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Engineering topics

Mayfield, Stephen P.

Publications and source records attributed to Mayfield, Stephen P..

12 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Engineering the Novel Extremophile Alga Chlamydomonas pacifica for High Lipid and High Starch Production as a Path to Developing Commercially Relevant Strains

Microalgae offer a compelling platform for the production of commodity products, due to their superior photosynthetic efficiency, adaptability to nonarable lands and nonpotable water, and their capacity to produce a versatile array of bioproducts, including biofuels and biomaterials. However, the scalability of microalgae as a bioresource has been hindered by challenges such as costly biomass production related to vulnerability to pond crashes during large-scale cultivation. This study presents a pipeline for the genetic engineering and pilot-scale production of biodiesel and thermoplastic polyurethane precursors in the extremophile species Chlamydomonas pacifica. This extremophile microalga exhibits exceptional resilience to high pH (>11.5), high salinity (up to 2% NaCl), and elevated temperatures (up to 42 °C). Initially, we evolved this strain to also have a high tolerance to high light intensity (>2000 μE/m 2 /s) through mutagenesis, breeding, and selection. We subsequently genetically engineered C. pacifica to significantly enhance lipid production by 28% and starch accumulation by 27%, all without affecting its growth rate. We demonstrated the scalability of these engineered strains by cultivating them in pilot-scale raceway ponds and converting the resulting biomass into biodiesel and thermoplastic polyurethanes. This study showcases the complete cycle of transforming a newly discovered species into a commercially relevant commodity production strain. This research underscores the potential of extremophile algae, including C. pacifica, as a key species for the burgeoning sustainable bioeconomy, offering a viable path forward in mitigating environmental challenges and supporting global bioproduct demands.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Renewable and Biodegradable Polyurethane Foams with Aliphatic Diisocyanates

The transition to renewable plastics will require the development of substitutes with existing industrial standards and manufacturing processes. Polyurethanes (PU), versatile plastics, are traditionally dominated by aromatic diisocyanates, which are challenging to derive from renewable sources. However, for higher biocontent, it is crucial to utilize aliphatic diisocyanates, which can be sourced from renewable plant or algae waste streams. Historically, PU foams relied on aromatic diisocyanates for essential hard segments, resulting in desired physical properties. Here, we report the generation of high-performance renewable and biodegradable PU foams utilizing aliphatic diisocyanates and aromatic polyols, translating hard segments into the polyester polyol component using biosourced furan dicarboxylic acid (FDCA) monomers. We demonstrate that an FDCA-based PU is suitable for foams with performance characteristics that meet commercial tolerances and can biodegrade under backyard compost conditions. Here, this demonstrates steps toward redesigning traditional petrochemical-based polymers to accommodate new biological monomers.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Rapid biodegradation of microplastics generated from bio-based thermoplastic polyurethane

The accumulation of microplastics in various ecosystems has now been well documented and recent evidence suggests detrimental effects on various biological processes due to this pollution. Accumulation of microplastics in the natural environment is ultimately due to the chemical nature of widely used petroleum-based plastic polymers, which typically are inaccessible to biological processing. One way to mitigate this crisis is adoption of plastics that biodegrade if released into natural environments. In this work, we generated microplastic particles from a bio-based, biodegradable thermoplastic polyurethane (TPU-FC1) and demonstrated their rapid biodegradation via direct visualization and respirometry. Furthermore, we isolated multiple bacterial strains capable of using TPU-FC1 as a sole carbon source and characterized their depolymerization products. Finally, to visualize biodegradation of TPU materials as real-world products, we generated TPU-coated cotton fabric and an injection molded phone case and documented biodegradation by direct visualization and scanning electron microscopy (SEM), both of which indicated clear structural degradation of these materials and significant biofilm formation.

09 BIOMASS FUELS↗
DOE OSTI2024

Harnessing genetic engineering to drive economic bioproduct production in algae

Our reliance on agriculture for sustenance, healthcare, and resources has been essential since the dawn of civilization. However, traditional agricultural practices are no longer adequate to meet the demands of a burgeoning population amidst climate-driven agricultural challenges. Microalgae emerge as a beacon of hope, offering a sustainable and renewable source of food, animal feed, and energy. Their rapid growth rates, adaptability to non-arable land and non-potable water, and diverse bioproduct range, encompassing biofuels and nutraceuticals, position them as a cornerstone of future resource management. Furthermore, microalgae’s ability to capture carbon aligns with environmental conservation goals. While microalgae offers significant benefits, obstacles in cost-effective biomass production persist, which curtails broader application. This review examines microalgae compared to other host platforms, highlighting current innovative approaches aimed at overcoming existing barriers. These approaches include a range of techniques, from gene editing, synthetic promoters, and mutagenesis to selective breeding and metabolic engineering through transcription factors.

09 BIOMASS FUELS↗
DOE OSTI2024

Isolation and characterization of microalgae strains able to grow on complex biomass hydrolysate for industrial application

Producing algae biomass economically remains one of the chief bottlenecks for commercializing algae products. The aim of this work is to identify new strains of algae that can grow on cost effective media derived from cellulosic waste streams, characterize the potential of these strains to produce compounds of high industrial value, and identify those strains capable of facile genetic transformation. Here we report, out of 45 strains initially isolated, three were selected based on their ability to efficiently grow on organic waste material (corn stover hydrolysate) as a carbon source; Chlorococcum sp., Desmodesmus sp., and Chlamydomonas debaryana. Untargeted metabolomics was performed on each strain, identifying several metabolites of high relative abundances that are of commercial interest, such as lactic acid, butane-2,3-diol, amino acids, tartaric acid, triacylglycerols, and lipid species containing different, mono- and polyunsaturated fatty acids, depending on the strain and growth condition. The strains also produced carbohydrates of industrial relevance. Chlorococcum sp. was found to be genetically transformable using standard simple transformation protocols. These results suggest that with further development, these strains could open the door to economic production of high value commercial compounds utilizing waste streams from cellulosic biomass.

09 BIOMASS FUELS↗
DOE OSTI2023

Advanced Algal Biofoundries for the Production of Polyurethane Precursors

The primary goal of the BEEPs project was to develop a process that could accelerate the development of algae as bioproduction platforms, from initial chemical product concept to an economically viable market supply. Under this program we elected to develop strains of algae that could generate polyurethane precursors, while simultaneously developing basic genetic tools to enable improved algal production systems. This program was specifically designed to incorporate National Laboratories as a means to utilize the expertise and facilities for new bio-production platforms. To that end, we designed a program to collaborate with the Agile BioFoundry at Lawrence Berkeley National Laboratory (LBNL) and computational platforms at Pacific Northwest National Laboratory (PNNL). In addition to these National Laboratory partners, we also had academic partners from UC Davis and Georgia Tech, as well as commercial partners Algenesis Materials and BASF. To achieve these goals, we initially focused on developing the genetic tools and high throughput screening technologies necessary to generate and assess production of polymer precursors (succinic acid) in algae and cyanobacteria, including advanced promoters and biosensors. In parallel, we computationally identified potential production bottlenecks and then used the developed genetic tools to increase production rates and yields. Constant feedback of data was used in conjunction with machine learning, high-throughput cell sorting, and synthetic biology, for additional targeted metabolic engineering. Multiple rounds of tool design, building, testing, and learning were supplied to partners at PNNL and LBNL to develop new models and tools that could expedite bioproduction platform development and increase yield performance. We targeted, and achieved, the FOA requirement yield metric of 20 g/L, as a milestone and deliverable from at least one of our engineered strains for the production of succinic acid.

09 BIOMASS FUELS↗
DOE OSTI2023

Utilization of lignocellulosic hydrolysates for photomixotrophic chemical production in Synechococcus elongatus PCC 7942

Abstract To meet the need for environmentally friendly commodity chemicals, feedstocks for biological chemical production must be diversified. Lignocellulosic biomass are an carbon source with the potential for effective use in a large scale and cost-effective production systems. Although the use of lignocellulosic biomass lysates for heterotrophic chemical production has been advancing, there are challenges to overcome. Here we aim to investigate the obligate photoautotroph cyanobacterium Synechococcus elongatus PCC 7942 as a chassis organism for lignocellulosic chemical production. When modified to import monosaccharides, this cyanobacterium is an excellent candidate for lysates-based chemical production as it grows well at high lysate concentrations and can fix CO 2 to enhance carbon efficiency. This study is an important step forward in enabling the simultaneous use of two sugars as well as lignocellulosic lysate. Incremental genetic modifications enable catabolism of both sugars concurrently without experiencing carbon catabolite repression. Production of 2,3-butanediol is demonstrated to characterize chemical production from the sugars in lignocellulosic hydrolysates. The engineered strain achieves a titer of 13.5 g L −1 of 2,3-butanediol over 12 days under shake-flask conditions. This study can be used as a foundation for industrial scale production of commodity chemicals from a combination of sunlight, CO 2 , and lignocellulosic sugars.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Fluctuating pH for efficient photomixotrophic succinate production

Cyanobacteria are attracting increasing attention as a photosynthetic chassis organism for diverse biochemical production, however, photoautotrophic production remains inefficient. Photomixotrophy, a method where sugar is used to supplement baseline autotrophic metabolism in photosynthetic hosts, is becoming increasingly popular for enhancing sustainable bioproduction with multiple input energy streams. In this study, the commercially relevant diacid, succinate, was produced photomixotrophically. Succinate is an important industrial chemical that can be used for the production of a wide array of products, from pharmaceuticals to biopolymers. In this system, the substrate, glucose, is transported by a proton symporter and the product, succinate, is hypothesized to be transported by another proton symporter, but in the opposite direction. Thus, low pH is required for the import of glucose and high pH is required for the export of succinate. Succinate production was initiated in a pH 7 medium containing bicarbonate. Glucose was efficiently imported at around neutral pH. Utilization of bicarbonate by CO 2 fixation raised the pH of the medium. As succinate, a diacid, was produced, the pH of the medium dropped. By repeating this cycle with additional pH adjustment, those contradictory requirements for transport were overcome. pH affects a variety of biological factors and by cycling from high pH to neutral pH processes such as CO 2 fixation rates and CO 2 solubility can vary. In this study the engineered strains produced succinate during fluctuating pH conditions, achieving a titer of 5.0gL -1 after 10 days under shake flask conditions. Finally, these results demonstrate the potential for photomixotrophic production as a viable option for the large-scale production of succinate.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Biodegradable waterborne polyurethane-urea dispersion adhesives with high biocontent

Non-biodegradable petroleum-based plastic wastes have become a leading environmental concern, and new efforts are underway to prepare biobased and biodegradable replacements. For this work, we have explored the preparation of adhesives suitable for use in consumer products, and here we report the development of waterborne, biodegradable adhesives from biobased monomers resulting in adhesives exceeding 70% biocontent. Using water as the polymer medium, viscosity challenges and the use of volatile organic solvents are avoided. Material properties of the polyurethane dispersions, resulting films, and laminates produced showed M w ranging between 56,000 and 124,000. Lastly, the biodegradability of films and laminates was evaluated. The resulting metrics indicate that the adhesives produced meet the desired mechanical and biodegradability targets, indicating that high renewability content solvent-free polyurethane dispersions are a viable solution for lamination adhesives.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Synthetic algal promoters

This invention provides synthetic promoters capable of promoting and/or initiating transcription of a polynucleotide in an algal cell, and methods of designing, producing and using such promoters.

Mayfield, Stephen P.↗
DOE OSTI2023

Developing algae as a sustainable food source

Current agricultural and food production practices are facing extreme stress, posed by climate change and an ever-increasing human population. The pressure to feed nearly 8 billion people while maintaining a minimal impact on the environment has prompted a movement toward new, more sustainable food sources. For thousands of years, both the macro (seaweed and kelp) and micro (unicellular) forms of algae have been cultivated as a food source. Algae have evolved to be highly efficient at resource utilization and have proven to be a viable source of nutritious biomass that could address many of the current food production issues. Particularly for microalgae, studies of their large-scale growth and cultivation come from the biofuel industry; however, this knowledge can be reasonably translated into the production of algae-based food products. The ability of algae to sequester CO 2 lends to its sustainability by helping to reduce the carbon footprint of its production. Additionally, algae can be produced on non-arable land using non-potable water (including brackish or seawater), which allows them to complement rather than compete with traditional agriculture. Algae inherently have the desired qualities of a sustainable food source because they produce highly digestible proteins, lipids, and carbohydrates, and are rich in essential fatty acids, vitamins, and minerals. Although algae have yet to be fully domesticated as food sources, a variety of cultivation and breeding tools exist that can be built upon to allow for the increased productivity and enhanced nutritional and organoleptic qualities that will be required to bring algae to mainstream utilization. Here we will focus on microalgae and cyanobacteria to highlight the current advancements that will expand the variety of algae-based nutritional sources, as well as outline various challenges between current biomass production and large-scale economic algae production for the food market.

59 BASIC BIOLOGICAL SCIENCES↗