Document Type

Dissertation - Open Access

Award Date


Degree Name

Doctor of Philosophy (PhD)

Department / School

Biology and Microbiology

First Advisor

Volker S. Brözel


Culture-based, Culture-independent, Diazotrophs, N-cycle, Nitrogenase, Streptomyces


Biological nitrogen fixation contributes to half of the global supply of nitrogen to the biosphere. It is carried out by a diverse group of prokaryotes called diazotrophs via the nitrogenase enzyme. Nitrogen fixation research is focused on the narrow group of symbiotic diazotrophs, and the vast majority of free-living diazotrophs which contribute significantly to fixed nitrogen are yet to be explored. The goal of this research was to access phylogeny of diazotrophs considering the most up-to-date genomic information and apply that knowledge to understand the diversity of free-living diazotrophs in a natural grassland ecosystem, both by culture dependent and independent methods. Phylogeny was reconstructed using the concatenated sequences of six core proteins of nitrogenase (NifHDKENB) from 963 prokaryotic genomes. The diversity of free-living diazotrophs in grassland was explored by isolation of putative diazotrophs on a solid nitrogen free medium (NFM) and diazotrophy confirmed by nifH PCR, acetylene reduction assay and 15N2 assimilation assay. Streptomyces, the most abundant bacteria, was further characterized by sequencing the genome of one prominent strain, and differential gene expression in nitrogen rich Vs nitrogen deficient medium. For culture independent study of nitrogen cycle activity, meta-transcriptomic sequencing of complete mRNA from a grassland soil sample was performed. Phylogeny of nif genes from the complete genomes of cultured isolates revealed that diazotrophs are distributed across Actinobacteria, Aquificae, Bacteroidetes, Chlorobi, Chloroflexi, Cyanobacteria, Deferribacteres, Firmicutes, Fusobacteria, Nitrospira, Proteobacteria, PVC group, and Spirochaetes, as well as the Euryarchaeota, providing a curated database of nif genes. Culturing yielded 474 bacterial isolates which belonged to the phyla Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes. However, only 81 (17%) of isolates yielded nifH, and the most dominant genus isolated on NFM, Streptomyces did not provide biochemical and genomic evidence of diazotrophy. The meta-transcriptomic study revealed nitrogen fixation and nitrification are the least and nitrate reduction is the most expressed pathway among various nitrogen cycling pathways. In conclusion, although the culture-based approach showed diverse free-living nitrogen fixing bacteria, diazotrophy should always be confirmed by biochemical and genetic evidence, and limitations to culture independent study due to primer bias in nifH PCR can be overcome by meta-transcriptomic study.

Library of Congress Subject Headings

Nitrogen-fixing microorganisms.
Nitrogen cycle.
Nitrogen -- Fixation.

Number of Pages



South Dakota State University

Included in

Microbiology Commons



Rights Statement

In Copyright