Reducing equivalents were extracted from in vitro photosynthesis and used to drive cell-free and enzyme-free biochemical reduction reactions in this research. To investigate photosynthetic electron flow, an algal extract dense in chloroplasts was made from the microalga Scenedesmus sp. A6. The algal extract was subjugated to a variety of environmental parameters and exogenous quinones in order to optimize electron extraction. To monitor electron extraction and donation to metabolites, a novel assay was created that monitored the chemiluminescence (CL) produced by superoxide radicals formed during the process. In particular, these formed when a reduced exogenous quinone oxidized spontaneously. Our studies found that calcium chloride improved the reduction of low redox potential mediators along with prolonged exposure to red light. Other salts and environmental conditions examined had diverse effects on the quinones based on structure, redox potential, and site of electron extraction. We next applied our assay for monitoring the reduction of different metabolites. The CL recorded for different metabolites was compared to the Gibbs free energy of reduction and a highly correlated relationship was found. The assay was then applied to the reduction of metabolites via the oxidation of glucose in an alkaline environment.
To exhibit the diverse application of the CL assay, urine of healthy individuals, patients with chronic kidney disease (CKD), and patients with bladder cancer (BCa) were characterized through their interactions with different quinones. The CL output was compared to that of SurineTM and urea followed by ANOVA analysis. Statistically significant differences were found for all quinones with 1,2-napthoquinone-4-sulfonate (NQS) producing significant differences between all groups examined.
Monitoring algal phenotypes for biofuels or photosynthetic output requires arduous protocols and advanced instrumentation. Both of these energy producing options were explored along with rapid, high-throughput protocols for measuring reduction reactions. To monitor the phenotypes and health of our microalgae, Raman microscopy was applied to algal cultures of Scenedesmus sp. A6 grown under stress. Statistically unique phenotypes were found based on environmental factors during cell growth. ANOVA analysis determined the effect of stressors that caused significant change to algal phenotypes related to photosynthesis and lipids. / Doctor of Philosophy / Photosynthesis is the process by which plants and algae harness sunlight to convert CO2 to plant mass. Photosynthesis is performed in the chloroplast which can excite electrons and use them to generate energy. Detecting how much energy a chloroplast can produce and what chemicals effect the chloroplast requires complex procedures with complicated instruments. In this thesis the chloroplast from the microalgae Scenedesmus sp. A6 were isolated to evaluate how they are affected by different chemicals in the environment using a new, rapid and robust assay. Then, a group of chemicals called quinones were used to steal electrons (plant energy), and this process was optimized in this research. The purpose of stealing this plant energy from photosynthesis was so it could be re-directed into synthesizing valuable chemicals that are normally produced from fossil fuels. A new sensor was also developed in this research that would "light-up" the environment whenever this plant energy (electron) stealing process was successful allowing us to measure the efficiency of this energy transfer. Once a quinone stole an electron, it would spontaneously give up the electron to oxygen, creating an unstable molecule that could then react with the chemical luminol, forming a strong luminescence (light) signal. We found that calcium chloride greatly enhanced a quinone's ability to harvest electrons from the chloroplast. We also reported unique effects caused by salt, magnesium, phosphate, a mild detergent, and changing the amount of light the chloroplast would receive. This information was then used to transfer electrons from the chloroplast to make new valuable chemicals. We found that electrons could be donated to multiple chemicals using a quinone, chloroplasts, and light. We were also able to take electrons from glucose with our quinones when glucose was in an environment with a high pH. Electrons from glucose could also be donated to chemicals of interest using quinones. In addition, Quinones were used once more to find differences in the urine composition of healthy individuals and those with chronic kidney disease or bladder cancer. The urine from healthy individuals produced a unique luminescence signal when interacting with the quinones. Thus, quinones could be used for rapidly detecting changes in a patient's kidney and bladder function.
We also developed a new method for detecting changes in the health of an algal culture. Algal cultures are used for producing biofuel, food, and pharmaceuticals, therefore it is imperative to track the growth of a culture to avoid contamination and algal death. Scenedesmus sp. A6 was exposed to chemicals harmful to algal health to see how these chemicals caused the algae to grow differently. Raman spectroscopy was used to collect data on algae grown under different conditions. The Raman spectra obtained then underwent statistical analysis to determine the chemicals that had the greatest impact on algal function. Methyl viologen, nickel sulfate, salt, and light exposure had the greatest impact on the algae.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/112232 |
| Date | 01 June 2021 |
| Creators | Scherr, David Michael |
| Contributors | Biological Systems Engineering, Senger, Ryan S., Collakova, Eva, Wright, Robert Clay, Chen, Juhong |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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