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Assessing Light Use Efficiencies (LUEs) Of Benthic Reef Communities For Spectral Modeling Applications

Description

Coral reefs are diverse marine ecosystems, where reef building corals provide both the structure of the habitat as well as the primary production through their symbiotic algae, and alongside algae

Coral reefs are diverse marine ecosystems, where reef building corals provide both the structure of the habitat as well as the primary production through their symbiotic algae, and alongside algae living on the reef itself, are the basis of the food web of the reef. In this way, coral reefs are the ocean's "forests" and are estimated to support 25% of all marine species. However, due to the large size of a coral reef, the relative inaccessibility and the reliance on in situ surveying methods, our current understanding of reefs is spatially limited. Understanding coral reefs from a more spatially complete perspective will offer insight into the ecological factors that contribute to coral reef vitality. This has become a priority in recent years due to the rapid decline of coral reefs caused by mass bleaching. Despite this urgency, being able to assess the entirety of a coral reef is physically difficult and this obstacle has not yet been overcome. However, similar difficulties have been addressed in terrestrial ecosystems by using remote sensing methods, which apply hyperspectral imaging to assess large areas of primary producers at high spatial resolutions. Adapting this method of remote spectral sensing to assess coral reefs has been suggested, but in order to quantify primary production via hyper spectral imaging, light-use efficiencies (LUEs) of coral reef communities need to be known. LUEs are estimations of the rate of carbon fixation compared to incident absorbed light. Here, I experimentally determine LUEs and report on several parameters related to LUE, namely net productivity, respiration, and light absorbance for the main primary producers in coral reefs surrounding Bermuda, which consist of algae and coral communities. The derived LUE values fall within typical ranges for LUEs of terrestrial ecosystems, with LUE values for coral averaging 0.022 ± 0.002 mol O2 mol photons-1 day-1 at a water flow rate of 17.5 ± 2 cm s^(-1) and 0.049 ± 0.011 mol O2 mol photons-1 day-1 at a flow rate of 32 ± 4 cm s^(-1) LUE values for algae averaged 0.0335 ± 0.0048 mol O2 mol photons-1 day-1 at a flow rate of 17.5 ± 2 cm s^(-1). These values allow insight into coral reef productivity and opens the door for future remote sensing applications.

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  • 2019-05

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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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  • 2016-12

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Knocking out the cytochrome bc complex in Heliobacterium modesticaldum

Description

The heliobacteria, a family of anoxygenic phototrophs, are significant to photosynthesis evolution research, as they possess the simplest known photosynthetic apparatus. Although they are photoheterotrophs in the light, the heliobacteria

The heliobacteria, a family of anoxygenic phototrophs, are significant to photosynthesis evolution research, as they possess the simplest known photosynthetic apparatus. Although they are photoheterotrophs in the light, the heliobacteria may also grow chemotrophically via pyruvate metabolism in the absence of light. In Heliobacterium modesticaldum, the cytochrome bc complex is responsible for oxidizing menaquinol and reducing cytochrome c553 in the electron flow cycle used for phototrophy. However, there is no known electron acceptor for cytochrome c553 other than the photosynthetic reaction center. Therefore, it was hypothesized that the cytochrome bc complex is necessary for phototrophy, but unnecessary for chemotrophic growth in the dark. Under this hypothesis, a mutant of H. modesticaldum lacking the cytochrome bc complex was predicted to be viable, but non-phototrophic. In this project, a two-step method for CRISPR-based genome editing was used in H. modesticaldum to delete the genes encoding the cytochrome bc complex. Genotypic analysis verified the deletion of the petC, B, D, and A genes encoding the catalytic components of complex. Spectroscopic studies revealed that re-reduction of cytochrome c553 after flash-induced photo-oxidation was ~130 to 190 times slower in the ∆petCBDA mutant compared to wildtype, phenotypically confirming the removal of the cytochrome bc complex. The resulting ∆petCBDA mutant was unable to grow phototrophically, instead relying on pyruvate metabolism to grow chemotrophically as does wildtype in the dark.

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  • 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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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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  • 2021-05

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Engineered Hydrogen Production in Heliobacteria using Clostridial Hydrogenase: A Probe for Understanding Cell Physiology

Description

Heliobacteria are an anaerobic phototroph that require carbon sources such as pyruvate, <br/>lactate, or acetate for growth (Sattley, et. al. 2008). They are known for having one of the <br/>simplest

Heliobacteria are an anaerobic phototroph that require carbon sources such as pyruvate, <br/>lactate, or acetate for growth (Sattley, et. al. 2008). They are known for having one of the <br/>simplest phototrophic systems, the central component of which is a Type I reaction center (RC) <br/>that pumps protons to generate the electrochemical gradient for making ATP. Heliobacteria <br/>preform cyclic electron flow (CEF) with the RC in the light but can also grow chemotropically in <br/>the dark. Many anaerobes like heliobacteria, such as other members of the class Clostridia, <br/>possess the capability to produce hydrogen via a hydrogenase enzyme in the cell, as protons can <br/>serve as an electron acceptor in anaerobic metabolism. However, the species of heliobacteria <br/>studied here, H. modesticaldum have been seen to produce hydrogen via their nitrogenase <br/>enzyme but not when this enzyme is inactive. This study aimed to investigate if the reason for <br/>their lack of hydrogen production was due to a lack of an active hydrogenase enzyme, possibly <br/>indicating that the genes required for activity were lost by an H. modesticaldum ancestor. This <br/>was done by introducing genes encoding a clostridial [FeFe] hydrogenase from C. thermocellum<br/>via conjugation and measuring hydrogen production in the transformant cells. Transformant cells <br/>produced hydrogen and cells without the genes did not, meaning that the heliobacteria ferredoxin <br/>was capable of donating electrons to the foreign hydrogenase to make hydrogen. Because the <br/>[FeFe] hydrogenase must receive electrons from the cytosolic ferredoxin, it was hypothesized <br/>that hydrogen production in heliobacteria could be used to probe the redox state of the ferredoxin <br/>pool in conditions of varying electron availability. Results of this study showed that hydrogen <br/>production was affected by electron availability variations due to varying pyruvate <br/>concentrations in the media, light vs dark environment, use acetate as a carbon source, and being <br/>provided external electron donors. Hydrogen production, therefore, was predicted to be an <br/>effective indicator of electron availability in the reduced ferredoxin pool.

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  • 2021-05

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The Effects of the PsaL Calcium Binding on the Oligomeric and Spectroscopic Properties in Synechocystis sp. PCC 6803

Description

In oxygenic photosynthesis, conversion of solar energy to chemical energy is catalyzed by the<br/>pigment-protein complexes Photosystem II (PSII) and Photosystem I (PSI) embedded within the<br/>thylakoid membrane of photoautotrophs. The function

In oxygenic photosynthesis, conversion of solar energy to chemical energy is catalyzed by the<br/>pigment-protein complexes Photosystem II (PSII) and Photosystem I (PSI) embedded within the<br/>thylakoid membrane of photoautotrophs. The function of these pigment-protein complexes are<br/>conserved between all photoautotrophs, however, the oligomeric structure, as well as the<br/>spectroscopic properties of the PSI complex, differ. In early evolving photoautotrophs, PSI<br/>exists in a trimeric organization, but in later evolving species this was lost and PSI exists solely<br/>as a monomer. While the reasons for a change in oligomerization are not fully understood, one<br/>of the 11 subunits within cyanobacterial PSI, PsaL, is thought to be involved in trimerization<br/>through the coordination of a calcium ion in an adjacent monomer. Recently published<br/>structures have demonstrated that PSI complexes are capable of trimerization without<br/>coordinating the calcium ion within PsaL.<br/>5 Here we explore the role the calcium ion plays in both<br/>the oligomeric and spectroscopic properties in PSI isolated from Synechocystis sp. PCC 6803.

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  • 2021-05

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Structural Analysis of the Spinach Rubisco Activase AAA+ Domain by Negative Stain Electron Microscopy

Description

Higher plant Rubisco activase (Rca) is a stromal ATPase responsible for reactivating Rubisco. It is a member of the AAA+ protein superfamily and is thought to assemble into closed-ring hexamers

Higher plant Rubisco activase (Rca) is a stromal ATPase responsible for reactivating Rubisco. It is a member of the AAA+ protein superfamily and is thought to assemble into closed-ring hexamers like other AAA+ proteins belonging to the classic clade. Progress towards modeling the interaction between Rca and Rubisco has been slow due to limited structural information on Rca. Previous efforts in the lab were directed towards solving the structure of spinach short-form Rca using X-ray crystallography, given that it had notably high thermostability in the presence of ATP-γS, an ATP analog. However, due to disorder within the crystal lattice, an atomic resolution structure could not be obtained, prompting us to move to negative stain electron microscopy (EM), with our long-term goal being the use of cryo-electron microscopy (cryo-EM) for atomic resolution structure determination. Thus far, we have screened different Rca constructs in the presence of ATP-γS, both the full-length β-isoform and truncations containing only the AAA+ domain. Images collected on preparations of the full-length protein were amorphous, whereas images of the AAA+ domain showed well-defined ring-like assemblies under some conditions. Procedural adjustments, such as the use of previously frozen protein samples, rapid dilution, and minimizing thawing time were shown to improve complex assembly. The presence of Mn2+ was also found to improve hexamer formation over Mg2+. Calculated class averages of the AAA+ Rca construct in the presence of ATP-γS indicated a lack of homogeneity in the assemblies, showing both symmetric and asymmetric hexameric rings. To improve structural homogeneity, we tested buffer conditions containing either ADP alone or different ratios of ATP-γS to ADP, though results did not show a significant improvement in homogeneity. Multiple AAA+ domain preparations were evaluated. Because uniform protein assembly is a major requirement for structure solution by cryo-EM, more work needs to be done on screening biochemical conditions to optimize homogeneity.

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Date Created
  • 2018-05

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An Infusion Approach to Optimizing the Mutagenesis of Rhodobacter sphaeroides

Description

Photosynthesis is the process by which plants, algae, and bacteria use light energy to synthesize organic compounds to use as energy. Among these organisms are a kind of purple photosynthetic

Photosynthesis is the process by which plants, algae, and bacteria use light energy to synthesize organic compounds to use as energy. Among these organisms are a kind of purple photosynthetic bacteria called Rhodobacter sphaeroides, a non-sulfur purple bacteria that grows aerobically in the dark by respiration. There have been many contributions throughout the history of this group of bacteria. Rhodobacter sphaeroides is metabolically very diverse as it has many different ways to obtain energy--aerobic respiration and anoxygenic photosynthesis being just a couple of the ways to do so. This project is part of a larger ongoing project to study different mutant strains of Rhodobacter and the different ways in which carries out electron transfer/photosynthesis. This thesis focused on the improvements made to protocol (standard procedure of site directed mutagenesis) through a more efficient technique known as infusion.

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Date Created
  • 2014-05

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Characterization of a Polyclonal Antibody Specific for Synechococcus WH8102 Plastoquinol Terminal Oxidase.

Description

Photosynthesis is a critical process that fixes the carbon utilized in cellular respiration. In higher plants, the immutans gene codes for a protein that is both involved in carotenoid biosynthesis

Photosynthesis is a critical process that fixes the carbon utilized in cellular respiration. In higher plants, the immutans gene codes for a protein that is both involved in carotenoid biosynthesis and plastoquinol oxidation (Carol et al 1999, Josse et al 2003). This plastoquinol terminal oxidase (PTOX) is of great interest in understanding electron flow in the plastoquinol pool. In order to characterize this PTOX, polyclonal antibodies were developed. Expression of Synechococcus WH8102 PTOX in E. coli provided a useful means to harvest the protein required for antibody production. Once developed, the antibody was tested for limit of concentration, effectiveness in whole cell lysate, and overall specificity. The antibody raised against PTOX was able to detect as low as 10 pg of PTOX in SDS-PAGE, and could detect PTOX extracted from lysed Synechococcus WH8102. The production of this antibody could determine the localization of the PTOX in Synechococcus.

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  • 2014-05