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Mayfield, Stephen

Publications and source records attributed to Mayfield, Stephen.

11 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Low-Cost Screening of Algae for Extreme Tolerance to pH, Temperature, Salinity, and Light

AbstractBioprospecting algae strains with tolerance to extreme conditions such as pH, temperature, salinity, and light is crucial for advancing biotechnology and environmental applications. However, traditional screening methods often involve significant costs and labor, restricting their accessibility and practical use. In this study, we developed and validated low-cost, high-throughput screening techniques, predominantly employing agar plates and liquid culture assays, to effectively differentiate tolerance levels among various algae strains. The methodologies were optimized using the model microalgaChlamydomonas reinhardtiiand its closely related speciesChlamydomonas incertaand the recently discovered extremophilicChlamydomonas pacifica. We systematically evaluated the algae for tolerance to extremes by establishing precise gradients of pH (acidic to alkaline conditions), salinity (0 to 5 M NaCl), temperature (34–42°C), and light intensity (40 to 2977 μE·m⁻²·s⁻¹). Our results demonstrated that these cost-effective, agar plate-based methods effectively distinguished algae strains exhibiting superior tolerance to extreme environmental conditions. These screening techniques not only provided clear differentiation among the closely related strains but also delivered reproducible outcomes suitable for scaling up to larger bioprospecting efforts. Furthermore, the affordability and simplicity of these methods facilitate their implementation in resource-limited laboratories, thereby broadening participation in algae bioprospecting endeavors. This study highlights the potential of low-cost, accessible screening techniques to significantly enhance the discovery and characterization of algal strains with extreme traits. Ultimately, these methods support the development of robust algae-based resources, driving innovation in diverse industrial processes and environmental solutions.Graphical Abstract

Saucedo, Barbara (ORCID:0009000008860839)↗
DOE OSTI2023

Development of Production Strains of Chlamydomonas Using Breeding Technologies

Chlamydomonas is a well-studied strain with excellent breeding capability. It can produce recombinant proteins in the raceway in the greenhouse. Increasing the pH of the culture resulted in a very stable cultivation process and cultures did not crash. This strain was not tolerant of high UV levels at KAF. This is can be probably easily overcome by breeding with a light tolerant strain. Overall Chlamydomonas is a very promising strain for production of recombinant proteins not only in the lab but also outdoors. It has excellent breeding capability and its progeny expressed the introduced protein for a long tiem. Chlamydomonas sp. produced lipids but overall lipid yield was low with highest lipid content of 15%. Chlamydomonas is a genus with a great potential for production of various specific proteins. It can be potentially cultivated outdoors and should be further investigated for production of specific molecules.

09 BIOMASS FUELS↗
DOE OSTI2023

Sustainable production of plastic degrading enzymes

The 21st century faces the challenge of transitioning to a climate-neutral economy. This hurdle will need to be addressed in a myriad of approaches, including all societal spheres, as highlighted in several UN and EU reports, including new and accessed technologies. Among them, biotechnology can play a core role by exploiting its environmental benefits (e.g., substituting fossil-based products and expected greenhouse emission reductions). In some cases, biotech can even outperform fossil counterparts' features (e.g., the biodegradability of some bioplastics). Microalga is an attractive bioplastic feedstock in this scenario since it directly substitutes petroleum and captures CO2. However, albeit its environmental benefits, the microalgal process still needs to be tipped economically and the net-positive ecological impact improved to drive adoption. To that end, we envisioned the use of a green alga as a source of bioplastic feedstock and industrial enzymes, combining natural traits of microalgae with engineered properties in a resourceful process to petroleum-based products replacement. As proof of concept, we demonstrated that we could use C. reinhardtii, a green algae, to produce plastic-degrading enzymes. We assemble a vector to secrete PHL7, a recently described PETase, capable of degrading post-consumer PET plastic. We successfully engineered the cell line to secrete the plastic-degrading enzyme. We confirmed the secretion of the enzyme by observing halos in agar plates supplemented with plastic dispersions, enzymatic assays, zymograms, and protein sequencing of supernatant samples. This approach allows using green algae to replace petrochemical-derived plastic while producing the tool necessary to recycle existing plastics sustainably.

Dutra Molino, Joao Vitor↗
DOE OSTI2023

Green Biodegradation: Analysis of Potential Polyurethane-degrading Enzymes and Their Secretion in Chlamydomonas reinhardtii

Plastic pollution is a critical global issue, with only 5% of plastics currently recycled. Polyester polyurethane (PU), a widely used plastic, can potentially be broken down using enzymatic biodegradation rather than mechanical processing that generates microplastics. This study focuses on PS5, a potential PU-degrading enzyme with esterase activity, capable of cleaving ester bonds found in PU structures. Researchers investigated the expression and secretion of PS5 in the green microalga Chlamydomonas reinhardtii, a sustainable production platform capable of generating PU precursors such as lipids and diacids while sequestering carbon. Experiments demonstrated that PS5 significantly outperformed commercial lipase in cleaving the FDA substrate, indicating high esterase activity. Algal transformants expressing PS5 also showed halo formation on Impranil®-agar plates, suggesting PU dispersion degradation. These findings highlight C. reinhardtii as a viable chassis for the production and secretion of plastic-degrading enzymes, offering a sustainable route to both upcycle and degrade PU products at the end of their lifecycle.

Diaz, Crisandra Jade↗
DOE OSTI2021

The algae revolution 2.0: the potential of algae for the production of food, feed, fuel and bioproducts – why we need it now

Algae made our world possible, and it can help us make the future more sustainable; but we need to change the way we live and adopt new more efficient production systems, and we need to do that now. When the world was new, the atmosphere was mainly carbon dioxide, and no animal life was possible. Along came algae with the process of photosynthesis, and things began to change. Ancient cyanobacteria algae turned carbon dioxide into enormous sums of lipids, proteins and carbohydrates, while they secreted oxygen into the atmosphere. Over a billion years, as oxygen filled the air and algae filled the seas, animal life became possible. Eventually all that algae biomass became petroleum and natural gas, which for eons sat undisturbed in vast underground reservoirs, holding enormous sums of untapped energy. Less than 200 years ago humans learned to tap these energy reserves to create the world we know today, but in so doing, we have released millions of years of stored CO 2 back into the atmosphere. Algae can again help make the world a better place, but this will require new thinking and new ways of producing our food, feed and fuels. We need an algae revolution 2.0.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Development of a cell surface display system in Chlamydomonas reinhardtii

Cell-surface display systems are biotechnological techniques used to express heterologous proteins on the cell surface. Their application depends directly on the cell system used, as well as on the anchoring point for the surface displayed protein. To meet most application demands an inexpensive, safe, and scalable production platform, that reduces the economic barriers for large scale use is needed. Toward this goal, we screened three possible cell surface anchoring points in the green algae Chlamydomonas by fusing mVenus to prospective anchors moieties. The vectors harboring mVenus:anchor were screened for mVenus fluorescence and tested for cellular localization by confocal laser scanning microscopy. This strategy allowed the identification of two functional anchors, one for the cytoplasmic membrane using the MAW8 GPI-anchor signal, and one for the cell wall using the GP1 protein. We also exploited GP1 chemical and biological traits to release the fused proteins efficiently during cell wall shedding. Our work provides a foundation for surface engineering of C reinhardtii supporting both cell biology studies and biotechnology applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Developing Advanced Genetic and Synthetic Biology Tools for Improved Algae Productivity

Under the UCSD-led Productivity Enhanced Algae and ToolKits (PEAK) project we sought to develop tools to enable the production of valuable co-products, as well as strategies to decrease the cost of cultivation, with an intended outcome of enabling the economic production of algae-based biofuel. We have developed new genetic tools and high throughput selection methods for eukaryotic green algae and used them to express a high value recombinant protein; a growth factor known as Osteopontin (OPN). Recombinant OPN protein expression levels were improved through a newly developed rapid breeding and mutagenesis strain improvement method to generate a robust strain capable of growing in outdoors raceway conditions in brackish water that tolerated high pH and large temperature swings. We further applied the new genetic tools to express recombinant protein in a recently bioprospected strain with even more robust growth characteristics. In response to the COVID-19 pandemic we used our improved workflow to rapidly express, purify, and characterize a recombinant SARS-CoV-2 spike protein Receptor Binding Domain (RBD) and demonstrate that it functionally interacts with its cognate human host receptor ACE2. The newly identified extremophile strain Chlamydomonas sp (402), both mating type (mt+) and mating type (mt-), have been deposited in the Chlamydomonas Resource Center (https://www.chlamycollection.org/), making them available world-wide with no restrictions. In addition, all of genetic tools are also deposited at this site and are also available world-wide with no restrictions.

09 BIOMASS FUELS↗
DOE OSTI2021

Oral delivery of a functional algal-expressed TGF-β mimic halts colitis in a murine DSS model

Inflammatory bowel disease (IBD) is a set of immunological disorders which can generate chronic pain and fatigue associated with the inflammatory symptoms. The treatment of IBD remains a significant hurdle with current therapies being only partially effective or having significant side effects, suggesting that new therapies that elicit different modes of action and delivery strategies are required. TGM1 is a TGF-β mimic that was discovered from the intestinal helminth parasite Heligmosomoides polygyrus and is thought to be produced by the parasite to suppress the intestinal inflammation response to help evade host immunity, making it an ideal candidate to be developed as a novel anti-inflammatory bio-therapeutic. Here we utilized the expression system of the edible green algae Chlamydomonas reinhardtii in order to recombinantly produce active TGM1 in a form that could be ingested. C. reinhardtii robustly expressed TGM1, and the resultant recombinant protein is biologically active as measured by regulatory T cell induction. When delivered orally to mice, the algal expressed TGM1 is able to ameliorate weight loss, lymphadenopathy, and disease symptoms in a mouse model of DSS-induced colitis, demonstrating the potential of this biologic as a novel treatment of IBD.

09 BIOMASS FUELS↗
DOE OSTI2021

Development of Algal Biomass Yield Improvements in an Integrated Process (Final Report)

This project built on the success of Global Algae Innovations Algae Biomass Yield Phase 1 project to accelerate the commercialization of algal biofuels through development of an integrated, economical, photosynthetic, open raceway system to produce algal oil. Two parallel pathways to a biofuel were investigated. In the algal crude oil pathway, highlighted in blue, the dewatered algal biomass slurry is used as a feed to hydrothermal liquefaction to produce an algae crude oil and recycle aqueous stream. In the algal lipid oil pathway, highlighted in green, the algal biomass slurry is dried, and the oil is extracted to produce an algal lipid oil biofuel intermediate and a high protein algae meal co-product. Upgrading to drop-in fuels has been demonstrated for the biofuel intermediates in both pathways. Since the algal lipid oil pathway requires lipid accumulation, the productivity is generally lower than the algal crude oil pathway, but the required productivity for economical algal biofuel production is also lower because the coproduct value is greater. The outdoor cultivation was performed at the Kauai Algae Farm. The project team has expertise across the full breadth strain development, advanced algal cultivation, open raceway contamination control, CO 2 supply, harvesting, dewatering, extraction, hydrothermal liquefaction, technoeconomic analysis and life cycle analysis: Hildebrand laboratory at Scripps Institution of Oceanography – diatom strain development. Mayfield laboratory at UCSD - green algae strain development. TSD Management Associates- CO 2 supply, algal harvesting and dewatering, and algal drying and extraction. Qualitas - large-scale test site for algal harvesting technology. Texas A&M University - algal drying and extraction technology. Pacific Northwest National Laboratory - hydrothermal liquefaction technology. GE Water and Power - membrane technology. National Renewable Energy Laboratory - algal techno-economic analysis. Phase 1 of the project resulted in tremendous productivity and pre-processing improvements in an integrated, large-scale, low-cost cultivation and pre-processing process that moved algal technology closer to economic viability for biofuels than ever before. Phase 2 accomplishments further facilitate development of a commercial algal biofuel industry by adding: several top performing strains and strain development tools including breeding; open-pond cultivation innovations that achieved a 30% improvement in overall productivity, an 83% reduction in cost, and a 95% reduction in energy use relative to conventional technology; further improvements in harvesting that led to the Zobi harvester ® as a universal, economical, low energy, commercially available microalgae harvesting solution; breakthroughs in drying and extraction including a new low energy drying process and several new low energy extraction unit operations that together with the Zobi harvester ® achieve a 90% reduction in cost and 97% reduction in energy use relative to conventional technology. These improvements are sufficient to enable economical production of algae biofuel and protein meal co-product if the process were scaled up to a commercial-scale 5,000-acre algae farm.

09 BIOMASS FUELS↗
DOE OSTI2020

Effects of the microalgae Chlamydomonas on gastrointestinal health

The effects of consuming whole-cell biomass of the green algae Chlamydomonas reinhardtii on gastrointestinal health in mice and humans was examined. A 14-day murine model of acute colitis revealed significantly less weight loss in mice that received C. reinhardtii biomass by oral gavage. Based on this result, human volunteers with varying gastrointestinal symptoms were asked to consume 1 or 3 g of C. reinhardtii daily for 30 days. Participants reported changes in their gastrointestinal health through a questionnaire and provided stool samples to analyze the composition of their gut microbiome. Those who typically experience frequent gastrointestinal symptoms reported significantly less bowel discomfort or diarrhea, significantly less gas or bloating, more regular bowel movements, and better stool consistency when regularly consuming C. reinhardtii. Analysis of participant stool samples suggested the gut microbiome composition in all groups remained complex, and no signs of dysbiosis or adverse effect on microbial composition were observed.

60 APPLIED LIFE SCIENCES↗