Biogeochemical Cycling of Nitro­gen | Microbiology

The key processes of biogeochemical cycling of nitro­gen are: 1. Nitrogen Fixation 2. Ammonification 3. Nitrification 4. Denitrification.

1. Nitrogen Fixation:

Nitro­gen fixation is a process of conver­sion of gaseous form of nitrogen (N₂) into combined forms i.e. ammo­nia or organic nitrogen by some bacteria and cyanobacteria. There are free-living as well as symbiotic microorganisms which fix N₂ into proteins. The nitrogen-fixing micro­organisms are called diazotrophs, and the phenomenon of this activity is known as diazotrophy.

2. Ammonification:

During the course of organic matter decomposition the complex form of organic nitrogen is rendered by microorganisms into the more labile inorganic form. This process is called nitrogen mineralization. As a result of mineralization ammonia and nitrate are formed and organic nitrogen disappears.

The process of formation of ammonia form organic compound is known as ammonification (Fig. 30.7) as represented below:

Mostly all the nitrogen found in surface soil horizon is in organic combination, the chemical composition of which is not fully understood. However, a few known combinations are free amino acids, amino-sugars (e.g. glucosamine and galactosamine) and several purines and pyrimidines derived from nucleic acids. Bound amino acids constitute about 20-50% of total nitrogen of humus, whereas 5-11% is amino-sugar nitrogen.

Moreover, the net change in the amount of inorganic nitrogen (N_i) is expressed as below:

N_i = Organic nitrogen mineralized – (N_a + N_p + N_l + N_d)

where, N_a = nitrogen assimilated by microorganisms

N_p = nitrogen removed by the plants

N_l = nitrogen lost by leaching

N_d = nitrogen volatilized by denitrification

Microbiology:

A diverse microflora liberates ammonia from organic nitrogen compounds. These include bacteria (e.g. Pseudomonas, Bacillus, Clostridium, Serratia, Micrococcus, etc.), fungi (e.g. Alternaria, Aspergil­lus, Mucor, Penicillium, Rhizopus, etc.), and actinomycetes. They synthesize extracellular proteolytic enzymes for the decompo­sition of proteins. The ammonifying population in­cludes aerobes and anaer­obes.

Proteins are con­verted to peptides and amino acids by extracellu­lar proteolytic enzymes.

Ammonia is produced after deamination (e.g. oxidative or reductive deamination) or the amino acids accord­ing to reactions as below:

NH₄ predominates in acidic and neutral environment with the increase of pH, therefore, NH₃ predominates and is released into the atmosphere.

NH₄ → NH₃ + H⁺

Ammonium is formed slowly at water levels slightly below the permanent wilting percentage. Ammonification is not eliminated by soil submergence and the process is rapid in wet paddy fields, where O₂ level is quite low.

Urea is found in soil which is made available as a decomoposed product of nitrogenous bases, as fertilizer and as animal excretory product. However, urea is readily hydrolysed, and transformed to ammonia.

3. Nitrification:

The process of oxidation of ammonium ions (oxidation level= – 3) to nitrite ions (oxidation level = +3) and subsequently to nitrate ions (oxidation- level = +5) is known as nitrification. Thus, ammonium (the most reduced form of inorganic nitrogen) acts as the starting point for nitrification. Nitrate is also produced (on addition to soil) in manure piles, during sewage processing, and marine environment.

In 1877, T. Schloesing and A. Muntz for the first time gave experimental evidence that nitrification is of biological origin. In 1890, S. Winogradsky isolated the responsible microorganisms from soil. However, L. Pasteur postulated earlier that the formation of nitrate was microbiological and analogous to the conversion of alcohol to vinegar.

There are certain chemical properties of the microbiological habitat that serve to change the magnitude of the transformation. Nitrification is proportional to the cation exchange capacity of soil.

In alkaline soil where the concentration of salt is high, nitrate production is declined probably due to low tolerance of nitrifiers. In environment having a near neutral reaction, formation of nitrate from ammonia is rapid, whereas in acidic soils nitrate is formed faster from organic material.

Microbiology:

The nitrifying bacteria are very important for plants because they affect nutrient availability. Nitric acid is formed from ammonia which drops the pH. This affect changes in concentration of soluble potassium, phosphate, magnesium, iron, manganese and calcium.

Nitrifi­cation is an example of aerobic respiration and appears to be present in a few autotrophic bacteria. The chemolithotrophs derive energy through these two processes (conversion of ammonia to nitrite, and nitrite to nitrate). These steps are carried out by two different types of nitrifying bacteria.

(a) Bacteria oxidizing ammonium to nitrite:

The nitrifying bacteria are Nitrosomonas, Nitrosococcus, Nitrosolobus, Nitrosospira, Nitrosovibrio, etc.

The reaction characterizing the chemoautotrophic bacteria of the first step of nitrification is as below:

NH₄⁺ + 1 ½ O₂ → NO₂^– + 2H⁺ + H₂O

(-3) (+3)

Here the oxidation state is changed from -3 of ammonia to +3 of nitrous acid through the removal of electrons.

The complete reaction is as below:

NH₃ → NH₂OH → (HNO?) NO → NO₂^–

Ammonium is first converted to hydroxylamine (NH₂OH) which in turn is changed to undefined metabolite (possible nitroxyl, HNO). This intermediate is oxidized to nitrite by the way of NO. Here accumulation of NH₂OH may lead toxicity to cells.

(b) Bacteria oxidizing nitrite to nitrate:

The example of such bacteria is Nitrobacter, Nitrospira and Nitrococcus.

They change nitrogen oxidation state from +3 to +5 as given below:

NO₂^– + H₂O → H₂O.NO₂^– → NO₃^– + 2H⁺

2H⁺+ ½ O₂^– → H₂O

Mostly nitrification is carried out by autotrophic bacteria but there are some heterotrophic bacteria and fungi which also take part in nitrification. The examples are strains of Nitrosomonas, Aspergillus flavus, etc.

4. Denitrification:

Nitrogen is converted into different forms by microorganisms.

When nitrate is added to soil, N₂, N₂O (nitrous oxide) and NO (nitric oxide) are evolved after reduction of nitrate as given below:

NO₃^– → NO₂^– → N₂O → N₂

Thus, microbial reduction of nitrate and nitrite with the liberation of molecular nitrogen and nitrous oxide is called denitrification. These are the volatile products and, therefore, are lost to the atmosphere and fail to enter the cell structure. Thus, denitrification is essentially a respiratory mechanism in which nitrate replaces the molecular oxygen.

Therefore, denitrification may be termed as nitrate respiration. There are three possible reactions through which volatilization of nitrogen occurs: non-biological losses of ammonia, chemical decomposition of nitrite, and microbial denitrification resulting in evolution of N₂, N₂O and NO into the atmosphere. For the purpose of crop production, nitrogen volatilization has deleterious effect because it depletes part of soil reserves of essential nutrients.

Microbiology:

Fungi and actinomycetes have not been found to be associated with denitrification. It is carried out only by certain bac­teria such as the genera: Pseudomonas, Bacillus, Paracoccus as well as oc­casionally Thiobacillus denitrificans, Chromobacterium, Corynebacterium, Hyphomicrobrium and Serratia species.

Denitrifying bacteria are abundant in arabic fields and count for about a mil­lion per gram soil. Their population is higher in rhizosphere soil. Denitrify­ing bacteria are aerobic but nitrate is used as the elec­tron donor for their growth in absence of O₂. In addi­tion, several chemoautotrophs (Thiobacillus denitrificans and Paracoccus denitrificans) are capable of reducing nitrate to nitrogen.

There are three microbiological reactions of nitrate: first, a complete reduction to ammonia, second an incomplete reduction and accumulation of nitrite, and third, a reduction to nitrite followed by the evolution of gases (i.e. denitrification).

The biochemical pathway of nitrate reduction and denitrification is given below:

(c) Anammox Bacteria:

In contrast to ammonia oxidizing bacteria, recently some chemoautotrophic ammonia oxidizing bacteria have been observed in soil, sewage, fresh and marine water, for example Nitrosomonas europea, Nitrococcus oceanus and Nitrospora briensis. Kuyper et al. (2003) isolated these bacteria from black sea which is the biggest anoxic basin in the world.

These flagellated bacteria reproduce by binary fission and also oxidize ammonia to nitrite as given below:

5NH₄⁺ + 3NO₂ → 4N₂ + 9H₂O +-2H⁺

The above reaction occurs in the absence of oxygen hence it is called ‘anammox reaction’.

The anammox bacteria are full of surprises. They contain membrane bound intracytoplasmic sacs like compartment called ‘anammoxosomes’ which helps in packing toxic hydrazine. Such complex structures are present in eukaryotic cell. Simpler prokaryotic cells including bacteria should lack them.

Only one kind of bacterium-the Planktomycetes, contains features of all three domains of life i.e. bacteria, eukaryotes and archaea. DNA studies place them with bacteria but their internal organelles resemble with eukaryotes.

These microbes lack the rigid polymer peptidoglycan in their cell wall, making them similar to the single celled archaea domain. Genetic analysis confirm these two anaerobic microbes and named Brocadia anammoxidans and Kuenema stuttgartiensis due to the unique property and their bright red color.

Recently, it has been observed that the anammox reaction occurs inside the membrane bag or anammoxosomes produced hydrazine as an intermediate. This high energy hydrazine is required to derive the anammox reaction. Hydrazine can easily diffuse through cell membrane so the bacterial membrane must be unusual.

The membrane’s lipids are made of five carbon based rings fused together to form a ladder. This ‘ladderin’ lipid is peculiar because it has a lot of energy built into it and is very unstable. This makes membrane exceptionally strong, and hence stops hydrazine i.e. N₂H₄ (rocket fuel) living into rest of cells.

The steps of chemical reaction are given below:

2HNO₂ + 4XH₂ → 2NH₂OH + 2H₂O + 4X

2NH₂OH + 2NH₃ → 2N₂H₄ + 2H₂O + 4X

2N₂H₂ + 4X → 2N₂ + 4X H₂

Applications:

The significance of anammox bacteria lies in waste water treatment, sewage plants and industrial process such as fertilizers manufacture and petroleum refining which generates millions of ammonia as waste. The conventional method is used by nitrifying bacteria to convert ammonia into nitrite and nitrate, and later gives rise to nitrogen. This process is uneconomical because it requires oxygen.

Electricity is also needed to aerate the sludge. The nitrifying bacteria need an energy source. Hence, the whole process becomes costly and is unsafe to the environment. On the other hand, anammox bacteria use ammonia as their fuel. They do not need oxygen; anammox bacteria consume carbon dioxide, so the method is eco-friendly.