Azotobacter
Encyclopedia
Azotobacter is a genus
of usually motile
, oval or spherical bacteria
that form thick-walled cyst
s and may produce large quantities of capsular slime
. They are aerobic, free-living soil microbes
which 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. Apart from being a model organism
, it is used by humans for the production of biofertilizer
s, food additive
s and some biopolymer
s. The first representative of the genus, Azotobacter chroococcum, was discovered and described in 1901 by the Dutch microbiologist
and botanist Martinus Beijerinck
. Azotobacter are Gram-negative bacteria. They are found in neutral and alkaline soils, in water and in association with some plants.
s of the genus Azotobacter are relatively large for bacteria (1–2 micrometres 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
. 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 microscope, 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. However, it was later demonstrated that the granules do not participate in the cell division. The colored grains are composed of volutin
whereas the colorless inclusions are drops of fat, which act as energy reserves.
, in particular, they are twice more resistant to UV
light. They are also resistant to drying, ultrasound
and gamma
and solar irradiation, but not to heating.
The formation of cysts is induced by changes in the concentration of nutrients in the medium and addition of some organic substances such as ethanol
, n-butanol
or β-hydroxybutyrate. Cysts are rarely formed in liquid media. The formation of cysts is induced by chemical factors and is accompanied by metabolic
shifts, changes in catabolism
, respiration
and biosynthesis
of macromolecule
s; it is also affected by aldehyde dehydrogenase
and the response regulator AlgR.
The cysts of Azotobacter are spherical and consist of the so-called central body – a reduced copy of vegetative cells with several vacuole
s – and the two-layer shell. The inner part of the shell is called intine and has a fibrous structure. The outer part has a hexagonal crystalline structure and is called exine. Exine is partially hydrolyzed by trypsin
and is resistant to lysozyme
, in contrast to the central body. The central body can be isolated in a viable state by some chelation
agents. The main constituents of the outer shell are alkylresorcinols
composed of long aliphatic
chains and aromatic
rings. Alkylresorcinols are also found in other bacteria, animals and plants.
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. Following the resumption of optimal environmental conditions, which include a certain value of pH
, temperature and source of carbon
, the cysts germinate, and the newly formed vegetative cells multiply via 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 is a slow process that takes about 4–6 hours. During germination, the central body grows and captures the granules of volutin, which were 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. 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 4 hours. The synthesis of protein
s and RNA
, occurs in parallel, but it intensifies only after 5 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, lipid
s and proteins and has almost the same volume as the central body. During germination of cysts, the intima hydrolyses
and is used by the cell for the synthesis its components.
reactions, using organic compounds as electron donor
s. Azotobacter can use a variety of carbohydrates, alcohol
s and salts of organic acid
s as sources of carbon and can fix at least 10 micrograms of nitrogen per gram of glucose consumed. Nitrogen fixation requires molybdenum
ions, but they can be partially replaced by vanadium
ions. The source of nitrogen can be nitrate
s, ammonium
ions or amino acid
s. 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 mixotroph
ically, in a 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 produce flat, slimy, paste-like colonies with a diameter of 5–10 mm, which may form films in liquid nutrient media. The colonies can have dark brown, green and other colors, or may be colorless, depending on the species. The growth is favored at a temperature of 20–30 °C.
s. 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 fluoresces
with a yellow-green light and a pigment with blue-white fluorescence.
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 plasmid
s.
and weakly basic 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 freshwater and brackish marshes. Several members are associated with plants and are found in the rhizosphere
, having certain relationships with the plant. Some strains are also found in the cocoons of the earthworm Eisenia fetida
.
species, they normally fix molecular nitrogen from the atmosphere without symbiotic
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 have a full range of enzymes needed to perform the 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
(ATP). Nitrogen fixation is highly sensitive to the presence of oxygen, and therefore Azotobacter developed a special defensive mechanism against oxygen, namely a significant intensification of metabolism that reduces the concentration of oxygen in the cells. There is also a special protein shethna, which protects nitrogenase and is involved in protecting the cells from oxygen. Mutant
s not producing this protein, are killed by oxygen during nitrogen fixation in the absence of a nitrogen source in the medium. Homocitrate
ions play a certain role in the processes of nitrogen fixation by Azotobacter.
; 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.
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
. The latter was previously used as an insecticide
, fungicide
and herbicide
but later found to have mutagen
ic and carcinogen
ic effects.
s such as azotobacterin. They are also used in production of alginic acid
(E400), which is applied in medicine as an antacid
, in the food industry as an additive to ice cream, puddings and creams, and in the biosorption of metals.
. He selected and described the species Azotobacter chroococcum – the first aerobic
, free-living nitrogen fixer.
In 1903, 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 of Azotobacter nigricans subsp. nigricans and Azotobacter nigricans subsp. achromogenes; in the same year, Thompson and Skerman described the . 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 , 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 Azotobacter vinelandii
belongs to the same clade
as the bacterium Pseudomonas aeruginosa
, and in 2007 it was suggested that the genera Azotobacter, Azomonas
and Pseudomonas
are related and might be synonyms
.
Genus
In biology, a genus is a low-level taxonomic rank used in the biological classification of living and fossil organisms, which is an example of definition by genus and differentia...
of usually motile
Motility
Motility is a biological term which refers to the ability to move spontaneously and actively, consuming energy in the process. Most animals are motile but the term applies to single-celled and simple multicellular organisms, as well as to some mechanisms of fluid flow in multicellular organs, in...
, oval or spherical bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
that form thick-walled cyst
Cyst
A cyst is a closed sac, having a distinct membrane and division on the nearby tissue. It may contain air, fluids, or semi-solid material. A collection of pus is called an abscess, not a cyst. Once formed, a cyst could go away on its own or may have to be removed through surgery.- Locations :* Acne...
s and may produce large quantities of capsular slime
Mucus
In vertebrates, mucus is a slippery secretion produced by, and covering, mucous membranes. Mucous fluid is typically produced from mucous cells found in mucous glands. Mucous cells secrete products that are rich in glycoproteins and water. Mucous fluid may also originate from mixed glands, which...
. They are aerobic, free-living soil microbes
Microorganism
A microorganism or microbe is a microscopic organism that comprises either a single cell , cell clusters, or no cell at all...
which play an important role in the nitrogen cycle
Nitrogen cycle
The nitrogen cycle is the process by which nitrogen is converted between its various chemical forms. This transformation can be carried out by both biological and non-biological processes. Important processes in the nitrogen cycle include fixation, mineralization, nitrification, and denitrification...
in nature, binding atmospheric nitrogen
Nitrogen
Nitrogen is a chemical element that has the symbol N, atomic number of 7 and atomic mass 14.00674 u. Elemental nitrogen is a colorless, odorless, tasteless, and mostly inert diatomic gas at standard conditions, constituting 78.08% by volume of Earth's atmosphere...
, which is inaccessible to plants, and releasing it in the form of ammonium
Ammonium
The ammonium cation is a positively charged polyatomic cation with the chemical formula NH. It is formed by the protonation of ammonia...
ions into the soil. Apart from being a model organism
Model organism
A model organism is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the organism model will provide insight into the workings of other organisms. Model organisms are in vivo models and are widely used to...
, it is used by humans for the production of biofertilizer
Biofertilizer
A bio-fertilizer is a substance which contains living microorganisms which, when applied to seed, plant surfaces, or soil, colonizes the rhizosphere or the interior of the plant and promotes growth by increasing the supply or availability of primary nutrients to the host plant.Bio-fertilizers add...
s, food additive
Food additive
Food additives are substances added to food to preserve flavor or enhance its taste and appearance.Some additives have been used for centuries; for example, preserving food by pickling , salting, as with bacon, preserving sweets or using sulfur dioxide as in some wines...
s and some biopolymer
Biopolymer
Biopolymers are polymers produced by living organisms. Since they are polymers, Biopolymers contain monomeric units that are covalently bonded to form larger structures. There are three main classes of biopolymers based on the differing monomeric units used and the structure of the biopolymer formed...
s. The first representative of the genus, Azotobacter chroococcum, was discovered and described in 1901 by the Dutch microbiologist
Microbiologist
A microbiologist is a scientist who works in the field of microbiology. Microbiologists study organisms called microbes. Microbes can take the form of bacteria, viruses, fungi, and protists...
and botanist Martinus Beijerinck
Martinus Beijerinck
Martinus Willem Beijerinck was a Dutch microbiologist and botanist. Born in Amsterdam, Beijerinck studied at the Technical School of Delft, where he was awarded the degree of Chemical Engineer in 1872. He obtained his Doctor of Science degree from the University of Leiden in 1877...
. Azotobacter are Gram-negative bacteria. They are found in neutral and alkaline soils, in water and in association with some plants.
Morphology
CellCell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
s of the genus Azotobacter are relatively large for bacteria (1–2 micrometres in diameter). They are usually oval, but may take various forms from rods
Bacillus (shape)
The word bacillus may be used to describe any rod-shaped bacterium, and such bacilli are found in many different taxonomic groups of bacteria. However, the name Bacillus, capitalized and italicized, refers to a specific genus of bacteria...
to spheres
Coccus
Coccus can be used to describe any bacterium that has a spherical shape. It is one of the three distinct types of bacteria shapes, the other two being bacillus and spirillum cells...
. In microscopic preparations, the cells can be dispersed or form irregular clusters or occasionally chains of varying lengths. In fresh cultures
Microbiological culture
A microbiological culture, or microbial culture, is a method of multiplying microbial organisms by letting them reproduce in predetermined culture media under controlled laboratory conditions. Microbial cultures are used to determine the type of organism, its abundance in the sample being tested,...
, cells are mobile due to the numerous flagella
Flagellum
A flagellum is a tail-like projection that protrudes from the cell body of certain prokaryotic and eukaryotic cells, and plays the dual role of locomotion and sense organ, being sensitive to chemicals and temperatures outside the cell. There are some notable differences between prokaryotic and...
. Later, the cells lose their mobility, become almost spherical and produce a thick layer of mucus
Mucus
In vertebrates, mucus is a slippery secretion produced by, and covering, mucous membranes. Mucous fluid is typically produced from mucous cells found in mucous glands. Mucous cells secrete products that are rich in glycoproteins and water. Mucous fluid may also originate from mixed glands, which...
, forming the cell capsule. The shape of the cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
is affected by the amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
glycine
Glycine
Glycine is an organic compound with the formula NH2CH2COOH. Having a hydrogen substituent as its 'side chain', glycine is the smallest of the 20 amino acids commonly found in proteins. Its codons are GGU, GGC, GGA, GGG cf. the genetic code.Glycine is a colourless, sweet-tasting crystalline solid...
which is present in the nutrient medium peptone.
Under microscope, the cells show inclusions, some of which are colored. In the early 1900s, the colored inclusions were regarded as "reproductive grains", or gonidia
Gonidium
A gonidium is an asexual reproductive cell or group of cells, especially in algae....
– a kind of embryo
Embryo
An embryo is a multicellular diploid eukaryote in its earliest stage of development, from the time of first cell division until birth, hatching, or germination...
cells. However, it was later demonstrated that the granules do not participate in the cell division. The colored grains are composed of volutin
Volutin granules
Volutin granules are an intracytoplasmic storage form of complexed inorganic polyphosphate, the production of which is used as one of the identifying criteria when attempting to isolate Corynebacterium diphtheriae on Löffler's medium....
whereas the colorless inclusions are drops of fat, which act as energy reserves.
Cysts
Cysts of the genus Azotobacter are more resistant to adverse environmental factors than the vegetative cellsVegetative reproduction
Vegetative reproduction is a form of asexual reproduction in plants. It is a process by which new individuals arise without production of seeds or spores...
, in particular, they are twice more resistant to UV
Ultraviolet
Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...
light. They are also resistant to drying, ultrasound
Ultrasound
Ultrasound is cyclic sound pressure with a frequency greater than the upper limit of human hearing. Ultrasound is thus not separated from "normal" sound based on differences in physical properties, only the fact that humans cannot hear it. Although this limit varies from person to person, it is...
and gamma
Gamma ray
Gamma radiation, also known as gamma rays or hyphenated as gamma-rays and denoted as γ, is electromagnetic radiation of high frequency . Gamma rays are usually naturally produced on Earth by decay of high energy states in atomic nuclei...
and solar irradiation, but not to heating.
The formation of cysts is induced by changes in the concentration of nutrients in the medium and addition of some organic substances such as ethanol
Ethanol
Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a...
, n-butanol
Butanol
Butanol or butyl alcohol can refer to any of the four isomeric alcohols of formula C4H9OH:*n-Butanol, butan-1-ol, 1-butanol, n-butyl alcohol;*Isobutanol, 2-methylpropan-1-ol, isobutyl alcohol;...
or β-hydroxybutyrate. Cysts are rarely formed in liquid media. The formation of cysts is induced by chemical factors and is accompanied by metabolic
Metabolism
Metabolism is the set of chemical reactions that happen in the cells of living organisms to sustain life. These processes allow organisms to grow and reproduce, maintain their structures, and respond to their environments. Metabolism is usually divided into two categories...
shifts, changes in catabolism
Catabolism
Catabolism is the set of metabolic pathways that break down molecules into smaller units and release energy. In catabolism, large molecules such as polysaccharides, lipids, nucleic acids and proteins are broken down into smaller units such as monosaccharides, fatty acids, nucleotides, and amino...
, respiration
Cellular respiration
Cellular respiration is the set of the metabolic reactions and processes that take place in the cells of organisms to convert biochemical energy from nutrients into adenosine triphosphate , and then release waste products. The reactions involved in respiration are catabolic reactions that involve...
and biosynthesis
Biosynthesis
Biosynthesis is an enzyme-catalyzed process in cells of living organisms by which substrates are converted to more complex products. The biosynthesis process often consists of several enzymatic steps in which the product of one step is used as substrate in the following step...
of macromolecule
Macromolecule
A macromolecule is a very large molecule commonly created by some form of polymerization. In biochemistry, the term is applied to the four conventional biopolymers , as well as non-polymeric molecules with large molecular mass such as macrocycles...
s; it is also affected by aldehyde dehydrogenase
Aldehyde dehydrogenase
Aldehyde dehydrogenases are a group of enzymes that catalyse the oxidation of aldehydes.- Function :Aldehyde dehydrogenase is a polymorphic enzyme responsible for the oxidation of aldehydes to carboxylic acids, which leave the liver and are metabolized by the body’s muscle and heart...
and the response regulator AlgR.
The cysts of Azotobacter are spherical and consist of the so-called central body – a reduced copy of vegetative cells with several vacuole
Vacuole
A vacuole is a membrane-bound organelle which is present in all plant and fungal cells and some protist, animal and bacterial cells. Vacuoles are essentially enclosed compartments which are filled with water containing inorganic and organic molecules including enzymes in solution, though in certain...
s – and the two-layer shell. The inner part of the shell is called intine and has a fibrous structure. The outer part has a hexagonal crystalline structure and is called exine. Exine is partially hydrolyzed by trypsin
Trypsin
Trypsin is a serine protease found in the digestive system of many vertebrates, where it hydrolyses proteins. Trypsin is produced in the pancreas as the inactive proenzyme trypsinogen. Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when...
and is resistant to lysozyme
Lysozyme
Lysozyme, also known as muramidase or N-acetylmuramide glycanhydrolase, are glycoside hydrolases, enzymes that damage bacterial cell walls by catalyzing hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between...
, in contrast to the central body. The central body can be isolated in a viable state by some chelation
Chelation
Chelation is the formation or presence of two or more separate coordinate bonds between apolydentate ligand and a single central atom....
agents. The main constituents of the outer shell are alkylresorcinols
Alkylresorcinols
Alkylresorcinols are phenolic lipids present in high amounts in the bran layer of wheat and rye...
composed of long aliphatic
Aliphatic compound
In organic chemistry, aliphatic compounds are acyclic or cyclic, non-aromatic carbon compounds.Thus, aliphatic compounds are opposite to aromatic compounds.- Structure :...
chains and aromatic
Aromaticity
In organic chemistry, Aromaticity is a chemical property in which a conjugated ring of unsaturated bonds, lone pairs, or empty orbitals exhibit a stabilization stronger than would be expected by the stabilization of conjugation alone. The earliest use of the term was in an article by August...
rings. Alkylresorcinols are also found in other bacteria, animals and plants.
Germination of cysts
A cyst of the genus Azotobacter is the resting form of a vegetativeVegetative reproduction
Vegetative reproduction is a form of asexual reproduction in plants. It is a process by which new individuals arise without production of seeds or spores...
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. Following the resumption of optimal environmental conditions, which include a certain value of pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
, temperature and source of carbon
Carbon
Carbon is the chemical element with symbol C and atomic number 6. As a member of group 14 on the periodic table, it is nonmetallic and tetravalent—making four electrons available to form covalent chemical bonds...
, the cysts germinate, and the newly formed vegetative cells multiply via a simple division
Asexual reproduction
Asexual reproduction is a mode of reproduction by which offspring arise from a single parent, and inherit the genes of that parent only, it is reproduction which does not involve meiosis, ploidy reduction, or fertilization. A more stringent definition is agamogenesis which is reproduction without...
. 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
Refraction
Refraction is the change in direction of a wave due to a change in its speed. It is essentially a surface phenomenon . The phenomenon is mainly in governance to the law of conservation of energy. The proper explanation would be that due to change of medium, the phase velocity of the wave is changed...
by cysts, which is detected with phase contrast microscopy
Phase contrast microscopy
Phase contrast microscopy is an optical microscopy illumination technique of great importance to biologists in which small phase shifts in the light passing through a transparent specimen are converted into amplitude or contrast changes in the image.A phase contrast microscope does not require...
. Germination of cysts is a slow process that takes about 4–6 hours. During germination, the central body grows and captures the granules of volutin, which were located in the intima
Tunica intima
The tunica intima is the innermost layer of an artery or vein. It is made up of one layer of endothelial cells and is supported by an internal elastic lamina...
(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
Oxygen
Oxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
and emit carbon dioxide
Carbon dioxide
Carbon dioxide is a naturally occurring chemical compound composed of two oxygen atoms covalently bonded to a single carbon atom...
; the rate of this process gradually increases and saturates after 4 hours. The synthesis of protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s and RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
, occurs in parallel, but it intensifies only after 5 hours after the addition of the carbon source. The synthesis of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
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, lipid
Lipid
Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins , monoglycerides, diglycerides, triglycerides, phospholipids, and others...
s and proteins and has almost the same volume as the central body. During germination of cysts, the intima hydrolyses
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...
and is used by the cell for the synthesis its components.
Physiological properties
Azotobacter respire aerobically, receiving energy from redoxRedox
Redox reactions describe all chemical reactions in which atoms have their oxidation state changed....
reactions, using organic compounds as electron donor
Electron donor
An electron donor is a chemical entity that donates electrons to another compound. It is a reducing agent that, by virtue of its donating electrons, is itself oxidized in the process....
s. Azotobacter can use a variety of carbohydrates, alcohol
Alcohol
In chemistry, an alcohol is an organic compound in which the hydroxy functional group is bound to a carbon atom. In particular, this carbon center should be saturated, having single bonds to three other atoms....
s and salts of organic acid
Organic acid
An organic acid is an organic compound with acidic properties. The most common organic acids are the carboxylic acids, whose acidity is associated with their carboxyl group –COOH. Sulfonic acids, containing the group –SO2OH, are relatively stronger acids. The relative stability of the conjugate...
s as sources of carbon and can fix at least 10 micrograms of nitrogen per gram of glucose consumed. Nitrogen fixation requires molybdenum
Molybdenum
Molybdenum , is a Group 6 chemical element with the symbol Mo and atomic number 42. The name is from Neo-Latin Molybdaenum, from Ancient Greek , meaning lead, itself proposed as a loanword from Anatolian Luvian and Lydian languages, since its ores were confused with lead ores...
ions, but they can be partially replaced by vanadium
Vanadium
Vanadium is a chemical element with the symbol V and atomic number 23. It is a hard, silvery gray, ductile and malleable transition metal. The formation of an oxide layer stabilizes the metal against oxidation. The element is found only in chemically combined form in nature...
ions. The source of nitrogen can be nitrate
Nitrate
The nitrate ion is a polyatomic ion with the molecular formula NO and a molecular mass of 62.0049 g/mol. It is the conjugate base of nitric acid, consisting of one central nitrogen atom surrounded by three identically-bonded oxygen atoms in a trigonal planar arrangement. The nitrate ion carries a...
s, ammonium
Ammonia
Ammonia is a compound of nitrogen and hydrogen with the formula . It is a colourless gas with a characteristic pungent odour. Ammonia contributes significantly to the nutritional needs of terrestrial organisms by serving as a precursor to food and fertilizers. Ammonia, either directly or...
ions or amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
s. The optimal pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
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 mixotroph
Mixotroph
A mixotroph is a microorganism that can use a mix of different sources of energy and carbon. Possible are alternations between photo- and chemotrophy, between litho- and organotrophy, between auto- and heterotrophy or a combination of it...
ically, in a nitrogen-free medium containing mannose
Mannose
Mannose is a sugar monomer of the aldohexose series of carbohydrates. Mannose is a C-2 epimer of glucose. It is not part of human metabolism, but is a component of microbial cell walls, and is therefore a target of the immune system and also of antibiotics....
; this growth mode is hydrogen dependent. Hydrogen is available in the soil, thus this growth mode may occur in nature.
While growing, Azotobacter produce flat, slimy, paste-like colonies with a diameter of 5–10 mm, which may form films in liquid nutrient media. The colonies can have dark brown, green and other colors, or may be colorless, depending on the species. The growth is favored at a temperature of 20–30 °C.
Pigments
Azotobacter produce pigmentPigment
A pigment is a material that changes the color of reflected or transmitted light as the result of wavelength-selective absorption. This physical process differs from fluorescence, phosphorescence, and other forms of luminescence, in which a material emits light.Many materials selectively absorb...
s. For example, Azotobacter chroococcum forms a dark-brown water-soluble pigment melanin
Melanin
Melanin is a pigment that is ubiquitous in nature, being found in most organisms . In animals melanin pigments are derivatives of the amino acid tyrosine. The most common form of biological melanin is eumelanin, a brown-black polymer of dihydroxyindole carboxylic acids, and their reduced forms...
. This process occurs at high levels of metabolism during the fixation of nitrogen and, is thought to protect the nitrogenase
Nitrogenase
Nitrogenases are enzymes used by some organisms to fix atmospheric nitrogen gas . It is the only known family of enzymes that accomplish this process. Dinitrogen is quite inert because of the strength of its N-N triple bond...
system from oxygen. Other Azotobacter species produce pigments from yellow-green to purple colors, including a green pigment which fluoresces
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
with a yellow-green light and a pigment with blue-white fluorescence.
Genome
The nucleotideNucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
sequence of chromosomes of Azotobacter vinelandii
Azotobacter vinelandii
Azotobacter vinelandii is diazotroph that can fix nitrogen while grown aerobically. It is a genetically tractable system that is used to study nitrogen fixation...
, strain AvOP is partially determined. This chromosome is a circular DNA molecule which contains 5,342,073 nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
pairs and 5,043 genes, of which 4,988 encode proteins. The fraction of guanine
Guanine
Guanine is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine . In DNA, guanine is paired with cytosine. With the formula C5H5N5O, guanine is a derivative of purine, consisting of a fused pyrimidine-imidazole ring system with...
+ cytosine
Cytosine
Cytosine is one of the four main bases found in DNA and RNA, along with adenine, guanine, and thymine . It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attached . The nucleoside of cytosine is cytidine...
pairs is 65 mole
Mole (unit)
The mole is a unit of measurement used in chemistry to express amounts of a chemical substance, defined as an amount of a substance that contains as many elementary entities as there are atoms in 12 grams of pure carbon-12 , the isotope of carbon with atomic weight 12. This corresponds to a value...
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 plasmid
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
s.
Distribution
Azotobacter are ubiquitous in neutralSoil pH
The soil pH is a measure of the acidity or basicity in soils. pH is defined as the negative logarithm of the activity of hydrogen ions in solution. It ranges from 0 to 14, with 7 being neutral. A pH below 7 is acidic and above 7 is basic. Soil pH is considered a master variable in soils as it...
and weakly basic soils
Alkali soils
Alkali, or alkaline, soils are clay soils with high pH , a poor soil structure and a low infiltration capacity. Often they have a hard calcareous layer at 0.5 to 1 metre depth. Alkali soils owe their unfavorable physico-chemical properties mainly to the dominating presence of sodium carbonate...
, 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 freshwater and brackish marshes. Several members are associated with plants and are found in the rhizosphere
Rhizosphere
The rhizosphere is the narrow region of soil that is directly influenced by root secretions and associated soil microorganisms. Soil which is not part of the rhizosphere is known as bulk soil. The rhizosphere contains many bacteria that feed on sloughed-off plant cells, termed rhizodeposition, and...
, having certain relationships with the plant. Some strains are also found in the cocoons of the earthworm Eisenia fetida
Eisenia fetida
Eisenia fetida , known under various common names such as redworm, brandling worm, tiger worm and red wiggler worm, red californian earth worm, is a species of earthworm adapted to decaying organic material. These worms thrive in rotting vegetation, compost, and manure; they are epigeal...
.
Nitrogen fixation
Azotobacter are free-living nitrogen-fixing bacteria, in contrast to RhizobiumRhizobium
Rhizobium is a genus of Gram-negative soil bacteria that fix nitrogen. Rhizobium forms an endosymbiotic nitrogen fixing association with roots of legumes and Parasponia....
species, they normally fix molecular nitrogen from the atmosphere without symbiotic
Symbiosis
Symbiosis is close and often long-term interaction between different biological species. In 1877 Bennett used the word symbiosis to describe the mutualistic relationship in lichens...
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 have a full range of enzymes needed to perform the nitrogen fixation: ferredoxin
Ferredoxin
Ferredoxins are iron-sulfur proteins that mediate electron transfer in a range of metabolic reactions. The term "ferredoxin" was coined by D.C. Wharton of the DuPont Co...
, hydrogenase
Hydrogenase
A hydrogenase is an enzyme that catalyses the reversible oxidation of molecular hydrogen . Hydrogenases play a vital role in anaerobic metabolism....
and an important enzyme nitrogenase
Nitrogenase
Nitrogenases are enzymes used by some organisms to fix atmospheric nitrogen gas . It is the only known family of enzymes that accomplish this process. Dinitrogen is quite inert because of the strength of its N-N triple bond...
. The process of nitrogen fixation requires an influx of energy in the form of adenosine triphosphate
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
(ATP). Nitrogen fixation is highly sensitive to the presence of oxygen, and therefore Azotobacter developed a special defensive mechanism against oxygen, namely a significant intensification of metabolism that reduces the concentration of oxygen in the cells. There is also a special protein shethna, which protects nitrogenase and is involved in protecting the cells from oxygen. Mutant
Mutant
In biology and especially genetics, a mutant is an individual, organism, or new genetic character, arising or resulting from an instance of mutation, which is a base-pair sequence change within the DNA of a gene or chromosome of an organism resulting in the creation of a new character or trait not...
s not producing this protein, are killed by oxygen during nitrogen fixation in the absence of a nitrogen source in the medium. Homocitrate
Homocitric acid
Homocitric acid is an organic compound with the formula HOC. This tricarboxylic acid occurs naturally as a component of the iron-molybdenum cofactor of certain nitrogenase proteins...
ions play a certain role in the processes of nitrogen fixation by Azotobacter.
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 vanadiumVanadium nitrogenase
Vanadium nitrogenase is a key enzyme for nitrogen fixation found in nitrogen-fixing bacteria, and is used as an alternative to molybdenum nitrogenase when molybdenum is unavailable. An important component of the nitrogen cycle, vanadium nitrogenase converts nitrogen gas to ammonia, thereby making...
; 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
Flavoprotein
Flavoproteins are proteins that contain a nucleic acid derivative of riboflavin: the flavin adenine dinucleotide or flavin mononucleotide ....
NifL which modulates the activation of gene transcription of nitrogen fixation by redox
Redox
Redox reactions describe all chemical reactions in which atoms have their oxidation state changed....
-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 synthesize some biologically active substances, including some phytohormones such as auxins, thereby stimulating plant growth. They also facilitate the mobility of heavy metals in the soil and thus enhance bioremediationBioremediation
Bioremediation is the use of microorganism metabolism to remove pollutants. Technologies can be generally classified as in situ or ex situ. In situ bioremediation involves treating the contaminated material at the site, while ex situ involves the removal of the contaminated material to be treated...
of soil from heavy metals, such as cadmium
Cadmium
Cadmium is a chemical element with the symbol Cd and atomic number 48. This soft, bluish-white metal is chemically similar to the two other stable metals in group 12, zinc and mercury. Similar to zinc, it prefers oxidation state +2 in most of its compounds and similar to mercury it shows a low...
, mercury
Mercury (element)
Mercury is a chemical element with the symbol Hg and atomic number 80. It is also known as quicksilver or hydrargyrum...
and lead
Lead
Lead is a main-group element in the carbon group with the symbol Pb and atomic number 82. Lead is a soft, malleable poor metal. It is also counted as one of the heavy metals. Metallic lead has a bluish-white color after being freshly cut, but it soon tarnishes to a dull grayish color when exposed...
. Some kinds of Azotobacter can also biodegrade chlorine
Chlorine
Chlorine is the chemical element with atomic number 17 and symbol Cl. It is the second lightest halogen, found in the periodic table in group 17. The element forms diatomic molecules under standard conditions, called dichlorine...
-containing aromatic compounds, such as 2,4,6-trichlorophenol
2,4,6-Trichlorophenol
2,4,6-Trichlorophenol, also known as TCP, phenaclor, Dowicide 2S, Dowcide 2S, omal, is a chlorinated phenol that has been used as a fungicide, herbicide, insecticide, antiseptic, defoliant, and glue preservative. It is a yellow solid with a strong, sweet odour...
. The latter was previously used as an insecticide
Insecticide
An insecticide is a pesticide used against insects. They include ovicides and larvicides used against the eggs and larvae of insects respectively. Insecticides are used in agriculture, medicine, industry and the household. The use of insecticides is believed to be one of the major factors behind...
, fungicide
Fungicide
Fungicides are chemical compounds or biological organisms used to kill or inhibit fungi or fungal spores. Fungi can cause serious damage in agriculture, resulting in critical losses of yield, quality and profit. Fungicides are used both in agriculture and to fight fungal infections in animals...
and herbicide
Herbicide
Herbicides, also commonly known as weedkillers, are pesticides used to kill unwanted plants. Selective herbicides kill specific targets while leaving the desired crop relatively unharmed. Some of these act by interfering with the growth of the weed and are often synthetic "imitations" of plant...
but later found to have mutagen
Mutagen
In genetics, a mutagen is a physical or chemical agent that changes the genetic material, usually DNA, of an organism and thus increases the frequency of mutations above the natural background level. As many mutations cause cancer, mutagens are therefore also likely to be carcinogens...
ic and carcinogen
Carcinogen
A carcinogen is any substance, radionuclide, or radiation that is an agent directly involved in causing cancer. This may be due to the ability to damage the genome or to the disruption of cellular metabolic processes...
ic effects.
Applications
Owing to its ability to fix molecular nitrogen and therefore increase the soil fertility and stimulate plant growth, Azotobacter are widely used in agriculture, particularly in nitrogen biofertilizerBiofertilizer
A bio-fertilizer is a substance which contains living microorganisms which, when applied to seed, plant surfaces, or soil, colonizes the rhizosphere or the interior of the plant and promotes growth by increasing the supply or availability of primary nutrients to the host plant.Bio-fertilizers add...
s such as azotobacterin. They are also used in production of alginic acid
Alginic acid
Alginic acid, also called algin or alginate, is an anionic polysaccharide distributed widely in the cell walls of brown algae, where it, through binding water, forms a viscous gum. In extracted form it absorbs water quickly; it is capable of absorbing 200-300 times its own weight in water. Its...
(E400), which is applied in medicine as an antacid
Antacid
An antacid is a substance which neutralizes stomach acidity.-Mechanism of action:Antacids perform a neutralization reaction, increasing the pH to reduce acidity in the stomach. When gastric hydrochloric acid reaches the nerves in the gastrointestinal mucosa, they signal pain to the central nervous...
, in the food industry as an additive to ice cream, puddings and creams, and in the biosorption of metals.
Taxonomy
The Azotobacter genus was discovered in 1901 by the Dutch microbiologist and botanist Martinus Beijerinck, who was one of the founders of environmental microbiologyEnvironmental microbiology
Environmental microbiology is the study of the composition and physiology of microbial communities in the environment. The environment in this case means the soil, water, air and sediments covering the planet and can also include the animals and plants that inhabit these areas...
. He selected and described the species Azotobacter chroococcum – the first aerobic
Aerobic organism
An aerobic organism or aerobe is an organism that can survive and grow in an oxygenated environment.Faculitative anaerobes grow and survive in an oxygenated environment and so do aerotolerant anaerobes.-Glucose:...
, free-living nitrogen fixer.
In 1903, Lipman described Azotobacter vinelandii
Azotobacter vinelandii
Azotobacter vinelandii is diazotroph that can fix nitrogen while grown aerobically. It is a genetically tractable system that is used to study nitrogen fixation...
, 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 of Azotobacter nigricans subsp. nigricans and Azotobacter nigricans subsp. achromogenes; in the same year, Thompson and Skerman described the . In 1991, Page and Shivprasad reported a microaerophilic and air-tolerant type which was dependent on sodium
Sodium
Sodium is a chemical element with the symbol Na and atomic number 11. It is a soft, silvery-white, highly reactive metal and is a member of the alkali metals; its only stable isotope is 23Na. It is an abundant element that exists in numerous minerals, most commonly as sodium chloride...
ions.
Earlier, representatives of the genus were assigned to the family Azotobacteraceae , 1933, but then were transferred to the family Pseudomonadaceae
Pseudomonadaceae
The Pseudomonadaceae is a family of bacteria that includes the genera Azomonas, Azomonotrichon, Azorhizophilus, Azotobacter, Cellvibrio, Mesophilobacter, Pseudomonas , Rhizobacter, Rugamonas, and Serpens...
based on the studies of nucleotide sequences 16S rRNA. In 2004, a phylogenetic
Phylogenetics
In biology, phylogenetics is the study of evolutionary relatedness among groups of organisms , which is discovered through molecular sequencing data and morphological data matrices...
study revealed that Azotobacter vinelandii
Azotobacter vinelandii
Azotobacter vinelandii is diazotroph that can fix nitrogen while grown aerobically. It is a genetically tractable system that is used to study nitrogen fixation...
belongs to the same clade
Clade
A clade is a group consisting of a species and all its descendants. In the terms of biological systematics, a clade is a single "branch" on the "tree of life". The idea that such a "natural group" of organisms should be grouped together and given a taxonomic name is central to biological...
as the bacterium Pseudomonas aeruginosa
Pseudomonas aeruginosa
Pseudomonas aeruginosa is a common bacterium that can cause disease in animals, including humans. It is found in soil, water, skin flora, and most man-made environments throughout the world. It thrives not only in normal atmospheres, but also in hypoxic atmospheres, and has, thus, colonized many...
, and in 2007 it was suggested that the genera Azotobacter, Azomonas
Azomonas
Azomonas are typically motile, oval to spherical, and secrete large quantities of capsular slime. They are distinguished from Azotobacter by their inability to form cysts, but like Azotobacter they can biologically fix nitrogen under aerobic conditions .-Etymology:The name Azomonas derives...
and Pseudomonas
Pseudomonas
Pseudomonas is a genus of gammaproteobacteria, belonging to the family Pseudomonadaceae containing 191 validly described species.Recently, 16S rRNA sequence analysis has redefined the taxonomy of many bacterial species. As a result, the genus Pseudomonas includes strains formerly classified in the...
are related and might be synonyms
Synonym (taxonomy)
In scientific nomenclature, a synonym is a scientific name that is or was used for a taxon of organisms that also goes by a different scientific name. For example, Linnaeus was the first to give a scientific name to the Norway spruce, which he called Pinus abies...
.