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Azotobacter salinestris - salinestris, comes from the Latin words “salinus,” meaning saline, and “-estris”, which means “living in”. It is a Gram-negative, nitrogen fixing bacteria. It can be found living in soil or water habitats as single cells or in chains of 6 to 8 cells. This organism is motile at younger stages but loses its flagella at older stages. This bacteria is known for its potential use in bioremediation.

Isolation
William J. Page and Shailaja Shivprasad isolated Azotobacter salinestris from saline soils of Western Canada and Egypt in 1991. The colonies used for their study were first taken from air-dried surface soil from Alberta, Canada. The soil was inoculated into a Burk nitrogen free mineral salt medium, which contained 1% glucose and 0.25 micrograms of copper chloride per milliliter of solution. The medium was incubated at 30oC and stored at 4oC. Colony formation was noted after 2-3 days of incubation. The same soil samples were also used to inoculate slant cultures, which were stored at room temperature. Azotobacter salinestris was found to lose viability in the slant cultures when stored at 4oC. Through these cultures and characterization tests, A. salinestris was found to share many of the general characteristics specific to the species Azotobacter. Originally, A. salinestris colonies were classified as Azotobacter chroococcum but were later identified as a separate species based on their salt dependent growth.

Morphology
Azotobacter salinestris is a Gram-negative, rod shaped organism. This organisms cells are approxiamately 2 x 4 microns in size when they are 18 hours old and can grow to be up to 5 microns in diameter. Older cells can also form cysts. A. salinestris colonies appear to be brown-black in color because they produce water-soluble catechol melanin.

Physiology
The bacteria that performed the most efficient atmospheric nitrogen fixation were from samples grown in 0.05% to 0.1% saline concentration soils. It was also observed that nitrogen fixation rates were not affected by the presence of oxygen. A. salinestris that grows in soils is a facultative anaerobe. Colonies growing in aquatic habitats were determined to be microaerophilic and very sensitive to the presence of hydrogen peroxide since they do not produce a catalase enzyme.

Metabolism
Azotobacter salinestris can use melibiose, galactose, mannitol, sucrose, glucose, and fructose as primary carbon sources. They prefer to use sodium ions as their electron acceptor, but will also use rubidium. Strains that do not have access to sodium ions produce acid a a product of the metabolism of their growth-promoting carbon substrate.

Ecology
All known Azotobacter salinestris samples were isolated from soils that had a neutral or slightly basic pH. The optimal growth pH of this species is 7.2-7.5. Soils must have at least one milli-molar concentration of saline for this organism to grow. Unlike other Azotobacter species, iron was absolutely required for growth. While most Azotobacter species are commonly found in soil, Azotobacter salinestris is unique due to being found in soils with high salt content and requiring the presence of iron to grow. These organisms can survive in aerobic and anaerobic conditions. Azotobacter salinestris is dependent on salinity so they can also be found in marine environments.

Azotobacter chroococcum is the most common species from Azotobacter to be isolated from soil samples. It is also a close relative to Azotobacter salinestris. All growth conditions used to isolate and determine optimum living conditions for Azotobacter salinestris were based on the optimal living conditions for Azotobacter chroococcum. The defining factor between these two species was the dependence on sodium ions to live. A. salinestris displayed a stronger dependence on sodium to live than A. chroococcum.

Genetics
While Page and Shivprasad are credited with the discovery and characterization of A. salinestris, Eydne and Wachter are credited with the sequencing of the bacteria’s 5S rRNA in 1987.

Although the results were never published, sequencing data placed this bacterial strain in the genus Azotobacter. The bacteria’s DNA has a melting point of 96.68 to 97.08 oC and the GC content was 67.73-67.8%. A separate sequencing of the 16S rRNA sequence, conducted by Moore and the University of Houston, confirmed that A. salinestris was indeed a separate species of the genus Azotobacter.

Importance
Azotobacter salinestris was the first prokaryote to show Na+/succinic acid efflux. It can tolerate up to 5% glyphosate, which is a pesticide used to kill weeds that compete with crops. Because the species is a common nitrogen fixer, it is important to the agricultural industry for the species to be able to survive in the presence of such a common pesticide. It can degrade endosulfan, which is an insecticide that is highly hazardous to human, mammal, and fish health. Endosulfan use was banned in 2012 by the United States, following a precedent established by New Zealand and the European Union. The decision to ban endosulfan use came after a study that showed the health risks to humans and wildlife were much higher than expected. It is similar to DDT (dichlorodiphenyltrichloroethane), causes birth defects, and is an estrogen analog. Therefore, the ability of A. salinestris to break down endosulfan is important for bioremediation to the environments where the substance was used.