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- All Subjects: Chlamydomonas Reinhardtii
- Creators: Redding, Kevin
Description
There is a critical need for the development of clean and efficient energy sources. Hydrogen is being explored as a viable alternative to fuels in current use, many of which have limited availability and detrimental byproducts. Biological photo-production of H2 could provide a potential energy source directly manufactured from water and sunlight. As a part of the photosynthetic electron transport chain (PETC) of the green algae Chlamydomonas reinhardtii, water is split via Photosystem II (PSII) and the electrons flow through a series of electron transfer cofactors in cytochrome b6f, plastocyanin and Photosystem I (PSI). The terminal electron acceptor of PSI is ferredoxin, from which electrons may be used to reduce NADP+ for metabolic purposes. Concomitant production of a H+ gradient allows production of energy for the cell. Under certain conditions and using the endogenous hydrogenase, excess protons and electrons from ferredoxin may be converted to molecular hydrogen. In this work it is demonstrated both that certain mutations near the quinone electron transfer cofactor in PSI can speed up electron transfer through the PETC, and also that a native [FeFe]-hydrogenase can be expressed in the C. reinhardtii chloroplast. Taken together, these research findings form the foundation for the design of a PSI-hydrogenase fusion for the direct and continuous photo-production of hydrogen in vivo.
ContributorsReifschneider, Kiera (Author) / Redding, Kevin (Thesis advisor) / Fromme, Petra (Committee member) / Jones, Anne (Committee member) / Arizona State University (Publisher)
Created2013
Description
There is an ever-increasing need in the world to develop a source of fuel that is clean, renewable and feasible in terms of production and implementation. Hydrogen gas presents a possible solution to these energy needs, particularly if given a way to produce hydrogen gas efficiently. Biological hydrogen (biohydrogen) production presents a potential way to do just this. It is known that hydrogenases are active in wild-type algal photosynthesis pathways but are only active in anoxic environments, where they serve as electron sinks and compete poorly for electrons from photosystem I. To circumvent these issues, a psaC-hydA1 fusion gene was designed and incorporated into a plasmid that was then used to transform hydrogenase-free Chlamydomonas reinhardtii mutants. Results obtained suggest that the psaC-hydA1 gene completely replaced the wild-type psaC gene in the chloroplast genome and the fusion was expressed in the algal cells. Western blotting verified the presence of the HydA1-PsaC fusion proteins in the transformed cells, P700 photobleaching suggested the normal assembly of FA/FB clusters in PsaC-HydA1, and PSII fluorescence data suggested that HydA1 protein limited photosynthetic electron transport flow in the fusion. Hydrogen production was measured in dark, high light, and under maximal reducing conditions. In all conditions, the wild-type algal strain (with a normal PsaC protein) exhibited higher rates of hydrogen production in the light over 2 hours than the WT strain, though both strains produced similar rates in the dark.
ContributorsSmith, Alec (Author) / Redding, Kevin (Thesis director) / Jones, Anne (Committee member) / Vermaas, Willem (Committee member) / School of Molecular Sciences (Contributor) / Sanford School of Social and Family Dynamics (Contributor) / Barrett, The Honors College (Contributor)
Created2017-12
Description
The oxygen sensitivity of hydrogenase is a large barrier in maximizing the efficiency of algal hydrogen production, despite recent efforts aimed at rewiring photosynthesis. This project focuses on the role of photosystem II (PSII) in extended hydrogen production by cells expressing the PSI-HydA1 chimera, with the goal of optimizing continuous production of photobiohydrogen in the green alga, Chlamydomonas reinhardtii. Experiments utilizing an artificial PSII electron
Therefore, it can be concluded that downstream processes are limiting the electron flow to the hydrogenase. It was also shown that the use of a PSII inhibitor, 3-(3,4-dichlorophenyl)-1,1- dimethylurea (DCMU), at sub-saturating concentrations under light exposure during growth temporarily improves the duration of the H2 evolution phase. The maximal hydrogen production rate was found to be approximately 32 nmol h-1 (µg Chl)-1. Although downregulation of PSII activity with DCMU improves the long-term hydrogen production, future experiments must be focused on improving oxygen tolerance of the hydrogenase as a means for higher hydrogen yields.
Therefore, it can be concluded that downstream processes are limiting the electron flow to the hydrogenase. It was also shown that the use of a PSII inhibitor, 3-(3,4-dichlorophenyl)-1,1- dimethylurea (DCMU), at sub-saturating concentrations under light exposure during growth temporarily improves the duration of the H2 evolution phase. The maximal hydrogen production rate was found to be approximately 32 nmol h-1 (µg Chl)-1. Although downregulation of PSII activity with DCMU improves the long-term hydrogen production, future experiments must be focused on improving oxygen tolerance of the hydrogenase as a means for higher hydrogen yields.
ContributorsO'Boyle, Taryn Reilly (Author) / Redding, Kevin (Thesis director) / Ghirlanda, Giovanna (Committee member) / Vermaas, Willem (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Arsenic contamination in groundwater is a serious problem both in local Arizonan communities and abroad: prolonged exposure to arsenic contamination can cause cancer, vascular damage, and liver failure. This project aims to engineer the microalgae Chlamydomonas reinhardtii to sequester arsenic out of water. Metallothionein, arsenate reductase, and ferritin were integrated into the microalgae via the pASapI plasmid. The plasmid rescues function of the photosystem II gene, leveraging the ability to photosynthesize as a selective trait. Metallothionein and ferritin bind the two most common forms of arsenic: arsenite and arsenate, respectively. Arsenate reductase catalyzes the reduction of arsenate to arsenite, allowing for the ultimate sequestration of the toxic metal to occur in the chloroplast. The algae was transformed using a biolistic device, to create three mutant strains, expressing Metallothionein (MT), Arsenate Reductase (ArsC)-HA, and MT-6xHIS plasmids respectively. When testing the fluorescence output of these three strains, they showed a maximum quantum yield of photosystem II comparable to that of the wildtype algae, indicating that the rescue gene had been incorporated into the chloroplast genome properly. Strains were exposed to arsenic-containing media at 50ppb and 500 ppb for 48 and 72 hours to determine the arsenic sequestration rate. Arsenic concentration in the supernatant was measured using ICP-MS analysis and sequestration rate was calculated in terms of arsenic concentration per fold growth of algae. The normalized arsenic sequestration rates of tagged protein expressing strains at 50 ppb were significantly higher than wildtype.
ContributorsLieberman, Emma (Author) / Bartelle, Benjamin (Thesis director) / Redding, Kevin (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2022-05