Azotobacter is a genus of usually Motility, oval or spherical bacteria that form thick-walled (and also has hard crust) and may produce large quantities of capsular Mucus. They are aerobic, free-living soil Microorganism that play an important role in the nitrogen cycle in nature, binding atmospheric nitrogen, which is inaccessible to plants, and releasing it in the form of ammonium ions into the soil (nitrogen fixation). In addition to being a model organism for studying , it is used by humans for the production of , , and some . The first representative of the genus, Azotobacter chroococcum, was discovered and described in 1901 by Dutch microbiologist and botanist Martinus Beijerinck. Azotobacter species are Gram-negative bacteria found in neutral and alkaline soils,
Biological characteristics
Morphology
Cells of the genus
Azotobacter are relatively large for bacteria (2–4 μm in diameter). They are usually oval but may take various forms from rods to
Coccus. In microscopic preparations, the cells can be dispersed or form irregular clusters or, occasionally, chains of varying lengths. In fresh cultures, cells are mobile due to the numerous
flagellum.
Later, the cells lose their mobility, become almost spherical, and produce a thick layer of
mucus, forming the cell capsule. The shape of the cell is affected by the
amino acid glycine, which is present in the nutrient medium
peptone.
Under magnification, the cells show inclusions, some of which are colored. In the early 1900s, the colored inclusions were regarded as "reproductive grains", or Gonidium – a kind of embryo cells.
Cysts
Cysts of the genus
Azotobacter are more resistant to adverse environmental factors than the vegetative cells; in particular, they are twice as resistant to
ultraviolet light. They are also resistant to drying,
ultrasound, and
Gamma ray and solar irradiation, but not to heating.
The formation of cysts is induced by changes in the concentration of nutrients in the medium and the addition of some organic substances such as ethanol, n-butanol, or β-hydroxybutyrate. Cysts are rarely formed in liquid media.
The cysts of Azotobacter are spherical and consist of the so-called "central body" – a reduced copy of vegetative cells with several – and the "two-layer shell". The inner part of the shell is called intine and has a fibrous structure.
Germination of cysts
A cyst of the genus
Azotobacter is the resting form of a vegetative cell; however, whereas usual vegetative cells are reproductive, the cyst of
Azotobacter does not serve this purpose and is necessary for surviving adverse environmental factors. When more favorable environmental conditions resume, which includes a certain value of pH, temperature, and source of
carbon, the cysts germinate, and the newly formed vegetative cells multiply by a simple division. During the germination, the cysts sustain damage and release a large vegetative cell. Microscopically, the first manifestation of spore germination is the gradual decrease in light
refraction by cysts, which is detected with phase contrast microscopy. Germination of cysts takes about 4–6 hours. During germination, the central body grows and captures the granules of volutin, which are located in the
Tunica intima (the innermost layer). Then, the exine bursts and the vegetative cell is freed from the exine, which has a characteristic horseshoe shape.
This process is accompanied by metabolic changes. Immediately after being supplied with a carbon source, the cysts begin to absorb
oxygen and emit
carbon dioxide; the rate of this process gradually increases and saturates after four hours. The synthesis of
and
RNA occurs in parallel, but it intensifies only after five hours after the addition of the carbon source. The synthesis of
DNA and nitrogen fixation are initiated 5 hours after the addition of glucose to a nitrogen-free nutrient medium.
Germination of cysts is accompanied by changes in the intima, visible with an electron microscope. The intima consists of carbohydrates, , and proteins and has almost the same volume as the central body. During germination of cysts, the intima undergoes hydrolysis and is used by the cell for the synthesis of its components.
Physiological properties
Azotobacter respires aerobically, receives energy from
redox reactions, using organic compounds as
, and can use a variety of carbohydrates, alcohols, and salts of
as sources of carbon.
Azotobacter can fix at least 10 μg of nitrogen per gram of glucose consumed. Nitrogen fixation requires molybdenum ions, but they can be partially or completely replaced by vanadium ions. If atmospheric nitrogen is not fixed, the source of nitrogen can alternatively be , Ammonia ions, or . The optimal pH for the growth and nitrogen fixation is 7.0–7.5, but growth is sustained in the pH range from 4.8 to 8.5.
While growing, Azotobacter produces flat, slimy, paste-like colonies with a diameter of 5–10 mm, which may form films in liquid nutrient media. The colonies can be dark-brown, green, or other colors, or may be colorless, depending on the species. The growth is favored at a temperature of 20–30°C.
Bacteria of the genus Azotobacter are also known to form intracellular inclusions of polyhydroxyalkanoates under certain environmental conditions (e.g. lack of elements such as phosphorus, nitrogen, or oxygen combined with an excessive supply of carbon sources).
Pigments
Azotobacter produces
. For example,
Azotobacter chroococcum forms a dark-brown water-soluble pigment
melanin. This process occurs at high levels of metabolism during the fixation of nitrogen and is thought to protect the
nitrogenase system from oxygen.
Other
Azotobacter species produce pigments from yellow-green to purple colors,
including a green pigment which
fluorescence with a yellow-green light and a pigment with blue-white fluorescence.
Genome
The
nucleotide sequence of chromosomes of
Azotobacter vinelandii, strain AvOP, is partially determined. This chromosome is a circular DNA molecule which contains 5,342,073
nucleotide pairs and 5,043 genes, of which 4,988 encode proteins. The fraction of
guanine +
cytosine pairs is 65 mole percent. The number of chromosomes in the cells and the DNA content increases upon aging, and in the stationary growth phase, cultures may contain more than 100 copies of a chromosome per cell. The original DNA content (one copy) is restored when replanting the culture into a fresh medium.
In addition to chromosomal DNA,
Azotobacter can contain
.
Distribution
Azotobacter species are ubiquitous in
Soil pH and weakly
Alkali soils, but not acidic soils.
They are also found in the Arctic and Antarctic soils, despite the cold climate, short growing season, and relatively low pH values of these soils.
In dry soils,
Azotobacter can survive in the form of cysts for up to 24 years.
Representatives of the genus Azotobacter are also found in aquatic habitats, including fresh water
Nitrogen fixation
Azotobacter species are free-living, nitrogen-fixing bacteria; in contrast to
Rhizobium species, they normally fix molecular nitrogen from the atmosphere without
symbiosis relations with plants, although some
Azotobacter species are associated with plants.
Nitrogen fixation is inhibited in the presence of available nitrogen sources, such as ammonium ions and nitrates.
Azotobacter species have a full range of enzymes needed to perform nitrogen fixation: ferredoxin, hydrogenase, and an important enzyme nitrogenase. The process of nitrogen fixation requires an influx of energy in the form of adenosine triphosphate. Nitrogen fixation is highly sensitive to the presence of oxygen, so Azotobacter developed a special defensive mechanism against oxygen, namely a significant intensification of metabolism that reduces the concentration of oxygen in the cells.
Nitrogenase
Nitrogenase is the most important enzyme involved in nitrogen fixation.
Azotobacter species have several types of nitrogenase. The basic one is molybdenum-iron nitrogenase.
An alternative type contains vanadium; it is independent of molybdenum ions
and is more active than the Mo-Fe nitrogenase at low temperatures. So it can fix nitrogen at temperatures as low as 5 °C and its low-temperature activity is 10 times higher than that of Mo-Fe nitrogenase.
An important role in maturation of Mo-Fe nitrogenase plays the so-called P-cluster.
Synthesis of nitrogenase is controlled by the
nif genes.
Nitrogen fixation is regulated by the
enhancer protein NifA and the "sensor"
flavoprotein NifL which modulates the activation of gene transcription of nitrogen fixation by
redox-dependent switching.
This regulatory mechanism, relying on two proteins forming complexes with each other, is uncommon for other systems.
Importance
Nitrogen fixation plays an important role in the nitrogen cycle.
Azotobacter also synthesizes some biologically active substances, including some
phytohormones such as
auxins,
thereby stimulating plant growth.
[ PDF copy ] They also facilitate the mobility of heavy metals in the soil, thus enhancing
bioremediation of soil from heavy metals, such as
cadmium, mercury and
lead.
Some kinds of
Azotobacter can also biodegrade
chlorine-containing aromatic compounds, such as 2,4,6-trichlorophenol, which was previously used as an
insecticide,
fungicide, and
herbicide, but later was found to have
and
effects.
Applications
Owing to their ability to fix molecular nitrogen and therefore increase the soil fertility and stimulate plant growth,
Azotobacter species are widely used in agriculture,
particularly in nitrogen
such as
azotobacterin. They are also used in production of
alginic acid,
which is applied in medicine as an
antacid, in the food industry as an additive to ice cream, puddings, and creams.
Taxonomy
The genus
Azotobacter was discovered in 1901 by Dutch microbiologist and botanist Martinus Beijerinck, who was one of the founders of environmental microbiology. He selected and described the species
Azotobacter chroococcum – the first
aerobic organism, free-living nitrogen fixer.
In 1909, Lipman described Azotobacter vinelandii, and a year later , which he named in honor of Beijerinck. In 1949, Russian microbiologist Nikolai Krasilnikov identified the species of which was divided in 1981 by Thompson Skerman into two subspecies – Azotobacter nigricans subsp. nigricans and Azotobacter nigricans subsp. achromogenes; in the same year, Thompson and Skerman described . In 1991, Page and Shivprasad reported a microaerophilic and air-tolerant type which was dependent on sodium ions.
Earlier, representatives of the genus were assigned to the family Azotobacteraceae Pribram, 1933, but then were transferred to the family Pseudomonadaceae based on the studies of nucleotide sequences 16S rRNA. In 2004, a Phylogenetics study revealed that A. vinelandii belongs to the same clade as the bacterium Pseudomonas aeruginosa,
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