Document Type

Dissertation - Open Access

Award Date


Degree Name

Doctor of Philosophy (PhD)


Biology and Microbiology

First Advisor

William R. Gibbons


Anabaena sp. PCC 7120, biofuel, cyanobacteria, hydrocarbons, life cycle analysis, photobioreactors


The objectives of this project were to improve the industrial potential of filamentous N2-fixing cyanobacteria by increasing its biofuel tolerance, and to evaluate the economic feasibility and environmental impacts of a theoretical, cyanobacteria-based biofuel production facility. To develop a method to quantify filamentous cyanobacteria in dilute culture media, a dual-stained fluorescence assay was evaluated. While the viable cell stain (SYTO® 9) was accurate, the non-viable cell stain (propidium iodide) also bound to viable cells. Additional non-viable cell stains were evaluated, but none were accurate at quantifying viability. Thus we concluded that the viable cell stain SYTO® 9 is a reliable assay and can be used in high-throughput assays. To develop cyanobacteria strains with increased tolerance to biofuels, directed evolution under the pressure of higher biofuel concentrations was used. As these biofuels are highly volatile, it was necessary to conduct experiments in sealed test-tubes. Thus, cyanobacteria growth in a sealed environment was optimized using BG11 as the basal medium supplemented with 0.5 g/L NaHCO3 as the carbon source. Subsequent directed evolution trials yielded 3 confirmed mutants with increased biofuel tolerance: Nostoc punctiforme ATCC 29133 with a 20% improvement in linalool tolerance, Anabaena variabilis ATCC 29413 with a 60% improvement in linalool tolerance, and Anabaena sp. PCC 7120 with a 220% improvement in farnesene tolerance. To determine the optimal nitrogen source, dinitrogen (N2 gas) was compared to various fixed nitrogen sources. Ammonium chloride was determined to be the preferred nitrogen source for large scale cyanobacteria cultivation based on growth rate and environmental impacts. Finally, an economic feasibility and a life cycle analysis were conducted on a theoretical limonene production facility that used a genetically engineered filamentous cyanobacteria strain. The facility was not economically feasible at current limonene productivity rates, but would be feasible if productivity can be increased 56.7-fold. The life cycle analysis showed that increasing limonene productivity worsens the environmental profile of the facility. While using filamentous N2-fixing cyanobacteria as industrial microorganisms is currently in its infancy, there is a great deal of potential for this microbe to become a significant contributor to renewable biofuels and high-value chemicals.

Library of Congress Subject Headings

Cyanobacteria -- Biotechnology

Biomass energy

Biomass conversion -- Testing

Renewable energy sources


Includes bibliographical references (page 277-330)



Number of Pages



South Dakota State University


© 2016 Tylor J. Johnson