Azotobacter

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 spheres. 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 flagella.{{cite journal

Under magnification, the cells show inclusions, some of which are colored. In the early 1900s, the colored inclusions were regarded as "reproductive grains", or gonidia – a kind of embryo cells.{{cite journal

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 and solar irradiation, but not to heating.{{cite journal

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.{{cite journal

The cysts of Azotobacter are spherical and consist of the so-called "central body" – a reduced copy of vegetative cells with several vacuoles – and the "two-layer shell". The inner part of the shell is called intine and has a fibrous structure.{{cite journal|author1=Pope L. M. |author2=Wyss O. |title= Outer Layers of the Azotobacter vinelandii Cyst

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 refractive 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 intima (the innermost layer). Then, the exine bursts and the vegetative cell is freed from the exine, which has a characteristic horseshoe shape.{{cite journal

Germination of cysts is accompanied by changes in the intima, visible with an electron microscope. The intima consists of carbohydrates, lipids, 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.{{cite journal

Physiological properties

Azotobacter respires aerobically, receives energy from redox reactions, using organic compounds as electron donors, and can use a variety of carbohydrates, alcohols, and salts of organic acids 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 nitrates, ammonium ions, or amino acids. 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. Azotobacter can also grow mixotrophically, in a molecular nitrogen-free medium containing mannose; this growth mode is hydrogen-dependent. Hydrogen is available in the soil, thus this growth mode may occur in nature.

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 pigments. 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.{{cite journal|author1=Shivprasad S. |author2=Page W. J. |title= Catechol Formation and Melanization by Na+-Dependent Azotobacter chroococcum: a Protective Mechanism for Aeroadaptation?

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.{{cite journal

Distribution

Azotobacter species are ubiquitous in neutral and weakly basic soils, but not acidic soils.{{cite journal|author1=Yamagata U. |author2=Itano A. |title= Physiological Study of Azotobacter chroococcum, beijerinckii and vinelandii types

Representatives of the genus Azotobacter are also found in aquatic habitats, including fresh water{{cite journal|doi = 10.2307/1936516|author = Johnstone D. B.

Nitrogen fixation

Main article: Nitrogen fixation

Azotobacter species are free-living, nitrogen-fixing bacteria; in contrast to Rhizobium species, they normally fix molecular nitrogen from the atmosphere without symbiotic relations with plants, although some Azotobacter species are associated with plants.{{cite journal|author1=Kass D. L. |author2=Drosdoff M. |author3=Alexander M. |title= Nitrogen Fixation by Azotobacter paspali in Association with Bahiagrass (Paspalum notatum)

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. Also, a special nitrogenase-protective protein protects nitrogenase and is involved in protecting the cells from oxygen. Mutants not producing this protein are killed by oxygen during nitrogen fixation in the absence of a nitrogen source in the medium.{{cite journal|doi = 10.1128/JB.182.13.3854-3857.2000|author1=Maier R. J. |author2=Moshiri F. |title= Role of the Azotobacter vinelandii Nitrogenase-Protective Shethna Protein in Preventing Oxygen-Mediated Cell Death|pmid=10851006|journal = Journal of Bacteriology|volume = 182

Nitrogenase

Main article: 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.{{cite journal |doi-access = free |access-date = 2018-11-04 |archive-date = 2017-12-02 |archive-url = https://web.archive.org/web/20171202133317/https://authors.library.caltech.edu/9607/1/HOWpnas06.pdf |url-status = live

Importance

Nitrogen fixation plays an important role in the nitrogen cycle. Azotobacter also synthesizes some biologically active substances, including some phytohormones such as auxins,{{cite journal |archive-url=https://web.archive.org/web/20100415114145/http://journals.tubitak.gov.tr/biology/issues/biy-05-29-1/biy-29-1-5-0410-1.pdf |archive-date=2010-04-15

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 biofertilizers such as azotobacterin. They are also used in production of alginic acid,{{cite journal |doi-access = free}}{{cite journal

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, 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.{{cite journal |doi-access=free

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 phylogenetic study revealed that A. vinelandii belongs to the same clade as the bacterium Pseudomonas aeruginosa,{{cite journal |doi-access = free |doi-access = free

References

References

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