NH , cell wall ria). In modern times, the anthropogenic input on land is by far larger than natural sources. It is estimated that >80 X 1012 g N/year is added through agricultural fertilizer usage (NH+, urea), with an additional 20 X 1012 g N/year from fossil fuel combustion. Natural N inputs are from litter and root decomposition, metabolic N excretion by saprotrophs and above-ground species (Tables 6.2 and 6.4). As the input N increases, the amount that is available to saprotrophs and plant roots also increases. The preferred form of nitrogen for uptake by osmotrophy is ammonia, although nitrate can be transported through cell membranes of many species. Some fungi cannot transport nitrate, but plant roots can transport ammonia and nitrate. As the N input amount increases, through biological transformations, the loss of soluble forms by leaching and gaseous forms by volatilization also increases. Because the activity of nitrogen fixation metabolic pathways depends to a large extent on the soil conditions, the activity varies with seasons and weather. It is therefore difficult to make generalization and predictions about the amount of nitrification and denitrification processes between field sites, without knowledge of the conditions at the time of sampling and of the food web composition.
The enzyme responsible for nitrogen fixation is nitrogenase (abbreviated nif and consists of two subunits. One subunit requires iron and the other requires both iron and molybdenum prosthetic groups. The enzyme requires ATP, reduced ferredoxin and other electron acceptors, and it is extremely sensitive to the oxygen concentration. Most nitrogenase enzymes are inactivated irreversibly by oxygen, so that the enzyme must be physiologically protected. The bacteria create a low oxygen microenvironment in the cell or wait for adequate conditions for nitrogenase activity. In most cases, exposure to oxygen is the primary limiting factor in nitrogen fixation, when all other parameters are favourable. However, there are both aerobic and anaerobic species that carry out nitrogen fixation (Fig. 6.6). The end-product of nitrogen fixation is ammonia, with hydrogen gas released as a by-product. The ammonia is used in cellular anabolic metabolism to synthesize organic molecules (ammonium assimilation) by transport through the cell membrane. Soil bacteria capable of fixing nitrogen are common, but those that form symbiotic associations with roots are more efficient at producing fixed nitrogenous compounds. In these symbioses, the N compounds are available to plant primary production and do not become part of the soil decomposition system until the plant biomass turns to litter. Only a small portion will be returned through root exudates. Legume is a general term given to crops that permit symbiosis with nitrogen-fixing bacteria. The bacteria form nodules inside root tissues in a species- or strain-specific manner. It is sometimes necessary to inoculate a field or crop with the adequate strains to induce nodule formation.
Nitrogen-fixing bacteria, especially non-symbiotic species, are an important source of nitrogen in the decomposition food web and often represent a keystone functional group. In nitrogen-limited soils, the rate of nitrogen fixation can be the rate-limiting step in decomposition.
Ammonification refers to the release of ammonia from organic molecules. A large portion of the ammonia is released as metabolic nitrogenous waste by various organisms (Tables 6.4 and 6.5). An important source of ammonia is from urea (excreted from various Animalia) through urease enzyme activity produced by certain primary sapro-trophs. The enzyme requires nickel as a prosthetic group. The presence of the enzyme in soils is crucial when urea-based fertilizers are used. The ammonia is more accessible than urea or organic N molecule monomers for cell membrane transport by osmotrophy.
Table 6.5. Glossary of the main biochemical transformations in the nitrogen cycle.
N fixation Biological transformation of N2 (g) into organic or inorganic compounds
Ammonium assimilation Cellular absorption of NH3 or NH+ from the environment for new cell biomass
Nitrification Oxidation of ammonia to NO,, and NO3 for chemical energy
Nitrate reduction Reduction of NO3 followed by its absorption into cell biomass
Ammonification Transformation of organic N to ammonia, e.g. from urea or uric acid
Denitrification Reduction of NO3 to any N oxides or N2(g)
There is a two-step process which involves the oxidation of ammonia to nitrite ions, followed by the oxidation of nitrite to nitrate ions, called nitrification (Table 6.5, Fig. 6.6). Each step is an energy-yielding process and occurs in a small number of autotrophic species. The two steps are often carried out by separate species of bacteria, but they occur in close proximity and simultaneously so that a net accumulation of nitrite is not usually observed. In soils, the most common species implicated are Nitrosolobus and Nitrosomonas, and in acid soils Nitrosospira (ammonia oxidation) and Nitrobacter (nitrite oxidation). The latter reaction is carried out by nitrite oxidoreductase and these species are capable of limited het-erotrophic growth, unlike the ammonia oxidizers which are strict autotrophs. Other species of primary saprotrophs are capable of a limited amount of ammonium oxidation. On their own, they probably do not contribute significantly to ammonium oxidation; however, if their pooled numbers are great, their contribution could be significant. Known heterotrophic nitrifiers are the fungus Aspergillus, and the bacteria Alcaligenes, Arthrobacter and several Actinobacteria. The importance of this pathway in nitrogen transformations depends on the clay mineralogy. If the clays are positively charged, the negative nitrite and nitrate ions are more likely to be bound and less accessible to osmotrophy. In the reverse situation, it would be more beneficial to transform the positive ammonium ions into the negatively charged oxidized ions. Nitrate ions are easily transported through cell membranes and assimilated into organic molecules (assimilatory nitrate reduction). It is the preferred form of nitrogen uptake for many species, especially of plants.
Soluble forms of nitrogen in the soil are transported through cell membranes by osmotrophy for assimilation into complex organic molecules by anabolic metabolism. These soluble forms include a variety of small organic molecules, ammonium, nitrate and nitrite ions (Table 6.4, Fig. 6.6). In the absence of oxygen, several pathways of nitrogen transformation become more dominant. Dissimilatory nitrate reduction becomes one important mechanism. It involves the oxidation of cell organic molecules utilizing nitrate ions as terminal electron acceptors, producing nitrite ions. This pathway is more efficient than the typical fermentation pathway. Two types of nitrate reduction are distinguished. In the first type, certain facultative anaerobic species can reduce nitrate to nitrite by transferring electrons from anabolic metabolism. Under certain conditions, these species will further transform the nitrite ions excreted into ammonia by nitrite ammonification. Under these conditions, the nitrate can be re-utilized as ammonia by osmotrophy and assimilated. Some soil genera implicated in this process include, Clostridium, Desulfovibrio, Enterobacter, Erwinia, Escherichia, Klebsiella, Nocardia, Pseudomonas, Spirillum and Vibrio.
In the second type, nitrate reduction is followed by further reduction of nitrite ions to NO, N2O and N2. This pathway is called denitrifi-cation because it produces gaseous N molecules which are not biologically useful. The main denitrifiers in the soil are Agrobacterium, Alcaligenes, Azospirillum, Bacillus, Bradyearhizobium, Cytophaga, Flavobacterium, Nitrosomonas, Propionibacterium, Pseudomonas, Rhizobium and Rhodopseudomonas. The enzymes for this process are close together in the cell membrane. Their activity is inhibited by oxygen and they require metal atom prosthetic groups. Nitrate is first transformed to nitrite by nitrate reductase (Nar), nitrite is transformed to nitric oxide by nitrite reductase (Nir), nitric oxide is transformed to nitrous oxide by nitric oxide reductase (Nor), and nitrous oxide is converted to dinitrogen by nitrous oxide reductase (Nos). Again here, nitrate reduction, ammonifica-tion and denitrification processes occur in close proximity in the soil. Peds are more aerobic on the periphery and more anaerobic in the centre. Bacteria are dispersed throughout the peds and utilize the available soil solution under the conditions they are in. Typically, nitrate assimilation at the periphery of peds is accompanied by nitrate reduction and denitrification in the anaerobic centre of peds and at anaerobic microsites. The exact balance depends on the combination of bacteria functioning in syntrophy at microsites or in peds, under soil aeration status. The amount of N osmotrophy and bacterivory by other soil organisms further protects from loss of nitrogen from the system. In aerobic conditions, ammonium oxidation is carried out by ammonia mono-oxygenase which has a broad substrate specificity, including methane and several halogenated organic molecules. Under anaerobic conditions, these species can also carry out further reduction of nitrite to N2O and thus contribute to denitrification. During a spell of anaerobic conditions, such as several days of flooded fields, a significant portion of the accumulated fixed nitrogen and dissolved N molecules can be lost to the atmosphere by denitrification reactions.
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