What are Nitrogen Fixing Bacteria?


As the name suggests, nitrogen-fixing bacteria are a type of bacteria capable of converting molecular/atmospheric nitrogen (dinitrogen) into fixed nitrogen (e.g. ammonia) which can be used by plants and several other organisms. Generally, these bacteria are capable of forming a mutually beneficial relationship with different types of hosts (e.g. plants, some arthropods, etc.) which allows easy access to nutrients (e.g. carbon) while the host benefits from fixed nitrogen.

However, some of the species are free-living and can be found in soil and other aquatic habitats. Therefore, because of their diversity, nitrogen-fixing bacteria are well distributed in different environments across the world. 




Importance of Nitrogen-Fixing Bacteria



Nitrogen Fixation


Nitrogen is an important element required for the production of proteins, nucleic acids and amino acids, etc. in many organisms. While some microorganisms (bacteria) are capable of using nitrogen in its molecular form, most plants and insects like termites cannot use it in this form.

For this reason, nitrogen fixation is required to convert atmospheric nitrogen into a form that these organisms can use. This section will focus on the importance of nitrogen-fixing bacteria for termites and plants.  


In Termites


Termites are cellulose-eating insects belonging to the Phylum Arthropoda. Nitrogen-fixing bacteria can be found in the gut of these insects which allows some of them (termites) to survive in areas characterized by a nitrogen-poor diet. Termites largely feed on wood material which lacks or have very little nitrogen than they require.

Based on different types of studies, nitrogen-fixing bacteria found in different types of termites have been shown to vary from aerobic species to facultative anaerobes. Regardless, they are all involved in the conversion of molecular nitrogen into a form that can be used by termites. 

 

* Nitrogen fixing bacteria provide about 60 percent of a termite's nitrogen needs. The rest of the nitrogen is obtained through recycling of uric acid.


* Spirochete bacteria with genes associated with nitrogen fixation have also been identified in human beings and the intestinal tract of cows. However, compared to spirochetes found in the gut of termites, these bacteria are incapable of nitrogen fixation. 


* The removal of spirochetes from the gut of termites result in a reduced lifespan- This is largely attributed to the fact that these bacteria make up about 50 percent of the total prokaryotes in the gut of termites.

 


While termites are incapable of using molecular nitrogen (atmospheric nitrogen), they contain a variety of nitrogen-fixing bacteria in their gut. A mentioned, spirochetes have been shown to be the most abundant microorganisms in this region of the termite. 

Like many other nitrogen-fixing bacteria, spirochetes found in the gut of these hosts contain the enzyme nitrogenase produced through the expression of the nIfH gene. This is an important enzyme that plays a viral role in splitting the triple bond of molecular nitrogen (the bond that links the two nitrogen atoms together). 

By breaking the triple bond, the enzyme also plays an important role in the reduction of nitrogen into nitrogen metabolites. Based on calculations, it's estimated that these bacteria fix nitrogen at the rate of between 0.06 to 0.41 ng per hour. 

Reduced atmospheric nitrogen is important for the synthesis of various molecules including amino acids. 

 

* For some fungus-growing termites and ants, researchers have suggested that nitrogen fixing bacteria play an important role in this relationship. By fixing molecular nitrogen into a form that can be used by the host (termites and ants), the nitrogenous waste produced is thought to promote the decomposition of various substrates thereby providing the fungus with essential nutrients. 



By propagating and defending the fungus, the insect also benefits from the nutrients that the fungus provides - This relationship between the three organisms is also thought to be one of the main reasons the concentration of nitrogen in fungus gardens is high compared to the concentration of nitrogen from other plant leaves.

 

* Flagellated protists (e.g. members of the families Teranymphidae and Eucomonympha) in the gut of termites which are cellulolytic protists responsible for breaking down cellulose, also harbor nitrogen-fixing bacteria.


* By proving nitrogen in the form that can be used by termites (and some insects) and protists (in the gut of termites), these bacteria indirectly contribute to the decomposition of plant material. 


 

Other examples of nitrogen-fixing bacteria found in the gut of termites include:

 

·        Enterobacter species - They can be found in the gut of termite species like Cryptotennes primus Hill, Nasutitermes graveolus, Coptotermes lacteus, and Heterotermes ferox


·        Citrobacter freundii - Commonly found in the gut of Nasititermes exitiosus and Coptotermes zactues


·        Klebsiella species - Found in Mactoterme termites


·        Desulfovibrio desulfuricans - Found in Reticulitermes santonensis


·        Desulfovibrio termitidis - Found in Heterotermes indicola


·        Klebsiella pneumoniae- Found in Coptotermes formosanus 


·        Clostridium species - Found in Mactotermes termites 

 


Plants


Like a number of other organisms, plants are unable to use atmospheric nitrogen (dinitrogen gas). For this reason, they have to rely on ammonium and nitrates in the soil for nitrogen. 

Although synthetic nitrogen fertilizers provide plants with the nitrogen they need for development, they have been shown to present several disadvantages. 

One of the biggest issues with these fertilizers (especially when used inappropriately) is that they may affect the concentration of nitrate in soil and groundwater thus presenting health risks. As well, the manufacturer of these fertilizers has been shown to consume significant amounts of energy when compared to several other types of fertilizers. 

Plants not supplied with nitrogen fertilizer would die as a result of nitrogen deficiency despite the fact that they are surrounded by about 78 percent of nitrogen in the atmosphere. Luckily, there are different types of bacteria (both free-living and symbiotic) in nature that can convert nitrogen gas in the atmosphere into a form (e.g. ammonia) that can be used by plants. 



One of the best examples of a close relationship (symbiotic relationship) between nitrogen-fixing bacteria and plants is found in nodules of leguminous plants (clovers, peas, beans, and peanuts plants). 

Normally, members of the genus Rhizobium (diazotrophic bacteria involved in nitrogen fixation) are found in soil as free-living organisms. However, when legumes release flavonoids in response to low levels of nitrogen in the soil, these bacteria are stimulated to release nodulation factors (Nod factors) which influence the development of more root hairs allowing for successful infection of the roots by the bacteria. In response to this infection, cells of these root hairs undergo rapid differentiation to form root nodules. 

As is the case with spirochetes found in the gut of insects like ants and termites, the conversion of nitrogen (N2) to ammonia or nitrates by Rhizobia bacteria in the nodules is a complex process that involves nitrogenases enzymes. 

In this reduction process, nitrogen (atmospheric nitrogen) gains electrons to produce ammonia and hydrogen gas. This is also an energy-dependent process in which ATP is converted to ADP through hydrolysis. 

 

* Between 21 and 25 ATP molecules are required for each nitrogen that is fixed.


* During nitrogen fixation, the enzyme (Nitrogenase) is not only involved in breaking the bonds between the nitrogen atoms, but also in binding each of the nitrogen atoms to three atoms of hydrogen to form two molecules of ammonia from each atmospheric nitrogen. 

 


The ammonia produced through nitrogen fixation process is then transported to the cells of the plant through the symbiosome membrane where they are used for the synthesis of amino acids like glutamine which is in turn used for protein production.

 

* Nitrogen is one of the main components of chlorophyll, the pigment that captures light energy during photosynthesis. For this reason, nitrogen is essential for photosynthesis and thus plant development. 

 


Some examples of nitrogen-fixing bacteria associated with plants include:

 

·        Sinorhizobium melilotii - Found in Alfalfa plant


·        Rhizobium tropici and Rhizobium legumninosarum - Found in plants like Clover and beans


·        Mesorhizobium loti - Found in the nodules of Lotus plant


·        Rhizobium fredii and Bradyrhizobium japonicum - Found in the nodules of Soy bean 


·        Azorhizobium caulinodans - Found in Sesbania flowering plants 





Free-living Nitrogen Fixing Bacteria



Free-living nitrogen-fixing bacteria can be found in soil and aquatic environments.


They are divided into several major groups that include:

 

·        Strict/obligate anaerobes - These are nitrogen-fixing bacteria that only survive in habitats with very little or no oxygen. Examples of these bacteria include some species in the genus Clostridium and Archaea like Methanococcus thermolithotrophicus


·        Facultative anaerobes - Unlike strict anaerobes, facultative anaerobes can survive in the presence or absence of oxygen. In the presence of oxygen, ATP is produced through aerobic respiration. However, in the absence of oxygen, they can switch to fermentation. Some examples of these bacteria include members of the genera Klebsiella and Azospirillum.


·        Microaerophiles - Nitrogen-fixing bacteria that survive in conditions with low oxygen concentrations 


·        Photosynthetic bacteria - Nitrogen-fixing bacteria capable of transforming light energy into chemical energy (they are autotrophs). These include species like Rhodospirillum.


·        Rubrum and Cyanobacteria like Anabaena 

 

* For the most part, nitrogen fixation by free-living bacteria occurs under anaerobic conditions (or in conditions with very little oxygen). This is due to the fact that oxygen can inhibit the enzyme nitrogenase (the enzyme required for nitrogen fixation) - Products of nitrogen fixation, ammonia, and nitrates, are then absorbed by plants from the soil through the roots.


* In various plantations (e.g. wheat plantations in Australia), free-living nitrogen-fixing bacteria have been shown to contribute to between 30 and 50 percent of the total nitrogen needs. 



Nitrogen Cycle



The nitrogen cycle refers to the process through which nitrogen is transformed into different forms as it circulates through living organisms and non-living matter, the soil and aquatic environments, and the atmosphere in a repeating cycle. 

Because nitrogen is an important component of various life processes (required for the synthesis of amino acids, proteins, DNA, and chlorophyll, etc.), it has to be converted into a form that can be used by various living organisms. Here, nitrogen-fixing bacteria convert diffused nitrogen into ammonia and nitrates which can be used by plants and other organisms. 

Higher animals, on the other hand, obtain nitrogen-based compounds when they consume plants. When plants and animals die, denitrifying bacteria, which use nitrates as a source of energy, convert nitrates to gaseous nitrogen which is released back into the atmosphere. 

 


Uses of Nitrogen Fixing Bacteria



Large Scale Production of Nitrogen-Fixing Bacteria


In 1888, Salfeld noticed that by growing leguminous crops using soil from which the same crop had been grown, they not only became nodulated but also had much better yields. This led to the development of new methods for growing rhizobia bacteria for use by farmers. 

While nitrogenous fertilizers provide the nitrogen that plants need for development, researchers also noticed that these products contribute to soil and groundwater contamination thus presenting a risk to human health. 

For this reason, the use of biofertilizers in the form of nitrogen-fixing bacteria has become a much better alternative not only because they provide plants with nitrogen, but also act as biocontrol agents. As biocontrol agents, these biofertilizers inhibit the growth of various pathogens that would otherwise cause damage to the plant. 

 

* For small farmers who may be unable to afford biofertilizers, one of the nodulation methods often used involves exposing seeds to nitrogen-fixing bacteria removed from nodules of other crops. 

Here, the process involved washing the seeds in water which causes them to swell before exposing them to the bacteria obtained from freshly crushed nodules. The swelling causes the outer coat to loosen making it easier for the bacteria to attach.

 


Engineering Nitrogen-Fixing Bacteria



Due to the benefits associated with nitrogen-fixing bacteria over various nitrogenous fertilizers, researchers have been working to manipulate various bacteria species into nitrogen-fixing organisms. In recent years, Escherichia coli is one of the bacteria that has received significant attention in this endeavor.

This has proved successful by transferring the nif genes from diazotrophic bacteria (bacteria species capable of nitrogen fixation). This induced the bacteria to produce the enzyme nitrogenase involved in the nitrogen fixation process.

With the many issues associated with nitrogenous fertilizer, success in engineering many other microorganisms presents several advantages including increased yields with minimized diseases. 


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References



Carole Santi, Didier Bogusz, Claudine Franche. (2013). Biological nitrogen fixation in non-legume plants. 

 

Panagiotis Sapountzis et al. (2016). Potential for Nitrogen Fixation in the Fungus-Growing Termite Symbiosis.

 

Satoko Noda, Daichi Shimizu, Masahiro Yuki, Osamu Kitade, and Moriya Ohkuma. (2018). Host-Symbiont Cospeciation of Termite-Gut Cellulolytic Protists of the Genera Teranympha and Eucomonympha and their Treponema Endosymbionts.

 

Sarannia Thanganathan and Kamariah Hasan. (2018). Diversity of Nitrogen Fixing bacteria Associated with Various Termite Species.

 

Links

 

https://cen.acs.org/articles/87/i47/Nitrogen-Fixing-Insects.html#:~:text=AAAS%2F%C2%A9%20Science-,Leaf%2Dcutter%20ants%20rely%20on%20nitrogen%2Dfixing%20bacteria%20to%20supply,living%20symbiotically%20with%20the%20insects.

https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/nitrogen-fixing-bacteria

https://www.nature.com/scitable/knowledge/library/biological-nitrogen-fixation-23570419/