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Description
Lipids and free fatty acids (FFA) from cyanobacterium Synechocystis can be used for biofuel (e.g. biodiesel or renewable diesel) production. In order to utilize and scale up this technique, downstream processes including culturing and harvest, cell disruption, and extraction were studied. Several solvents/solvent systems were screened for lipid extraction from Synechocystis. Chloroform + methanol-based Folch and Bligh & Dyer methods were proved to be "gold standard" for small-scale analysis due to their highest lipid recoveries that were confirmed by their penetration of the cell membranes, higher polarity, and stronger interaction with hydrogen bonds. Less toxic solvents, such as methanol and MTBE, or direct transesterification of biomass (without pre-extraction step) gave only slightly lower lipid-extraction yields and can be considered for large-scale application. Sustained exposure to high and low temperature extremes severely lowered the biomass and lipid productivity. Temperature stress also triggered changes of lipid quality such as the degree of unsaturation; thus, it affected the productivities and quality of Synechocystis-derived biofuel. Pulsed electric field (PEF) was evaluated for cell disruption prior to lipid extraction. A treatment intensity > 35 kWh/m3 caused significant damage to the plasma membrane, cell wall, and thylakoid membrane, and it even led to complete disruption of some cells into fragments. Treatment by PEF enhanced the potential for the low-toxicity solvent isopropanol to access lipid molecules during subsequent solvent extraction, leading to lower usage of isopropanol for the same extraction efficiency. Other cell-disruption methods also were tested. Distinct disruption effects to the cell envelope, plasma membrane, and thylakoid membranes were observed that were related to extraction efficiency. Microwave and ultrasound had significant enhancement of lipid extraction. Autoclaving, ultrasound, and French press caused significant release of lipid into the medium, which may increase solvent usage and make medium recycling difficult. Production of excreted FFA by mutant Synechocystis has the potential of reducing the complexity of downstream processing. Major problems, such as FFA precipitation and biodegradation by scavengers, account for FFA loss in operation. Even a low concentration of FFA scavengers could consume FFA at a high rate that outpaced FFA production rate. Potential strategies to overcome FFA loss include high pH, adsorptive resin, and sterilization techniques.
ContributorsSheng, Chieh (Author) / Rittmann, Bruce E. (Thesis advisor) / Westerhoff, Paul (Committee member) / Vermaas, Willem (Committee member) / Arizona State University (Publisher)
Created2011
Description
Photosynthesis converts sunlight to biomass at a global scale. Among the photosynthetic organisms, cyanobacteria provide an excellent model to study how photosynthesis can become a practical platform of large-scale biotechnology. One novel approach involves metabolically engineering the cyanobacterium Synechocystis sp. PCC 6803 to excrete laurate, which is harvested directly.
This work begins by defining a working window of light intensity (LI). Wild-type and laurate-excreting Synechocystis required an LI of at least 5 µE/m2-s to sustain themselves, but are photo-inhibited by LI of 346 to 598 µE/m2-s.
Fixing electrons into valuable organic products, e.g., biomass and excreted laurate, is critical to success. Wild-type Synechocystis channeled 75% to 84% of its fixed electrons to biomass; laurate-excreting Synechocystis fixed 64 to 69% as biomass and 6.6% to 10% as laurate. This means that 16 to 30% of the electrons were diverted to non-valuable soluble products, and the trend was accentuated with higher LI.
How the Ci concentration depended on the pH and the nitrogen source was quantified by the proton condition and experimentally validated. Nitrate increased, ammonium decreased, but ammonium nitrate stabilized alkalinity and Ci. This finding provides a mechanistically sound tool to manage Ci and pH independently.
Independent evaluation pH and Ci on the growth kinetics of Synechocystis showed that pH 8.5 supported the fastest maximum specific growth rate (µmax): 2.4/day and 1.7/day, respectively, for the wild type and modified strains with LI of 202 µE/m2-s. Half-maximum-rate concentrations (KCi) were less than 0.1 mM, meaning that Synechocystis should attain its µmax with a modest Ci concentration (≥1.0 mM).
Biomass grown with day-night cycles had a night endogenous decay rate of 0.05-1.0/day, with decay being faster with higher LI and the beginning of dark periods. Supplying light at a fraction of daylight reduced dark decay rate and improved overall biomass productivity.
This dissertation systematically evaluates and synthesizes fundamental growth factors of cyanobacteria: light, inorganic carbon (Ci), and pH. LI remains the most critical growth condition to promote biomass productivity and desired forms of biomass, while Ci and pH now can be managed to support optimal productivity.
This work begins by defining a working window of light intensity (LI). Wild-type and laurate-excreting Synechocystis required an LI of at least 5 µE/m2-s to sustain themselves, but are photo-inhibited by LI of 346 to 598 µE/m2-s.
Fixing electrons into valuable organic products, e.g., biomass and excreted laurate, is critical to success. Wild-type Synechocystis channeled 75% to 84% of its fixed electrons to biomass; laurate-excreting Synechocystis fixed 64 to 69% as biomass and 6.6% to 10% as laurate. This means that 16 to 30% of the electrons were diverted to non-valuable soluble products, and the trend was accentuated with higher LI.
How the Ci concentration depended on the pH and the nitrogen source was quantified by the proton condition and experimentally validated. Nitrate increased, ammonium decreased, but ammonium nitrate stabilized alkalinity and Ci. This finding provides a mechanistically sound tool to manage Ci and pH independently.
Independent evaluation pH and Ci on the growth kinetics of Synechocystis showed that pH 8.5 supported the fastest maximum specific growth rate (µmax): 2.4/day and 1.7/day, respectively, for the wild type and modified strains with LI of 202 µE/m2-s. Half-maximum-rate concentrations (KCi) were less than 0.1 mM, meaning that Synechocystis should attain its µmax with a modest Ci concentration (≥1.0 mM).
Biomass grown with day-night cycles had a night endogenous decay rate of 0.05-1.0/day, with decay being faster with higher LI and the beginning of dark periods. Supplying light at a fraction of daylight reduced dark decay rate and improved overall biomass productivity.
This dissertation systematically evaluates and synthesizes fundamental growth factors of cyanobacteria: light, inorganic carbon (Ci), and pH. LI remains the most critical growth condition to promote biomass productivity and desired forms of biomass, while Ci and pH now can be managed to support optimal productivity.
ContributorsNguyen, Binh Thanh (Author) / Rittmann, Bruce E. (Thesis advisor) / Krajmalnik-Brown, Rosa (Committee member) / Westerhoff, Paul (Committee member) / Arizona State University (Publisher)
Created2015
Description
The unicellular cyanobacterium Synechocystis sp. PCC 6803 contains a NiFe-type bidirectional hydrogenase that is capable of using reducing equivalents to reduce protons and generate H¬2. In order to achieve sustained H2 production using this cyanobacterium many challenges need to be overcome. Reported H2 production from Synechocystis is of low rate and often transient. Results described in this dissertation show that the hydrogenase activity in Synechocystis is quite different during periods of darkness and light. In darkness, the hydrogenase enzyme acts in a truly bidirectional way and a particular H2 concentration is reached that depends upon the amount of biomass involved in H2 production. On the other hand, in the presence of light the enzyme shows only transient H2 production followed by a rapid and constitutive H2 oxidation. H2 oxidation and production were measured from a variety of Synechocystis strains in which components of the photosynthetic or respiratory electron transport chain were either deleted or inhibited. It was shown that the light-induced H2 oxidation is dependent on the activity of cytochrome b6f and photosystem I but not on the activity of photosystem II, indicating a channeling of electrons through cytochrome b6f and photosystem I. Because of the sequence similarities between subunits of NADH dehydrogenase I in E. coli and subunits of hydrogenase in Synechocystis, NADH dehydrogenase I was considered as the most likely candidate to mediate the electron transfer from hydrogenase to the membrane electron carrier plastoquinone, and a three-dimensional homology model with the associated subunits shows that structurally it is possible for the subunits of the two complexes to assemble. Finally, with the aim of improving the rate of H2 production in Synechocystis by using a powerful hydrogenase enzyme, a mutant strain of Synechocystis was created in which the native hydrogenase was replaced with the hydrogenase from Lyngbya aestuarii BL J, a strain with higher capacity for H2 production. H2 production was detected in this Synechocystis mutant strain, but only in the presence of external reductants. Overall, this study emphasizes the importance of redox partners in determining the direction of H2 flux in Synechocystis.
ContributorsDatta, Īpsitā (Author) / Vermaas, Willem Fj (Thesis advisor) / Garcia-Pichel, Ferran (Committee member) / Rittmann, Bruce (Committee member) / Jones, Anne K (Committee member) / Arizona State University (Publisher)
Created2015
Description
Synechocystis sp. PCC 6803 is a readily transformable cyanobacteria used to study cyanobacterial genetics, as well as production of biofuels, polyesters, and other industrial chemicals. Free fatty acids are precursors to biofuels which are used by Synechocystis cells as a means of energy storage. By genetically modifying the cyanobacteria to expel these chemicals, costs associated with retrieving the products will be reduced; concurrently, the bacteria will be able to produce the products at a higher concentration. This is achieved by adding genes encoding components of the Escherichia coli AcrAB-TolC efflux system, part of the resistance-nodulation-division (RND) transporter family, to Synechocystis sp. PCC 6803. AcrAB-TolC is a relatively promiscuous multidrug efflux pump that is noted for expelling a wide range of substrates including dyes, organic solvents, antibiotics, and free fatty acids. Adding components of the AcrAB-TolC multidrug efflux pump to a previously created high free fatty acid producing strain, SD277, allowed cells to move more free fatty acids to the extracellular environment than did the parent strain. Some of these modifications also improved tolerance to antibiotics and a dye, rhodamine 6G. To confirm the function of this exogenous efflux pump, the genes encoding components of the AcrAB-TolC efflux pump were also added to Synechocystis sp. PCC 6803 and shown to grow on a greater concentration of various antibiotics and rhodamine 6G. Various endogenous efflux systems have been elucidated, but their usefulness in expelling products currently generated in Synechocystis is limited. Most of the elucidated pumps in the cyanobacteria are part of the ATP-binding cassette superfamily. The knowledge of the resistance-nodulation-division (RND) family transporters is limited. Two genes in Synechocystis sp. PCC 6803, slr2131 and sll0180 encoding homologs to the genes that encode acrB and acrA, respectively, were removed and the modifications resulted in changes in resistance to various antibiotics and a dye and also had an impact on free fatty acid secretion. Both of these deletions were complemented independently with the homologous E. coli gene and the resulting cyanobacteria strains had some of the inherent resistance restored to chloramphenicol and free fatty acid secretion was modified when compared to the wild-type and a high free fatty acid producing strain.
ContributorsBellefleur, Matthew Paul Allen (Author) / Curtiss, III, Roy (Thesis advisor) / Nielsen, David R (Committee member) / Wang, Xuan (Committee member) / Rittmann, Bruce E. (Committee member) / Arizona State University (Publisher)
Created2018
Description
Creating sustainable alternatives to fossil fuel resources is one of the greatest
challenges facing mankind. Solar energy provides an excellent option to alleviate modern dependence on fossil fuels. However, efficient methods to harness solar energy are still largely lacking. Biomass from photosynthetic organisms can be used as feedstock to produce traditional fuels, but must be produced in great quantities in order to meet the demands of growing populations. Cyanobacteria are prokaryotic photosynthetic microorganisms that can produce biomass on large scales using only sunlight, carbon dioxide, water, and small amounts of nutrients. Thus, Cyanobacteria are a viable option for sustainable production of biofuel feedstock material. Photobioreactors (PBRs) offer a high degree of control over the temperature, aeration, and mixing of cyanobacterial cultures, but cannot be kept sterile due to the scales necessary to meet domestic and global energy demands, meaning that heterotrophic bacteria can grow in PBRs by oxidizing the organic material produced and excreted by the Cyanobacteria. These heterotrophic bacteria can positively or negatively impact the performance of the PBR through their interactions with the Cyanobacteria. This work explores the microbial ecology in PBR cultures of the model cyanobacterium Synechocystis sp. PCC6803 (Synechocystis) using microbiological, molecular, chemical, and engineering techniques. I first show that diverse phylotypes of heterotrophic bacteria can associate with Synechocystis-based PBRs and that excluding them may be impossible under typical PBR operating conditions. Then, I demonstrate that high-throughput sequencing can reliably elucidate the structure of PBR microbial communities without the need for pretreatment to remove Synechocystis 16S rRNA genes, despite the high degree of polyploidy found in Synechocystis. Next, I establish that the structure of PBR microbial communities is strongly influenced by the microbial community of the inoculum culture. Finally, I show that maintaining available phosphorus in the culture medium promotes the production and enrichment of Synechocystis biomass in PBRs by reducing the amount of soluble substrates available to heterotrophic bacteria. This work presents the first analysis of the structure and function of microbial communities associated with Synechocystis-based PBRs.
challenges facing mankind. Solar energy provides an excellent option to alleviate modern dependence on fossil fuels. However, efficient methods to harness solar energy are still largely lacking. Biomass from photosynthetic organisms can be used as feedstock to produce traditional fuels, but must be produced in great quantities in order to meet the demands of growing populations. Cyanobacteria are prokaryotic photosynthetic microorganisms that can produce biomass on large scales using only sunlight, carbon dioxide, water, and small amounts of nutrients. Thus, Cyanobacteria are a viable option for sustainable production of biofuel feedstock material. Photobioreactors (PBRs) offer a high degree of control over the temperature, aeration, and mixing of cyanobacterial cultures, but cannot be kept sterile due to the scales necessary to meet domestic and global energy demands, meaning that heterotrophic bacteria can grow in PBRs by oxidizing the organic material produced and excreted by the Cyanobacteria. These heterotrophic bacteria can positively or negatively impact the performance of the PBR through their interactions with the Cyanobacteria. This work explores the microbial ecology in PBR cultures of the model cyanobacterium Synechocystis sp. PCC6803 (Synechocystis) using microbiological, molecular, chemical, and engineering techniques. I first show that diverse phylotypes of heterotrophic bacteria can associate with Synechocystis-based PBRs and that excluding them may be impossible under typical PBR operating conditions. Then, I demonstrate that high-throughput sequencing can reliably elucidate the structure of PBR microbial communities without the need for pretreatment to remove Synechocystis 16S rRNA genes, despite the high degree of polyploidy found in Synechocystis. Next, I establish that the structure of PBR microbial communities is strongly influenced by the microbial community of the inoculum culture. Finally, I show that maintaining available phosphorus in the culture medium promotes the production and enrichment of Synechocystis biomass in PBRs by reducing the amount of soluble substrates available to heterotrophic bacteria. This work presents the first analysis of the structure and function of microbial communities associated with Synechocystis-based PBRs.
ContributorsZevin, Alexander Simon (Author) / Rittmann, Bruce E. (Thesis advisor) / Krajmalnik-Brown, Rosa (Thesis advisor) / Vermaas, Willem Fj (Committee member) / Arizona State University (Publisher)
Created2015
Description
One solution to mitigating global climate change is using cyanobacteria or single-celled algae (collectively microalgae) to replace petroleum-based fuels and products, thereby reducing the net release of carbon dioxide. This work develops and evaluates a mechanistic kinetic model for light-dependent microalgal growth. Light interacts with microalgae in a variety of positive and negative ways that are captured by the model: light intensity (LI) attenuates through a microalgal culture, light absorption provides the energy and electron flows that drive photosynthesis, microalgae pool absorbed light energy, microalgae acclimate to different LI conditions, too-high LI causes damage to the cells’ photosystems, and sharp increases in light cause severe photoinhibition that inhibits growth. The model accounts for all these phenomena by using a set of state variables that represent the pooled light energy, photoacclimation, PSII photo-damage, PSII repair inhibition and PSI photodamage. Sets of experiments were conducted with the cyanobacterium Synechocystis sp. PCC 6803 during step-changes in light intensity and flashing light. The model was able to represent and explain all phenomena observed in the experiments. This included the spike and depression in growth rate following an increasing light step, the temporary depression in growth rate following a decreasing light step, the shape of the steady-state growth-irradiance curve, and the “blending” of light and dark periods under rapid flashes of light. The LI model is a marked improvement over previous light-dependent growth models, and can be used to design and interpret future experiments and practical systems for generating renewable feedstock to replace petroleum.
ContributorsStraka, Levi (Author) / Rittmann, Bruce E. (Thesis advisor) / Fox, Peter (Committee member) / Torres, César I (Committee member) / Arizona State University (Publisher)
Created2017
Description
Like most other phototrophic organisms the cyanobacterium Synechocystis sp. PCC 6803 produces carotenoids. These pigments often bind to proteins and assume various functions in light harvesting, protection from reactive oxygen species (ROS) and protein stabilization. One hypothesis was that carotenoids bind to the surface (S-)layer protein. In this work the Synechocystis S-layer protein was identified as Sll1951 and the effect on the carotenoid composition of this prokaryote by disruption of sll1951 was studied. Loss of the S-layer, which was demonstrated by electron microscopy, did not result in loss of carotenoids or changes in the carotenoid profile of the mutant, which was shown by HPLC and protein analysis. Although Δsll1951 was more susceptible to osmotic stress than the wild type, the general viability of the mutant remained unaffected. In a different study a combination of mutants having single or multiple deletions of putative carotenoid cleavage dioxygenase (CCD) genes was created. CCDs are presumed to play a role in the breakdown of carotenoids or apo-carotenoids. The carotenoid profiles of the mutants that were grown under conditions of increased reactive oxygen species were analyzed by HPLC. Pigment lifetimes of all strains were estimated by 13C-labeling. Carotenoid composition and metabolism were similar in all strains leading to the conclusion that the deleted CCDs do not affect carotenoid turnover in Synechocystis. The putative CCDs either do not fulfill this function in cyanobacteria or alternative pathways for carotenoid degradation exist. Finally, slr0941, a gene of unknown function but a conserved genome position in many cyanobacteria downstream of the δ-carotene desaturase, was disrupted. Initially, the mutant strain was impaired in growth but displayed a rather normal carotenoid content and composition, but an apparent second-site mutation occurred infrequently that restored growth rates and caused an accumulation of carotenoid isomers not found in the wild type. Based on the obtained data a role of the slr0941 gene in carotenoid binding/positioning for isomerization and further conversion to mature carotenoids is suggested.
ContributorsTrautner, Christoph (Author) / Vermaas, Willem Fj (Thesis advisor) / Chandler, Douglas E. (Committee member) / Misra, Rajeev (Committee member) / Bingham, Scott E (Committee member) / Arizona State University (Publisher)
Created2011
Description
Transmission electron microscopy has been used to identify poly-3-hydroxybutyrate (PHB) granules in cyanobacteria for over 40 years. Electron-transparent (sometimes containing a slightly electron-dense area in the inclusions) or slightly electron-dense spherical inclusions found in transmission electron micrographs of cyanobacteria are often assumed to be PHB granules. The aim of this study was to test this assumption in Synechocystis sp. PCC 6803, and to determine whether all inclusions of this kind are indeed PHB granules. Based on the results gathered, it is concluded that not all of the slightly electron-dense spherical inclusions are PHB granules in Synechocystis sp. PCC 6803. This result is potentially applicable to other cyanobacteria.
ContributorsTsang, Tin Ki (Author) / Vermaas, Willem F. J. (Thesis director) / Nielsen, David (Committee member) / Roberson, Robert (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2013-05
Description
With global warming becoming a more serious problem and mankind's alarming dependency on fossil fuels, the need for a sustainable and environmentally friendly fuel source is becoming more important. Biofuels produced from photosynthetic microorganisms like algae or cyanobacteria offer a carbon neutral replacement for petroleum fuel sources; however, with the technology and information available today, the amount of biomass that would need to be produced is not economically feasible. In this work, I examined a possible factor impacting the growth of a model cyanobacterium, Synechocystis sp. PCC6803, which is heterotrophic bacteria communities accompanying the cyanobacteria. I experimented with three variables: the type of heterotrophic bacteria strain, the initial concentration of heterotrophic bacteria, and the addition of a carbon source (glucose) to the culture. With experimental information, I identified if given conditions would increase Synechocystis growth and thus increase the yield of biomass. I found that under non-limiting growth conditions, heterotrophic bacteria do not significantly affect the growth of Synechocystis or the corresponding biomass yield. The initial concentration of heterotrophic bacteria and the added glucose also did not affect the growth of Synechocystis. I did see some nutrient recycling from the heterotrophic bacteria as the phosphate levels in the growth medium were depleted, which was apparent from prolonged growth phase and higher levels of reactive phosphate in the media.
ContributorsCahill, Brendan Robert (Author) / Rittmann, Bruce (Thesis director) / Krajmalnik-Brown, Rosa (Committee member) / W. P. Carey School of Business (Contributor) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2015-12
Description
Cyanobacteria and microalgae help reduce the environmental impact of human energy consumption by playing a vital role in carbon and nitrogen cycling. They are also used in various applications like biofuel production, food, medicine, and bioremediation. Understanding how these organisms respond to stress is important for efficient recovery strategies and sustainable outcomes. This study investigated the effects of low-level bleaching and thermal stress on cyanobacteria and microalgae, specifically Synechocystis, Chlorella, and Scenedesmus. The role of ferroptosis, an iron-dependent form of cell death, in the degradation of cellular components under these stressors was examined. Flow cytometry and spectrophotometry were used to measure changes in cellular health and viability. The results showed that temperature influences the type of cell death mechanism and can impact photosynthetic organisms. When treated with Liproxstatin-1, an inhibitor of ferroptosis, both Synechocystis and Chlorella experienced a decrease in oxidative damage, suggesting a potential protective role for the compound. Further investigation into ferroptosis and other forms of cell death, as well as identifying additional inhibitory molecules, could lead to strategies for mitigating oxidative stress and enhancing the resilience of cyanobacteria and microalgae.
ContributorsRayes, Rammy (Author) / Rittmann, Bruce (Thesis director) / Eustance, Everett (Committee member) / Lewis, Christine (Committee member) / Khdour, Omar (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2023-05