Matching Items (29)

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Photosynthesis under Rocks: Hypolith Distribution across the Namib Desert Rainfall Gradient

Description

“Extremophile” is used to describe life that has adapted to extreme conditions and the conditions they live in are often used to understand the limits of life. In locations with

“Extremophile” is used to describe life that has adapted to extreme conditions and the conditions they live in are often used to understand the limits of life. In locations with low precipitation and high solar radiation, photosynthetic cyanobacteria can colonize the underside of quartz fragments, forming ‘hypoliths.’ The quartz provides protection against wind, reduces solar radiation, and slows the rate of evaporation following infrequent rain or fog events. In most desert systems, vascular plants are the main primary producers. However, hypoliths might play a key role in carbon fixation in hyperarid deserts that are mostly devoid of vegetation. I investigated hypolith distribution and carbon fixation at six sites along a rainfall and fog gradient in the central Namib Desert in Namibia. I used line point intersect transects to assess ground cover (bare soil, colonized quartz fragment, non-colonized quartz fragment, non-quartz rock, grass, or lichen) at each site. Additionally, at each site I selected 12 hypoliths and measured cyanobacteria colonization on quartz and measured CO2 flux of hypoliths at five of the six sites.
Ground cover was fairly similar among sites, with bare ground > non-colonized quartz fragments > colonized quartz fragments > non-quartz rocks. Grass was present only at the site with the highest mean annual precipitation (MAP) where it accounted for 1% of ground cover. Lichens were present only at the lowest MAP site, where they accounted for 30% of ground cover. The proportion of quartz fragments colonized generally increased with MAP, from 5.9% of soil covered by colonized hypoliths at the most costal (lowest MAP) site, to 18.7% at the most inland (highest MAP) site. There was CO2 uptake from most hypoliths measured, with net carbon uptake rates ranging from 0.3 to 6.4 μmol m-2 s-1 on well hydrated hypoliths. These carbon flux values are similar to previous work in the Mojave Desert. Our results suggest that hypoliths might play a key role in the fixation of organic carbon in hyperarid ecosystems where quartz fragments are abundant, with MAP constraining hypolith abundance. A better understanding of these extremophiles and the niche they fill could give an understanding of how microbial life might exist in extraterrestrial environments similar to hyperarid deserts.

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Date Created
  • 2016-12

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Engineering High Yield Production of L-Serine in Cyanobacterium Synechococcus sp. PCC 7002

Description

Cyanobacteria have the potential to efficiently produce L-serine, an industrially important amino acid, directly from CO2 and sunlight, which is a more sustainable and inexpensive source of energy as compared

Cyanobacteria have the potential to efficiently produce L-serine, an industrially important amino acid, directly from CO2 and sunlight, which is a more sustainable and inexpensive source of energy as compared to current methods. The research aims to engineer a strain of Cyanobacterium Synechococcus sp. PCC 7002 that increases L-serine production by mutating regulatory mechanisms that natively inhibit its production and encoding an exporter. While an excess of L-serine was not found in the supernatant of the cell cultures, with further fine tuning of the metabolic pathway and culture conditions, high titers of L-serine can be found. With the base strain engineered, the work can be extended and optimized by deleting degradation pathways, tuning gene expression levels, optimizing growth conditions, and investigating the effects of nitrogen supplementation for the strain.

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Date Created
  • 2020-05

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Genetic Engineering of Cyanobacteria to Improve Photosynthetic Yield

Description

Increasing energy and environmental problems describe the need to develop renewable chemicals and fuels. Global research has been targeting using microbial systems on a commercial scale for synthesis of valuable

Increasing energy and environmental problems describe the need to develop renewable chemicals and fuels. Global research has been targeting using microbial systems on a commercial scale for synthesis of valuable compounds. The goal of this project was to refactor and overexpress b6-f complex proteins in cyanobacteria to improve photosynthesis under dynamic light conditions. Improvement in the photosynthetic system can directly relate to higher yields of valuable compounds such as carotenoids and higher yields of biomass which can be used as energy molecules. Four engineered strains of cyanobacteria were successfully constructed and overexpressed the corresponding four large subunits in the cytochrome b6-f complex. No significant changes were found in cell growth or pigment titer in the modified strains compared to the wild type. The growth assay will be performed at higher and/or dynamic light intensities including natural light conditions for further analysis.

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  • 2020-05

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Analysis of Acyl Carrier Protein in the cyanobacterium Synechocystis sp. PCC 6803

Description

Acyl Carrier Protein (ACP) is a small, acidic protein that plays an essential role in fatty acid synthesis by elongating fatty acid chains. ACP was isolated from an extract of

Acyl Carrier Protein (ACP) is a small, acidic protein that plays an essential role in fatty acid synthesis by elongating fatty acid chains. ACP was isolated from an extract of a modified strain of Synechocystis sp. PCC 6803 that contains a thioesterase and from which the acyl-ACP synthetase has been deleted. Using ammonium sulfate precipitation to isolate a crude protein fraction containing ACP, immunoblot analysis was performed to determine relative amounts of free and acylated-ACP in the cell. The nature of fatty acids attached to ACP was determined by creating butylamide derivatives that were analyzed using GC/MS. Immunoblot analysis showed a roughly 1:1 ratio of acylated ACP to free ACP in the cell depending on the nutritional state of the cell. From GC/MS data it was determined that palmitic acid was the predominate component of acyl groups attached to ACP. The results indicate that there is a significant amount of acyl-ACP, a feedback inhibitor of early steps in the fatty acid biosynthesis pathway, in the cell. Moreover, the availability of free ACP may also limit fatty acid biosynthesis. Most likely it is necessary for ACP to be overexpressed or to have the palmitic acid cleaved off in order to synthesize optimal amounts of lauric acid to be used for cyanobacterial biofuel production.

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Date Created
  • 2016-05

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Methyl Laurate Production in Synechocystis

Description

To efficiently produce biofuels and meet the planet’s rising energy demands, different biofuel production methods need to be developed and improved. One of the ways is to produce fatty acid

To efficiently produce biofuels and meet the planet’s rising energy demands, different biofuel production methods need to be developed and improved. One of the ways is to produce fatty acid methyl esters (FAMEs) in Synechocystis sp. PCC 6803, a versatile strain of cyanobacteria. In this thesis, Synechocystis was engineered to produce and excrete methyl laurate. In this pathway, first, lauroyl-ACP from fatty acid biosynthesis is converted to laurate by a thioesterase (TE) from Umbellularia californica. Then, the laurate is methylated to methyl laurate by a juvenile hormone acid O-methyltransferase (DmJHAMT) from Drosophila melanogaster. The TE/∆slr1609 strain of Synechocystis sp. PCC 6803 contains the TE gene and lacks the slr1609 gene encoding an acyl–acyl carrier protein synthetase, which functions in free fatty acid reuptake. The DmJHAMT gene was introduced into this strain for FAME production.
The DmJHAMT gene was cloned into a vector that contains neutral sites from the Synechocystis genome, making it suitable for homologous recombination, and a kanamycin resistance gene, for selection. The obtained plasmid was verified using restriction digests and Sanger sequencing. The sequence analysis and comparison of the cDNA in the obtained plasmid and the mRNA transcript of the same gene revealed three amino acid differences. Subsequent comparison with homologous genes in other Drosophila species revealed the differences in the cDNA match those of the other species, and thus, the gene most likely is functional.
The plasmid was transformed into Synechocystis, and PCRs were used to confirm proper integration and segregation. The TE/∆slr1609/DmJHAMT strain produced 62 mg/L methyl laurate in 12 days under a light intensity of 150 µmol photons m-2 s-1, bubbled with 0.5% CO2 at a rate of 30 mL/min, and supplemented with 0.5 mM methionine. The laurate levels did not decrease over time, but instead, remained stagnant after day 3. When the strain was grown in the same conditions without methionine, the laurate concentrations continued to increase above 400 µM, suggesting minimal methyl laurate production and thus a strong need for methionine supplementation. This work provides further evidence of the viability and success of the introduced FAME production pathway, and improved efficiency may be gained in the future.

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Date Created
  • 2020-05

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Marine Aggregation interactions of Prochlorococcus and Marinobacter adhaerens

Description

The changes in marine ecological conditions brought on by warming and stratification of the oceans have radically shifted many marine environments around the globe. This project aimed to better characterize

The changes in marine ecological conditions brought on by warming and stratification of the oceans have radically shifted many marine environments around the globe. This project aimed to better characterize the aggregation behavior of the abundant picocyanobacterium Prochlorococcus marinus, which is hypothesized to dominate over other phytoplankton as the primary autotroph in increasingly warmer and nutrient poor oceans. This aggregation, believed to be mediated through the secretion of sticky Transparent Exopolymeric Substances (TEP), might be key for Prochlorococcus to sink throughout the ocean and serve as a source of carbon to other communities within its environment. Considering the relatively low concentration of TEP secreted by Prochlorococcus when on its own, this study explored the synergistic effect that heterotrophic bacteria and inorganic minerals in the surrounding seawater may have on the aggregation of P. marinus. This was done by inoculating P. marinus and the model heterotroph Marinobacter adhaerens HP15 individually and mixed in cylindrical roller tanks with the addition of ballasting clay minerals into roller tanks to simulate constant sinking for 7 days. The aggregates which formed after rolling were quantified and their sinking velocities and excess densities were measured. Our results indicate that the most numerous and densest aggregates formed when Prochlorococcus was in the presence of both M. adhaerens and kaolinite clay particles. I will discuss how methodology, particularly cell number, may play a role in the enhanced aggregation that I found when Prochlorococcus was cultured together with the Marinobacter.

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Date Created
  • 2020-05

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Studies of Engineered and Native Cyanobacterial Strains for Increased Growth Rate

Description

The production of sustainable biochemicals has been a major topic of discussion in recent years. Using microbial cells for their production through genetic engineering has been a major topic of

The production of sustainable biochemicals has been a major topic of discussion in recent years. Using microbial cells for their production through genetic engineering has been a major topic of research. Cyanobacteria have been considered as a viable candidate for such production. However, the slow growth rate of the cells presents a challenge for the possibility of scaling for use in industrial settings. This project focuses on two different solutions for this problem. The first is using four different engineered strains of Synechocystis sp. PCC 6803 that overexpress the proteins in the b6f complex to improve photosynthetic efficiency. It was found that the strains PetB and PetD showed an increase in growth rate compared to wild type cells. This was especially true under mixotrophic conditions and with a light intensity of 100 µmol photons*m-2s-1 for 3 days. The second solution is by using a newly discovered marine strain of cyanobacteria, Synechococcus sp. PCC 11901, which has a higher reported growth rate. Higher growth rates were achieved for this strain when it was grown mixotrophically with glycerol, and when grown in bubble cultures with aeration.

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Date Created
  • 2021-05

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Controlling Light Intensity in Outdoor Photobioreactors

Description

This thesis focused on the development of a system that can sense light intensity and then control a smart film to provide the optimal light intensity for cyanobacteria. The overarching

This thesis focused on the development of a system that can sense light intensity and then control a smart film to provide the optimal light intensity for cyanobacteria. The overarching goal of this project is to further the study of biofuels as an alternative energy source by increasing growth rates. If more algae or cyanobacteria can be grown per day, then the cost to produce the biofuel will decrease. To achieve this goal, PDLC (polymer dispersed liquid crystal) film was selected to be controlled due to its unique properties. It can be controlled with electricity and has variable states, in other words, not restricted to simply on or off. It also blocks 80% ultraviolet light and reduces thermal heat gain by 40% which is an important consideration for outdoor growing situations. To control the film, a simple control system was created using an Arduino Uno, SainSmart 8 channel relay board, an inverter, and a power supply. A relay board was utilized to manage the 40 volts required by the PDLC film and protected the electronics on the Arduino Uno. To sense the light intensity, the Arduino Uno was connected to a photoresistor, which changes resistance with light intensity. A 15 day test of two flasks of Cyanobacteria Synechocycstis sp. 6803, one shaded by the PDLC film, and the other unshaded, yielded 65% difference in optical densities. Overall, the experiment showed promise for controlling light intensity for photobioreactors. Ideally, this research will help to optimize light intensities when growing cyanobacteria or algae outdoors or it will help to discover what an ideal light intensity is by allowing a researcher unprecedented control.

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Date Created
  • 2015-05

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Optimization of the Toxin MazF as a Counter-Selection Marker in the Cyanobacterium Synechocystis sp. PCC 6803

Description

Traditional methods of genetic engineering are often limited to relatively few rounds of gene additions, deletions, or alterations due to a lack of additional available antibiotic resistance markers. Counter-selection marker

Traditional methods of genetic engineering are often limited to relatively few rounds of gene additions, deletions, or alterations due to a lack of additional available antibiotic resistance markers. Counter-selection marker methods can be used to remove and reuse marker genes as desired, resulting in markerless engineered strains and allowing for theoretically unlimited rounds of genetic modifications. The development of suitable counter-selection markers is vital for the development of model organisms such as cyanobacteria as biotechnological platforms.
In the hopes of providing other researchers with a new tool for markerless genetic engineering of cyanobacteria, the toxin MazF from E. coli was developed as a counter-selection marker in the most widely used cyanobacterium, Synechocystis sp. PCC 6803. The mazF gene from E. coli was cloned and inserted into a plasmid vector for downstream transformation of Synechocystis. The plasmid construct also contained two homologous flanking regions for integration of the insert into the Synechocystis genome, a nickel-inducible response regulator and promoter to control MazF expression, and a kanamycin resistance gene to serve as the antibiotic marker. In order to ensure the mazF plasmids could be cloned in a MazF-sensitive E. coli host even with slight promoter leakage, MazF expression was toned down by decreasing the efficiency of translation initiation by inserting base pairs between the ribosome binding site and the start codon of the mazF gene. Following successful cloning by E. coli, the mazF plasmids were then used to transform Synechocystis to create mazF mutant strains. Genomic analysis confirmed the successful transformation and segregation of mazF mutant strains containing the desired marker cassette. Phenotypic analysis revealed both growth arrest and production of mazF transcripts in mazF mutant strains following the addition of nickel to the cell cultures, indicating successful nickel-induced MazF expression as desired.

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Date Created
  • 2019-05

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Prochlorococcus Marinus Cell Growth, Aggregate Formation and TEP Production under Nutrient Limited Conditions

Description

Prochlorococcus marinus (MED4), a genus of marine picocyanobacteria that proliferates in open oligotrophic ocean, is one of the most abundant photosynthetic microbes in the world, estimated to contribute up to

Prochlorococcus marinus (MED4), a genus of marine picocyanobacteria that proliferates in open oligotrophic ocean, is one of the most abundant photosynthetic microbes in the world, estimated to contribute up to 10% of the ocean’s primary production. The productivity of these microorganisms is controlled by macronutrient availability in the surface waters. The ratio of macronutrients in the ocean was defined, by Alfred Redfield, as an elemental ratio of 106C:16N:1P. However, the C:N:P ratio varies based on region, season, temperature and irradiance, as well as the composition of the primary producers. In oligotrophic gyres, these nutrient ratios are elevated from the Redfield stoichiometry, but whether this ratio exerts influence on the growth rate of the organism has not been investigated. Elemental stoichiometry of available nutrients can affect the aggregation of organic carbon and exportation of the particles to the ocean depths. The purpose of this study was to investigate the effects of nutrient limitation on aggregation and transparent exopolymeric particle (TEP) production which aids in aggregation. My findings suggested that nutrient limitation reduces TEP production and does not increase aggregate volume concentration. With continued warming, certain regions of the ocean will become more oligotrophic, which further decreases the nutrient supply available for Prochlorococcus. My research shows that this could lead to decreased exportation of organic carbon matter to the depths of the sea.

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Date Created
  • 2019-05