotrophs are similar to those of free-living forms. The primary advantage for associative types is that close proximity to the plant may lessen somewhat the energy source limitations. However, the diazotrophs must also compete with a multitude of other heterotrophs in the rhizoplane/sphere for this energy.
On balance the amounts of N fixed by associative forms is greater than that of free-living diazotrophs but relatively small compared to that produced through symbiotic process (Table 4).
Thus, the relative importance of associative BNF for any given ecosystem depends on whether or not plants forming symbiotic BNF associations are abundant. For example, in grasslands that have few inputs of fixed N other than that produced associative these relatively small amounts may be significant.

2. Symbiotic forms

BNF through symbiotic associations produces the greatest amount of fixed N (Table 4). This has been estimated to be as much as 100 million tons y^-1 in soils, and account for 65% of the N used in agriculture.
The symbiotic association responsible for this N input is that between legumes and bacteria belonging the rhizobia group. Also agriculturally important is the symbiosis between waterferns and the cyanobacteria, which produces much of the N used in rice production. We'll discuss this later in conjunction with phototrophic diazotrophs.
Another widespread BNF symbiosis is that between Gram-positive bacteria of the genus Frankia and a wide variety of "actinorhizal" plants; as a group the latter have little more in common than the symbiotic association with Frankia.
Basic features of all symbioses are that: 1.) both partners benefit, 2.) the interaction is specific and requires mutual recognition, and 3.) following recognition one or both partners undergo structural/physiological differentiation to facilitate the symbiosis.

a.) Legume-rhizobia symbioses

i.) Nodule development and function

Rhizobia in soil attach to root hairs and elicit curling to "shepards crook" (FIG. 4A). Rhizobia begin infecting root, but don't enter plant cells per se: penetration is contained within and directed by a plant-derived "infection thread" (FIG. 4B).
The rhizobia are then released into cortical cells, differentiate into "bacteroids" and are surrounded by plant-derived peribacteroid membrane. Plant cells in the adjoining region then swell to give the structures observable as "nodules."
Bacteroids are distinct from their free-lining predecessors. Structurally, bacteroids may grow without dividing thus producing enlarged cells (sometimes with multiple chromosomes). Physiologically, bacteroids form new e- transport systems and contain up to 10% of their total protein as Nase.
Bacteroids are supplied by the plant with energy (photosynthate) and leghemoglobin, which is an oxygen carrier (gives nodules pink/red color) that helps protect Nase from O₂ inactivation. The bacteroids in turn excrete NH₃, which is then incorporated into amino acids and exported from the nodules to the rest of the plant (FIG. 5).
The bacteroid state is not irreversible; bacteroids released from decaying nodules can revert to free-living forms and take up residence in soil until the next opportunity arises.

ii.) Rhizobia infectiveness and effectiveness

There are many species of rhizobia, each exhibits specificity with respect to the legume(s) it will infect and form nodules. This phenomenon is referred to as "infectiveness" and in practice is not based just on a property of the rhizobia, but rather on the outcome of a series of biochemical signals between the plant and bacteria.
The specificity of rhizobia for infecting certain legumes had formed the basis for distinguishing between different types of Rhizobium, which was the genus name previously applied to all legume nodulators. By this system, organisms infecting a given legume comprised an "inoculation group" and the genus name of the host was used to designate the species of Rhizobium (TABLE 6). However, more detailed biochemical and genetic analyses conducted over the past 15 years has supported separation of Rhizobium into four genera (TABLE 6 ).
"Effectiveness" refers to the fact that once settled in the nodules, the amounts of N fixed by rhizobia vary between genera, between species, and even within species (i.e., between "strains").

iii.) Rhizobia ecology

In soils where a given legume has been grown, it's partner rhizobia persist at relatively low levels (ca. 10³ to 10⁴ g^-1). Typically, these indigenous rhizobia are highly infective (i.e., form many nodules per plant) but as a group are only moderately effective.
The latter characteristic of indigenous rhizobia has driven efforts to isolate and/or develop superior N fixers, which could then be inoculated into soil to enhance legume productivity. Unfortunately, this approach has yielded little success as inoculant strains typically form a minority (e.g., < 20%) of the nodules on a plant, and thus do not significantly increase it's N supplies.
The poor performance of inoculants in nodulating their hosts reflects the fact that these strains are less "competitive" in soil than the indigenous rhizobia. Competitiveness is an ill-defined phenomenon that likely reflects the interactions of physiological/genetic characteristics of the rhizobia with the host plant and the soil environment.

iv.) Soil factors affecting the legume-rhizobia symbiosis

Soil mineral N levels are a primary factor effecting this symbiosis. Plants and diazotrophs will utilize inorganic N from the soil in preference to obtaining N through BNF. High levels of NH₄ and/or NO₃ in relation to available carbon supplies thus tend to reduce nodulation.
Soil acidity have been widely examined as a factor limiting rhizobia survival and the nodulation process. This is important because acid soils (pH <5) are common in tropical areas where BNF may be heavily relied upon for N inputs.
Rhizobia as a group not generally tolerant of low soil pH, this may reflect Mn/Al toxicity or deficiencies of Ca, P, or Mo. However, rhizobia do occur naturally in acid soils, and these indigenous strains have not been demonstrated to be acid-tolerant. Thus, poor performance of rhizobia-legume symbiosis in acid soils probably attributable to a combination of soil properties effecting both the plant and bacteria.

3. Phototrophic diazotrophs

At the most general level phototrophic diazotrophs can be categorized as to whether they grow as aerobes or anaerobes. Organisms comprising the latter group belong to the order Rhodospirillales (example genera: Rhodospirillum, Chlorobium), carry out anoxygenic photosynthesis, and colonize shallow anoxic waters/sediments exposed to sunlight.
Attention here will focus on the aerobic group, which is comprised of the cyanobacteria (aka blue-green algae). Cyanobacteria are similar to plants and eukaryotic algae in that they carry out oxygenic photosynthesis. Three billion years ago, cyanobacteria (or similar forms) are considered to have caused the greatest pollution event in the history of the planet; transformation of the atmosphere from anaerobic to aerobic.
Cyanobacteria are most abundant in aquatic environments but are also common inhabitants of surface soils. In either environment, they may be free-living or grow in symbiotic associations. Most, but not all, cyanobacteria are diazotrophic (FIG. 6)

In soils, lichens are a widespread symbiotic association of fungi and cyanobacteria. The unique association allows lichens to colonize barren mineral surfaces, and thus serve as pioneering organisms that facilitate further soil development processes.
An agriculturally important symbiosis in aquatic environments is that between waterferns (Azolla) and cyanobacteria (Anabaena)(FIG. 7) This symbioses provides the major N input for rice production (FIG. 8), and thus plays a critical role in providing food for much of the world's population.
As phototrophs, BNF by cyanobacteria is not limited by carbon and energy supplies as it is for heterotrophic diazotrophs. However, the unique problem cyanobacteria face is protecting the Nase complex from the oxygen they generate through photosynthesis.
Filamentous cyanobacteria resolve this dilemma by restricting N fixation to special cells called "heterocysts" (FIG. 9), which lack the oxygen-generating portion of the photosynthesis process (photosystem II). Cyanobacteria thus physically segregate energy generation from N fixation .

III. Organic N dynamics (N ammonification and immobilization) Top

Ammonification is the process in which organic N is released from biopolymers and from SOM into the mineral pool, which is accessible to all microbes and plants.
Proteins contain the greatest amount of N, other major N sources are cell wall polymers (murein, chitin) and nucleic acids (FIG. 10). In almost all N occurs in the NH₂ and is released as ammonia.
Biodegradation of N containing polymers is initiated outside the cells; monomers may be further degraded releasing NH₃ or may be taken up by the cell (Table 7). Inside the cells, monomers may be directly incorporated into new polymers, or broken down to release NH₃ for other cell uses.
Given that the SOM pool contains most of the soil N, the amount and rate at which it is released can significantly change the available mineral N pool. Ammonification rates from SOM are difficult to determine, and in the NC US these are estimated at 5% year^-1.

B. Immobilization

Immobilization is the uptake of N and assimilation in microbial biopolymers. This N is "immobilized" from the view that the N is removed from the active N pool and no longer accessible to plants.
N Immobilization occurs when the amount of carbon available for growth of microbial biomass exceeds the amount of available N. These situations may occur when residues contain a high amount of C relative to N (i.e, have high C/N ratios). The net short term effect is that microbes consume all of the N contained in the residue and may even deplete the mineral pool (FIG. 11).
An example of how C/N ratios may be used to evaluate the potential for N mineralization/immobilization is given in FIG. 12.

IV. Nitrification Top

Nitrification is the two-stage oxidation of NH₄⁺ to NO₂^-, and then NO₂^- to NO₃^-. These reactions are mediated by two distinct physiological groups of bacteria (collectively referred to as nitrifying bacteria), the ammonia-oxidizers (named "Nitroso", e.g., Nitrosomonas) and the nitrite-oxidizers (named Nitro", e.g., Nitrobacter)

Nitrosomonas NH₄⁺ + 1.5O₂ ---> NO₂^- + 2H⁺ + H₂O G'o = -14 kJ
Nitrobacter NO₂^- + 0.5O₂ ---> NO₃^- G'o = -4 kJ
Nitrifying bacteria are obligate aerobic chemolithotrophs. Single organisms that oxidize NH₄⁺ to NO₃^- are not known.
Of all the reactions and microorganisms involved in the N cycle, nitrification and nitrifying bacteria are the most specialized: All known nitrifiers are contained within the Proteobacteria group, a relatively small corner of the microbial world.

B. Biochemistry of nitrifying bacteria.

The energetics of NH₄ oxidation to NO₂ and NO₂ to NO₃ are such that organisms using these reactions to support growth are obligate aerobes, and provide the organisms with relatively small energy yields (FIG. 13).
The first step of nitrification is mediated by ammonia monooxygenase (AMO; FIG. 14). AMO is similar to MMO the two enzymes oxidize a number of the same substrate (Table 8). AMO may also accidentally oxidize pollutant compounds.
Nitrifiers are autotrophic and use CO₂ as their source of cell C. However, the electrons nitrifiers obtain from oxidation of NH₄ and NO₂ lack the necessary power to reduce CO₂ to biomass (FIG. 13).
To overcome this obstacle, nitrifiers put energy into reversing electron transport to push relatively low energy e- from NH₄ and NO₂ uphill to a level with sufficient energy to reduce CO₂ (FIG. 13).
Collectively, the low energy yields of NH₄/NO₂ and NO₂/NO₃ oxidation coupled with the high energy demand of reversed electron transport results in very low biomass production.

C. Ecology of nitrifiers

In soil nitrifiers are typically present in low numbers (hundreds to thousands per gram) because they consume relatively large amounts of NH₄ or NO₂ and produce little biomass. Furthermore, nitrifiers must compete with plants and heterotrophic bacteria for NH₄ so their growth is further limited by N availability
Nitrifiers sensitive to pH, at high pH may there may be problems with NH₃ while at low pH the formation of acid (HNO₃) may be inhibitory (FIG. 15) The practical significance is that the process may be slowed (but not totally inhibited) in acidic soils (pH < 5).

D. Nitrification inhibitors

Nitrification may be undesirable from an agronomic standpoint as it represents conversion of available N into the mobile NO₃^- form, which may be easily leached from the root zone by precipitation or irrigation. This loss is significant because it may reduce potential plant yields and increase operation costs (lost fertilizer). Furthermore, there are environmental concerns about groundwater pollution with nitrate.
Nitrification inhibitors have been developed to block activity of ammonia-oxidizers (Nitrosomonas) by inhibiting AMO. A widely used inhibitor is nitrapyrin (N-serve), which chelates the Cu⁺² needed by AMO for its function.

V. Nitrate reduction Top

Microbes may reduce nitrate as part of either assimilatory pathways (incorporation of elements into bipolymers to form cells) or dissimilatory pathways (generation of energy). The discussion here will focus on the latter

A. Reduction to ammonium (DNRA)

Many obligate anaerobes can utilize NO₃ as an TEA but reduce the compound only as far as NH₄. This is probably a minor reaction in most cases because bacteria capable of DNRA become active only after NO₃ is depleted by facultative anaerobes.
A scenario in which DNRA might be significant would be if NO₃ were added to a soil or groundwater where methanogenic conditions had existed for some time.

B. Reduction to N₂O, N₂ (Denitrification)

A more common nitrate reduction reaction is:
NO₃^- ----> NO₂^- ----[NO]---> N₂O (gas) ----> N₂ (gas)
This process is what is commonly referred to as denitrification and is mediated by facultative anaerobes (i.e., organisms that would preferentially respire oxygen). The role of NO as an intermediate in this pathway and mechanism(s) of N₂O formation are unresolved.
For bacteria to respire NO₃ requires the formation of four additional electron transport proteins (FIG. 16): nitrate reductase (Nar), nitrite reductase (Nir), nitric oxide reductase (Nor), and nitrous oxide reductase (Nos).
Denitrifying bacteria are found in essentially all groups within the Bacteria domain. Most denitrifiers are heterotrophs and couple nitrate reduction to organic matter oxidation. However, there are also chemolithotrophic denitrifiers, that respire NO₃ while using sulfur (HS^-, S), Fe⁺², or H₂ as energy sources.

C. Factors affecting denitrification
1. Primary
a.) Oxygen

For bacteria to respire NO₃ requires the formation of additional enzymes (reductases). However, because the denitrifying bacteria can obtain somewhat greater energy respiring O₂ rather than NO₃ the presence of O₂ prevents the formation of (represses) denitrifying enzymes (FIG. 17)
In general "derepression" of reductases does not occur until O₂ levels drop below 0.5 ppm (15 M). This is relatively little O₂ considering that water exposed to air contains 6 to 8 ppm at ambient temperatures. Sequential derepression of N reductases may be reflected in transient accumulation of intermediates (FIG. 18)

b.) NO₃^- concentration

Denitrification rates are directly proportional to the level of nitrate over the range of zero to ca. 20 mg NO₃^-N L^-1 (i.e., first order with respect to nitrate). Above 20 mg NO₃^-N L^-1 , denitrification rates are not affected by the nitrate concentration (i.e., zero order with respect to nitrate).
The nitrate concentration may also affect the predominant endproduct. At high levels (> 20 mg L^-1) the process tends to go completely to N₂, at low NO₃ levels N₂O accumulates as the major endpoint.

c.) Available C

Because heterotrophs are the dominant denitrifiers in most soils, nitrate reduction will be favored in cases where there is a relatively high amount of available organic matter.

2. Secondary
a.) Soil water content

₂

b.) Soil pH

Acidic pH tends to inhibit (but not prevent) denitrification; soil pH < 5 favors N₂O product rather than N₂.

c.) Temperature

At lower temperatures denitrification is slower and tends to culminate with N₂O as the terminal product. As temperatures increase, denitrification accelerates and tends to go completely to N₂ (FIG. 20).

D. Environmental concerns and variability in denitrification.

From an agricultural standpoint, denitrification is unfavorable as it results in loss of N that might otherwise contribute to crop production. Denitrification can also have adverse environmental impacts in that the gaseous N-oxides produced may diffuse to the stratosphere and participate in chain reactions that result in ozone destruction and acid rain formation (FIG. 21A,B).
Studying denitrification in the environment is complicated by the extremely high degree of temporal and spatial variability inherent to the process (FIGs 22-24).