Document Type

Thesis - Open Access

Award Date

2016

Degree Name

Master of Science (MS)

Department

Biology and Microbiology

First Advisor

Volker S. Brozel

Keywords

bacteriology, soil biology, Bradyrhizobium, nif genes, free-living nitrogen fixing systems

Abstract

Biological Nitrogen Fixation is carried out by a highly diverse group of members of the domains Bacteria and Archaea which express a nitrogenase enzyme complex and are termed diazotrophs. The best known diazotrophs form symbiotic associations with plants, but free-living nitrogen fixing bacteria also contribute a substantial amount of nitrogen to ecosystems, including arid lands. The aim of this study was to isolate free-living diazotrophs from the soil crusts of sod tables of South Dakota Badlands. Samples were obtained from sod tables and the surrounding base (matrix) in spring and fall of 2014. A culture dependent approach was used to isolate diazotrophs on solid nitrogen free medium (NMF) under hypoxic conditions. The 16srRNA and nifH genes were amplified and sequenced. This was complemented using a culture independent approach, amplifying nifH from soil DNA extracts. The 16S rRNA gene data indicated a diversity of putative free-living diazotrophs across 4 phyla (Actinomycetes, Proteobacteria, Bacteroidetes, and Firmicutes), but ~50% of these clustered with Streptomyces. The Streptomyces isolates grew in liquid NFM in an ammonia-depleted environment in a sealed container. Only 5 of these yielded a nifH gene product using the PolF/PolR primer set. Of these four aligned with nifH of the cyanobacterium Scytonema (NCBI BLAST) and the other one aligned with nifH of Bradyrhizobium. All six selected Streptomyces isolates, three of which were nifH positive, indicated 15N2 incorporation providing strong support of nitrogen fixation. The culture independent method yielded a low diversity. All the nifH amplicons from soil DNA extract resembled cyanobacteria by NCBI BLAST. This is the first known report of a Streptomyces that is diazotrophic, other than the thermophilic, autotrophic S. thermoautotrophicus. The nifH genes of these Streptomyces were related to those from cyanobacteria. Whether the cyanobacteria-like nifH amplicons obtained from soil DNA were associated with Streptomyces, is intriguing to speculate.

Library of Congress Subject Headings

Nitrogen -- Fixation -- Badlands (S.D. and Neb.)
Nitrogen-fixing microorganisms -- Badlands (S.D. and Neb.)

Description

Includes bibliographical references (page 70-98)

Format

application/pdf

Number of Pages

117

Publisher

South Dakota State University

Rights

Copyright © 2016 Bibha Dahal

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