Diversity of Nitrogen Fixing Microorganisms in Soil


Carbon and nitrogen are the two main elements essential for life because they are both part of DNA and RNA bases. Nitrogen is also an important part of amino acids, amino sugars and numerous cofactors. The average bacterial cell is approximately 13 percent nitrogen, and relies on this element to create and repair its nucleic acids and proteins. Though many use ammonia to satisfy this requirement, and some are able to use nitrates, one group of bacteria has the unique ability to reduce atmospheric nitrogen from the air into ammonia. These bacteria are found throughout nature and are even able to live and thrive in environments where ammonia and nitrates are limited or not available for consumption [5]. This research review evaluates nitrogen fixation by diazotrophs by surveying recent scientific literature to foster an understanding of nitrogen-fixation as it relates to soil microbe diversity.

Nitrogen Fixation

Nitrogen is the most abundant gas in Earth’s atmosphere, but as N2, it is unusable for the many plants that rely on its availability for growth. Fortunately, a diverse range of microbes can reduce atmospheric N2 into the usable form ammonia [9]. This occurs because these specialized microbes, called diazotrophs, have a dinitrogenase and dinitrogenase reductase enzyme complex called nitrogenase. The dinitrogenase reductase part of this enzyme group must be protected from exposure to O2, which would immediately render it inactive. Some microbes accomplish this through symbiotic relationships with plants like legumes, and others have specialized protective mechanisms like slime layers that keep the enzyme from contacting atmospheric oxygen [5].

These diazotrophs either use the product, ammonia, for their own metabolism and production of nucleic acids and proteins, or they share it through a symbiotic relationship with a plant. Plants convert this usable form of nitrogen into further growth of their own systems, which eventually allows the nitrogen to move up the food chain to animals and humans [9].

Soil Microbes

Diazotrophs are found across the planet in aquatic and terrestrial environments, but are most abundant and most diverse in soil. Some are free-living and others are symbiotic organisms. Diazotrophs identified from an Amazon rain forest soil sample include bacteria in the following phyla: Actinobacteria, Bacteriodetes, Firmicutes, and Proteobacteria. Researchers identified these free-living, nitrogen-fixing bacteria by growing them on a nitrogen-free medium. After this process, isolates were identified using 16s rRNA gene sequencing data. Bacteria identified include: 78 percent Pseudomonas, 8 percent Rahnella, 4 percent Brevundimonas, 3 percent Flavobacterium, 2 percent Enterobacter, 2 percent Rhizobium, 2 percent Xanthomonas, and 1 percent Burkholderia. Pseudomonas showed a high level of competition when plated with other diazotrophs, which may account for the high percentage of these bacteria on the final plate [10].

One way to identify diazotrophs is to identify any present nif genes. These genes code for the aforementioned enzyme complex that gives the bacteria the ability to fix nitrogen [3]. The many varied members of the soil microbiome display a wide array of effects on associated plant life – from positive growth promotion to detrimental pathogenic activities, and the microbes in turn depend on specific plant species to create an environment in which they can flourish [1]. But plant/microbe dependence is not always tied as closely together as it is with Rhizobia and legume root nodules [6]. Sometimes this dependence is more about what each entity contributes to the evolving soil chemistry and how that contribution affects future plant life, thereby influencing the diversity of future bacterial colonies [1].

One of the most commonly discussed microbes that fixes atmospheric nitrogen is the species Rhizobium. Associated with legumes, for example Phaseolus vulgaris or the common bean, Rhizobium takes up residence in the plants roots, creating nodules in which oxygen levels remain low enough to not inactivate dinitrogenase reductase. Mnasri et al. sequenced the 16s rRNA genetic information from a sampling of nodules to assess the diversity of microbes within the plant and compared it to the GenBank repository of data [6], maintained by the National Institute of Health’s National Center for Biotechnology Information [8].

Kumar et al. uncovered a high diversity of diazotrophs in both arid and semi-arid Indian soils, and identified them based on ribosomal DNA sequences. They found one strain of alpha Proteobacteria: Agrobacterium tumefaciens MM10, and one strain of and gamma Proteobacteria: Stenotrophomonas sp. MM35, as well as eight strains of Firmicutes: Paenibacillus sp. MM38, Bacillus firmus NFB28, Bacillus sp. NFB4, Bacillus pumilus MM15, Bacillus sp. MM80, Bacillus sp. MDB 10(3), Bacillus pumilus DB 15(3), and Bacillus pumilus NFB1. Notable characteristics of these bacteria include that: 80 percent of the species were Gram-positive, none of them are able to convert tryptophan into indole, none have the enzyme urease to break down urea into ammonia and carbon dioxide, 80 percent are esculin positive, and only 20 percent can reduce nitrate [4].

As with most plants, rice crop growth is limited by the amount of available nitrogen. To compensate, in the case of Oryza alta (O. alta) wild rice, it has developed symbiotic relationships with a number of nitrogen-fixing bacteria including Acetobacter diazotrophicus, Acinetobacter johnsonii, Alcaligenes spp., Azospirillum spp., Bradyrhizobium spp., Burkholderia fungorum, Enterobacter cloacae, Herbaspirillum spp., Ideonella spp., Klebsiella oxytoca, and Pseudomonas oleovorans. Chaudry et al. grew bacteria from the rice sample on nitrogen-free plates and were able to find the nifH gene, thereby proving that these bacteria sourced from the roots of O. alta are diazotrophs. The research team also identified a new species, which they called Acinetobacter oryzae for the rice on which it was first identified [2].

Researchers in Malaysia identified 38 diazotrophs in rice and rice adjacent soil samples. In the soil they found mostly beta-Proteobacteria including Bacillus spp and Burkholderia sp., which were found on and in the roots of the rice samples. Area rice field soil samples showed a mixture of nitrogen-fixing species, including: Bacillus, Burkholderia vietnamiensis, Rhizobium, Stenotrophomonas maltophilia [7].

Diversity of bacteria, even diazotrophs, is directly related to the environment in which it is found. An alteration of plant species or nutrients inevitably causes an alignment of the accompanying microbes. In the case of nitrogen-fixing bacteria, which can be found free-living or in symbiotic relationships with plants, a lack of nitrogen allows it to thrive above other organisms which cannot reduce atmospheric N2 into a usable form [1]. Describing the diversity of diazotrophic organisms present is limited by the number of different environments scientists assess. As in the case of Chaudhary et al., the more researchers look for these microbes, the more they find – naming additional species and learning more about how the nitrogen fixation process benefits the entirety of the food chain [2].


  1. Bakker, M. G., Schlatter, D. C., Otto-Hanson, L., & Kinkel, L. L. (2014). Diffuse symbioses: roles of plant-plant, plant-microbe and microbe-microbe interactions in structuring the soil microbiome. Molecular Ecology, 23(6), 1571-1583. doi:10.1111/mec.12571
  2. Chaudhary, H. J., Peng, G., Hu, M., He, Y., Yang, L., Luo, Y., & Tan, Z. (n.d). Genetic Diversity of Endophytic Diazotrophs of the Wild Rice, Oryza alta and Identification of the New Diazotroph, Acinetobacter oryzae sp nov. Microbial Ecology, 63(4), 813-821.
  3. Gaby, J. C., & Buckley, D. H. (n.d). A global census of nitrogenase diversity. Environmental Microbiology, 13(7), 1790-1799.
  4. Kumar, V., Kayasth, M., Chaudhary, V., & Gera, R. (n.d). Diversity of diazotrophs in arid and semi-arid regions of Haryana and evaluation of their plant growth promoting potential on Bt-cotton and pearl millet. Annals Of Microbiology, 64(3), 1301-1313.
  5. Madigan, M. T., Martinko, J. M., Stahl, D. A., and Clark, D. P. (2012). Brock Biology of Microorganisms. San Francisco, CA. Benjamin Cummings.
  6. Mnasri, B., Liu, T. Y., Saidi, S., Chen, W. F., Chen, W. X., Zhang, X. X., & Mhamdi, R. (2014). Rhizobium azibense sp. nov., a nitrogen fixing bacterium isolated from root-nodules of Phaseolus vulgaris. International Journal Of Systematic And Evolutionary Microbiology, 64(Pt 5), 1501-1506. doi:10.1099/ijs.0.058651-0
  7. Naher, U. A., Othman, R., Mohammad Abdul, L., Qurban Ali, P., Megat Amaddin, P. A., & Zulkifli H., S. (2013). Biomolecular Characterization of Diazotrophs Isolated from the Tropical Soil in Malaysia. International Journal Of Molecular Sciences, 14(9), 17812-17829. doi:10.3390/ijms140917812
  8. National Institutes of Health. (2014). Web. <www.ncbi.nlm.nih.gov/genbank> Retrieved 22 Nov. 2014.
  9. Philippot, L. & Germon, J. G. (2005). Chapter 8:Contribution of Bacteria to Initial Input and Cycling of Nitrogen in Soils. Microorganisms in Soils: Roles in Genesis and Functions. New York, NY: Springer Berlin Heidelberg New York.
  10. Mirza, B. S., & Rodrigues, J. M. (n.d). Development of a Direct Isolation Procedure for Free-Living Diazotrophs under Controlled Hypoxic Conditions. Applied and Environmental Microbiology, 78(16), 5542-5549.

Author: tatumlyles

Tatum Lyles Flick is a public relations practitioner, news and science writer, photographer, graphic designer and website designer with experience in industry, the news media, and academia.

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