In bioenergy production systems, nitrogen is a critical macronutrient for algal growth and is mostly supplied as inorganic nitrogen molecules derived from the Haber-Bosch process. The Haber-Bosch process creates ammonia (NH 3) from N2 and H2 under high temperature and pressure, and with high CO2 emissions, making it a costly although necessary process for algae bioenergy production. Unfortunately, current algae production systems do not recycle nutrients like nitrogen and phosphorus efficiently. These nutrients are lost externally as waste biomass although attempts have been made to internally recycle nutrients back into the algae production system using methods like hydrothermal liquefaction and anaerobic digestion. The ability of many algae to survive on the generated organic and inorganic nitrogen sources are unknown, so two marine microalgae, Dunaliella viridis and Nannochloropsis oceanica, were tested for utilization of organic nitrogen sources.
D. viridis and N. oceanica are promising bioenergy sources with robust growth and high starch and lipid accumulation. However, little was known about nitrogen source usage of D. viridis and N. oceanica, especially amino acids, which are the major nitrogen-containing molecules in organisms. Mining of D. viridis and N. oceanica transcriptomes and genomes revealed intact inorganic nitrogen assimilation pathways and 10 and 27 putative amino acid transporters, respectively, for D. viridis and N. oceanica. However, growth tests showed that only D. viridis could uptake and utilize a single amino acid, histidine. Both algae showed growth on glutamine, tryptophan, and cysteine, but these amino acids naturally degrade into carbon skeleton and ammonia, a viable nitrogen source. Since amino acids were poor nitrogen sources for both algae two different research paths were pursued.
Ammonia is toxic to D. viridis even at low concentration (>1 mM) but can be provided slowly by degradation of glutamine. This was a convenient system to explore the differences in metabolism and gene transcription in D. viridis temporally using metabolic assays and RNA Sequencing. Cells grown on glutamine (ammonia) adopted a partial nitrogen starvation metabolic phenotype but maintained the same cell density as cells grown on nitrate. Analysis of the transcriptome revealed that rather than entering a different metabolic state due to ammonia, the cells were nitrogen starved as the metabolic data had suggested. The degradation of glutamine was too slow and thus the cells become starved.
Although nitrogen can be provided by degrading amino acids, genetic engineering with characterized amino acid transporters could allow for utilization of all amino acids. Since N. oceanica cannot utilize any amino acids, it was an ideal candidate for genetic modification. Eight amino acid transporter genes from yeast, Arabidopsis, and D. viridis were selected as candidates for expression in N. oceanica. Uncharacterized transporters were first screened for amino acid uptake in a mutant yeast strain with deficient amino acid transport. No transport was seen in the yeast lines with the D. viridis transporters, while the osmophilic yeast transporter INDA1 showed uptake of 13 amino acids. Two yeast transporters and the Arabidopsis transporter were transformed into N. oceanica. When grown on amino acids as a sole nitrogen source, increased growth was not demonstrated in the transgenic lines. This suggests that either the transporters were insufficient to allow growth on amino acids, or perhaps that the transgenes localized to a membrane other than the plasma membrane.
This work reveals a common transcriptional response to nitrogen starvation in D. viridis and hints at novel alkali-tolerant responses. It broadened the knowledge of the capability of algae to utilize nitrogen derived from exogenously supplied amino acids. Although modifying membrane transport is a complex process in algae, characterization of these transporters will lead to a deeper understanding of how algae interact with the environment.
|School:||North Carolina State University|
|School Location:||United States -- North Carolina|
|Source:||DAI-B 79/12(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Botany, Plant sciences|
|Keywords:||Amino acid transporter, Bioenergy, Marine, Microalgae, Nitrogen recycling, Transcriptome|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be