Document Type

Thesis - Open Access

Award Date


Degree Name

Master of Science (MS)


Biology and Microbiology

First Advisor

Volker S. Brozel


bacillus, bacteria co-culture, free-living diazotrophs, natural prairie, nitrogen fixation, streptomyces


There are only two natural ways by which molecular nitrogen can be available to support life, either by free-living or by symbiotic nitrogen fixing bacteria. The best studied diazotrophs form symbiotic associations with plants, primarily legumes and certain tree species, but little is known about how non-leguminous plants such as grasses obtain nitrogen in their environment. Natural prairies have few legumes, thus have less symbiotic nitrogen fixer interaction. This indicates presence of free-living nitrogen (FLN) fixation activity towards the balance of the N cycle. The objective of this study was to characterize the culturable diversity of free-living diazotrophs in native prairie. I isolated bacteria using Nitrogen Free Medium (NFM). Bacteria growing on NFM were subcultured with repeated steps to obtain single isolates. The phylogeny tree of 458-putative diazotrophs was determined using the 16S rRNA gene. The PolF/PolR primer set was used for nifH PCR to confirm the presence of nitrogenase, required for the reduction of molecular nitrogen to ammonia. Due to poor performance of multiple nifH primer sets various approaches to improve amplification efficiency were evaluated focusing on addition of Dimethyl Sulfoxide and Bovine Serum Albumin, and glycerol alone or combination. Of 458 putative diazotrophs, 96 yielded nifH by PCR. The sequences were analyzed using ARB and R. High diversity was found among FLN fixers, including alpha-, beta-, and gamma – Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria, but the largest group were Streptomyces. Out of 115 Streptomyces, 40 yielded nifH amplicons aligning with those of unrelated taxa such Mesorhizobium, Paenibacillus, and Herbaspirillium. Whole genome sequencing of three Streptomyces revealed presence of Bacillus, indicating co-culture. Purity of the isolates obtained was confirmed using phylum-specific 16S rRNA gene primers for Actinobacteria and Firmicutes as well as alpha and beta – Proteobacteria. This revealed that many of the putative pure cultures had a second strain belonging to a different phylum. In conclusion, there is a considerable diversity in the free-living nitrogen fixing bacterial community in prairie soil. Additionally, our data indicate apparent bacterial bi-cultures associated with nitrogen cycling. These findings could help us understand the dynamics of nitrogen cycling in naturally sustained prairie grassland ecosystem.

Library of Congress Subject Headings

Nitrogen-fixing microorganisms.
Nitrogen -- Fixation.
Nitrogen cycle.
Prairie ecology -- Big Sioux River Valley (S.D. and Iowa)
Soil ecology -- Big Sioux River Valley (S.D. and Iowa)
Prairie ecology -- South Dakota.
Soil ecology -- South Dakota.


Includes bibliographical references



Number of Pages



South Dakota State University


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Microbiology Commons