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At least 235 records · Page 13

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DOE OSTI2024

RuBisCO activity assays: a simplified biochemical redox approach for in vitro quantification and an RNA sensor approach for in vivo monitoring

Abstract Background Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant soluble protein in nature. Extensive studies have been conducted for improving its activity in photosynthesis through approaches like protein engineering. Concurrently, multiple biochemical and radiolabeling assays have been developed for determining its activity. Although these existing assays yield reliable results, they require addition of multiple external components, rendering them less convenient and expensive. Therefore, in this study, we have developed two relatively cheaper, convenient, and easily reproducible assays for quantitative and qualitative estimation of RuBisCO activity. Results We simplified a contemporary NADH based spectrophotometric RuBisCO assay by using cyanobacterial cell lysate as the source for Calvin cycle enzymes. We analyzed the influence of inorganic carbon substrates, CO 2 and NaHCO 3 , and varying protein concentrations on RuBisCO activity. Ribulose-1,5-bisphosphate (RuBP) consumption rates for the cultures grown under 5% CO 2 were 5–7 times higher than the ones grown with 20 mM NaHCO 3 , at different protein concentrations. The difference could be due to the impaired activity of carbonic anhydrase in the cell lysate, which is required for the conversion of HCO 3 − to CO 2 . The highest RuBisCO activity of 2.13 nmol of NAD + / µg of Chl-a/ min was observed with 50 µg of protein and 5% CO 2 . Additionally, we developed a novel RNA-sensor based fluorescence assay that is based on the principle of tracking the kinetics of ATP hydrolysis to ADP during the conversion of 3-phosphoglycerate (3-PG) to 1,3-bisphosphoglycerate (1,3-BPG) in the Calvin cycle. Under in vitro conditions, the fluorometric assay exhibited ~ 3.4-fold slower reaction rate (0.37 min −1 ) than the biochemical assay when using 5% CO 2 . We also confirmed the in vivo application of this assay, where increase in the fluorescence was observed with the recombinant strain of Synechocystis sp. PCC 6803 (SSL142) expressing the ADP-specific RNA sensor, compared to the WT. In addition, SSL142 exhibited three-fold higher fluorescence when supplemented with 20 mM NaHCO 3 as compared to the cells that were grown without NaHCO 3 supplementation. Conclusions Overall, we have developed a simplified biochemical assay for monitoring RuBisCO activity and demonstrated that it can provide reliable results as compared to the prior literature. Furthermore, the biochemical assay using 5% CO 2 (100% relative activity) provided faster RuBP consumption rate compared to the biochemical assay utilizing 20 mM NaHCO 3 (30.70% relative activity) and the in vitro fluorometric assay using 5% CO 2 (29.64% relative activity). Therefore, the absorbance-based biochemical assay using 5% CO 2 or higher would be suitable for in vitro quantification of the RuBisCO activity. On the other hand, the RNA-sensor based in vivo fluorometric assay can be applied for qualitative analysis and be used for high-throughput screening of RuBisCO variants. As RuBisCO is an enzyme shared amongst all the photoautotrophs, the assays developed in this study can easily be extended for analyzing the RuBisCO activities even in microalgae and higher plants.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Reclassification of Botryococcus braunii chemical races into separate species based on a comparative genomics analysis

The colonial green microalga Botryococcus braunii is well known for producing liquid hydrocarbons that can be utilized as biofuel feedstocks. B. braunii is taxonomically classified as a single species made up of three chemical races, A, B, and L, that are mainly distinguished by the hydrocarbons produced. We previously reported a B race draft nuclear genome, and here we report the draft nuclear genomes for the A and L races. A comparative genomic study of the three B. braunii races and 14 other algal species within Chlorophyta revealed significant differences in the genomes of each race of B. braunii. Phylogenomically, there was a clear divergence of the three races with the A race diverging earlier than both the B and L races, and the B and L races diverging from a later common ancestor not shared by the A race. DNA repeat content analysis suggested the B race had more repeat content than the A or L races. Orthogroup analysis revealed the B. braunii races displayed more gene orthogroup diversity than three closely related Chlamydomonas species, with nearly 24-36% of all genes in each B. braunii race being specific to each race. This analysis suggests the three races are distinct species based on sufficient differences in their respective genomes. We propose reclassification of the three chemical races to the following species names: Botryococcus alkenealis (A race), Botryococcus braunii (B race), and Botryococcus lycopadienor (L race).

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Engineering the green algae Chlamydomonas incerta for recombinant protein production

Chlamydomonas incerta , a genetically close relative of the model green alga Chlamydomonas reinhardtii , shows significant potential as a host for recombinant protein expression. Because of the close genetic relationship between C. incerta and C. reinhardtii , this species offers an additional reference point for advancing our understanding of photosynthetic organisms, and also provides a potential new candidate for biotechnological applications. This study investigates C. incerta ’s capacity to express three recombinant proteins: the fluorescent protein mCherry, the hemicellulose-degrading enzyme xylanase, and the plastic-degrading enzyme PHL7. We have also examined the capacity to target protein expression to various cellular compartments in this alga, including the cytosol, secretory pathway, cytoplasmic membrane, and cell wall. When compared directly with C. reinhardtii , C. incerta exhibited a distinct but notable capacity for recombinant protein production. Cellular transformation with a vector encoding mCherry revealed that C. incerta produced approximately 3.5 times higher fluorescence levels and a 3.7-fold increase in immunoblot intensity compared to C. reinhardtii . For xylanase expression and secretion, both C. incerta and C. reinhardtii showed similar secretion capacities and enzymatic activities, with comparable xylan degradation rates, highlighting the industrial applicability of xylanase expression in microalgae. Finally, C. incerta showed comparable PHL7 activity levels to C. reinhardtii , as demonstrated by the in vitro degradation of a polyester polyurethane suspension, Impranil® DLN. Finally, we also explored the potential of cellular fusion for the generation of genetic hybrids between C. incerta and C. reinhardtii as a means to enhance phenotypic diversity and augment genetic variation. We were able to generate genetic fusion that could exchange both the recombinant protein genes, as well as associated selectable marker genes into recombinant offspring. These findings emphasize C. incerta ’s potential as a robust platform for recombinant protein production, and as a powerful tool for gaining a better understanding of microalgal biology.

cell membranes↗

DOE OSTI2020

DISCOVR: Development of Integrated Screening, Cultivar Optimization, and Verification Research

The DISCOVR consortium has developed and is applying an integrated platform and workflow for standardized, deep characterization of highly productive and resilient microalgae strains. New strains are screened using three consecutive tiers in a conceptual down-selection funnel to arrive at the most promising strains. At tier I, strains are screened on gradient incubators to determine their temperature and salinity tolerance range, and to evaluate their resistance to infectious agents (e.g., chytrids). At tier II, the strains’ winter and summer season biomass productivities are quantified in Laboratory Environmental Algae Pond Simulator (LEAPS) photobioreactors, and biomass composition is determined under N-replete and N-deplete conditions. At tier III, the best winter and summer season strains are evaluated in indoor crash ponds to determine their resilience to induced infections, and strains are tested in outdoor raceway ponds to confirm high biomass productivity and culture stability. The top strains are then evaluated in seasonal SOT outdoor pond culture trials at AzCATI, the dedicated Department of Energy SOT (State of Technology) testbed. Additional research is carried out on evaluating growth promoting molecules, reducing the impact of oxygen inhibition, optimizing pond operational conditions, measuring compositional shift dynamics, developing integrated pest management strategies, analysing culture health using 96 well-plate tools, implementing spectroradiometric monitoring and using machine learning to predict culture crashes.

Huesemann, Michael H↗

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 OSTI2024

Marine Circular Bioeconomy Workshop

Funds were used to support a Marine Circular Bioeconomy (MCB) Workshop. The purpose of the workshop was to explore the potential of marine macroalgae and microalgae production to meet the global climate, energy, and food challenges confronting society during the 21st century.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2022

Identification and Quantification of Photosynthetic Pigments in Algae (Laboratory Analytical Procedure (LAP))

The Laboratory Analytical Procedure (LAP) outlined here describes a method to quantitatively extract phytopigments from microalgae biomass, as well as to identify and quantify individual pigments based on separation and detection with a High-Performance Liquid Chromatography (HPLC) system coupled to a Diode Array Detector (DAD). Pigments were extracted with greater than 95 % extraction efficiency. Chromatographic conditions allowed for isomeric resolution between pigments and identification based on UV/Vis spectra and comparison to analytical standards.

09 BIOMASS FUELS↗

DOE OSTI2022

Success Through Synergy (STS): Increasing Cultivation Yield and Stability with Rationally Designed Consortia

Consortia of functionally diverse microbial assemblages hold potential to enhance the production of microalgae for biofuels and bioproducts. In laboratory experiments, increases in algal yield have been achieved through the amendment of algal cultures with growth-promoting bacteria, as well as the rational design of algal-algal consortia (i.e., combining strains with complementary traits). Despite this evidence, commercial systems typically rely on monocultures because of the technical challenges that multi-taxa cultures impose. In this project, we aimed to generate algae-bacteria and algae-algae consortia in the lab and test the performance of these consortia against monoculture counterparts in the field. We also aimed to generate toolkits that could be used by the scientific community and algal farmers to advance the cultivation of consortia.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2020

TAILORED BIOBLENDSTOCKS WITH LOW ENVIRONMENTAL IMPACT TO OPTIMIZE MCCI ENGINES

The overall objective of the project is to develop and demonstrate a microalgae bio-blendstock with greater than 60% greenhouse gas reduction potential relative to petroleum diesel, that can reduce sooting propensity, increase cetane number and improve engine thermal efficiency relative to a baseline diesel engine operating on conventional fuel. Specific objectives include: (1) development of a new framework for both LCA and TEA that explicitly considers temporal variation in productivity and the frequency of crop loss; (2) determination of how fuel compounds that can be produced from the algal biomass can be ‘bio-tailored’ based on the species composition and biological production process, and subsequent processing via HTL to biocrude, and upgrading of the biocrude; (3) execution of a feedback loop (algae production  biocrude refining  combustion optimization  feedback to refining stage), for optimization of fuels for MCCI combustion; (4) optimization of MCCI combustion and emissions performance, accounting first for the biological processes that dictate the chemical composition of biocrude oil, and second for the subsequent chemical processes that comprise mixing controlled compression ignition combustion; (6) simulation of MCCI engine combustion processes to demonstrate the incorporation of relevant fuel chemistry that captures the specific impacts of optimized algal fuels.

09 BIOMASS FUELS↗

DOE OSTI2023

Tailored Bioblendstocks With Low Environmental Impact To Optimize MCCI Engines

The prohect goal is to develop and demonstrate a microalgae bio-blendstock with greater than 60% greenhouse gas reduction potential relative to petroleum diesel, that can reduce sooting propensity, increase cetane number and improve engine thermal efficiency relative to a baseline diesel engine operating on conventional fuel.

09 BIOMASS FUELS↗

DOE OSTI2021

Tailored Bioblendstocks With Low Environmental Impact To Optimize MCCI Engines

This project seeks to overcome impediments to expansion of algae cultivation and conversion into fuels to displace petroleum and reduce greenhouse gas (GHG) emissions. The overall objective of the project is to develop and demonstrate a microalgae bio-blendstock with greater than 60% greenhouse gas reduction relative to petroleum diesel, reduce sooting propensity, increase cetane number, improve engine thermal efficiency relative to a baseline diesel engine operating on conventional fuel. These objectives are being accomplished by: Applying cultivation of algae polycultures to achieve robustness and productivity, Converting whole algae to bio-hydrocarbons via Hydrothermal Liquefaction (HTL) to make a biocrude and up-grading the biocrude via hydroprocessing to obtain tailored bioblendstocks for diesel fuel, Optimizing diesel (MCCI) combustion through blending model compounds into diesel fuel to represent the tailored bioblendstocks through experiments and numerical simulation.

09 BIOMASS FUELS↗

DOE OSTI2023

Utilizing data-based modeling with low life cycle GHG emissions algae biofuels for engine optimization

Aquatic microalgae are a highly promising feedstock for the production of biocrude and tailored biofuels, with distinct advantages over traditional terrestrial crops, such as reduced land use and avoidance of food production competition. However, unlocking their full potential requires the development of biofuels with low life cycle greenhouse emissions biofuels, such as algae biofuels, which can significantly reduce the environmental impact of the transportation systems without requiring a complete overhaul of existing engine technology. In this study, we employ cutting-edge data-based AI modeling techniques to optimize the performance of heavy-duty engines, with a focus on transitioning towards biofuels with low life cycle greenhouse emissions biofuels. Our methodology offers significant advantages over traditional sweep testing, enabling efficient and accurate optimization of engine performance with minimal time and resources consumption. Our findings demonstrate the potential of utilizing this approach, with up to 55% NOx emissions reductions and up to 2% reduction in fuel consumption compared to the baseline optimized point. Moving forward, we plan to utilize a 30% blend of algae biofuels with diesel fuel, with the ultimate goal of achieving up to 60% lifecycle GHG emissions. Lastly, we plan to compare the results with 100% renewable biodiesel to add an additional dimension of investigating the impact of fuel chemistry on engine optimization. Overall, this study underscores the vital importance of biofuels for reducing the carbon footprint of the transportation sector and supporting a sustainable future. By harnessing the power of data-based AI modeling with low life cycle greenhouse emissions biofuels, we can accelerate the adoption of more environmentally friendly transportation systems and reduce their impact on the planet. Our findings contribute to this transition and offer insights for developing efficient and effective strategies for addressing global climate change.

09 BIOMASS FUELS↗

DOE OSTI2023

Tailored Bioblendstocks with Low Environmental Impact to Optimize MCCI Engines (Final Technical Report)

09 BIOMASS FUELS↗

DOE OSTI2024

Biomanufacturing and bioprocessing of lunar regolith

Microbial biomanufacturing is important to accelerate lunar construction because it can leverage lunar material and waste streams as feedstocks to create a circular production system. In-space bio-mining and biomanufacturing using moon and asteroidal source material will enable the creation of infrastructure, produce industrial fuels and lubricants, and enable recovery of actinides and rare-earth elements (REEs) present in trace concentrations. Moreover, biomanufacturing in closed-loop systems (recycling and reuse of resources toward the establishment of a circular economy) will enable long-term lunar activities by recycling waste (CO 2 , gray water) and producing oxygen and biomaterials. Our response focuses on the use of lunar regolith and waste streams as feedstocks for protein and microbial-enabled biomining and bioprocessing to extract actinides and REEs, and to create biocomposites for lunar infrastructure. We envision an enclosed process that initiates with (1a) extracting actinides and REEs from lunar regolith using immobilized proteins, followed by (1b) creating biocomposites from the post-extracted lunar regolith for infrastructure, and (1c) cultivating diatoms and other microalgae on waste streams to harvest silica shells for incorporating into biocomposites and to generate O 2 for human respiration and/or producing refinable feedstocks. LLNL has significant expertise in all three processes and provides facilities, personnel, and expertise at the intersection of metal (lanthanide, actinide, transition) separations, purifications, biohydrometallurgy, radiobiochemistry, synthetic and systems biology, and materials science and engineering. Importantly, all three processes are relatively well-studied for Earth-based workflows and can be derisked for demonstration on the lunar surface by 2029.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

'Omics and Big Data in Harmful Algal Bloom Research

Phytoplankton, a group including eukaryotic microalgae and cyanobacteria, play a crucial climate role converting CO 2 into organic carbon through global primary production. They support a wide range of life, both freshwater and marine, from zooplankton to fish and mammals. While they are essential in nutrient cycles, certain phytoplankton species can proliferate excessively under favorable conditions, leading to harmful algal blooms (HABs) that pose significant threats to human and ecosystem health through the toxins they produce.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Air Carbon for Algae Production (AirCAP) – Expanding algae resource potential via direct (in-pond) air-CO2 capture.

Microalgae biofuel production potential in the United States is constrained by a lack of suitable cultivation locations within a close proximity to CO2 point-sources. Initial estimates suggest that removing the requirement of a concentrated CO2 source expands resource potential ten-fold, allowing the projection of production levels that approach levels of current U.S. diesel consumption. Decoupling cultivation locations from concentrated CO2 point-sources requires direct capture of air-CO2 into the pond bulk at a rate sufficient to meet maximum expected carbon assimilation rates (10 g C/m2-day and higher). Enhancement of the ingassing rate via chemical reaction, the direct reaction of dissolved CO2 with hydroxide ions, has been proposed as a mechanism to increase the ingassing rate of air-CO2. The central objectives of this project are to validate whether chemically enhanced air-CO2 ingassing rates in large raceway ponds can meet or exceed the level required to support high levels of algae productivity, then subsequently identify alkaliphilic strains able to withstand such conditions while maintaining a biomass productivity that enables process economics. Full scale ponds (~1 acre, unlined) will be used to validate ingassing rates as a function of pH expected at scale in a series of abiotic experiments. To date, mass-transfer dynamics have been characterized in ponds ranging from 1 to 1,000 m2. Initial results will be presented describing the mass transfer coefficient in 1-acre (~4,000 m2), unlined ponds. The maximum expected carbon ingassing rate will be measured during ingassing trials, in which pond pH is displaced from equilibrium via the addition of a strong base, with the subsequent decrease in pH (and increase in dissolved inorganic carbon) corresponding to the air-CO2 absorption rate. A mass-transfer model, validated with experimental results, will be used to project ingassing rates expected at pond scales (10 acres) required for production of commodity chemicals.

09 BIOMASS FUELS↗

DOE OSTI2025

2024 Case Study: Hydrothermal Liquefaction of Biomass Sources at a Wastewater Treatment Facility

A case study was developed for the fiscal year 2024 to examine the potential of wastewater-grown microalgae as a feedstock for hydrothermal liquefaction (HTL) to produce fuels and other products. In this year’s case study, scenarios are investigated in which the algae cultivation process is placed at different points within the water resource recovery facility (WRRF). Additionally, the supply of biomass for HTL is boosted by blending with wastewater solids collected at different locations within the WRRF.

09 BIOMASS FUELS↗

DOE OSTI2025

Whole Algae Hydrothermal Liquefaction and Upgrading: A review of progress and challenges and insight into the future

This report summarizes the research at Pacific Northwest National Laboratory (PNNL) to evaluate the economic viability and environmental impact of using microalgae to produce fuels and other products via hydrothermal liquefaction (HTL). Over the past several years, PNNL has examined key aspects of feedstock cost and availability, formatting and conversion techniques, and the utilization of all HTL products. Investigations of feedstock cost led to opportunities to work with cost-advantaged algal feedstocks that can be provided at minimal cost for HTL processing. Cost-advantaged algae include wastewater-grown algae and harvested algal blooms. Although farm-cultivated algae offer the best possible biomass composition and scalability for HTL processing, the cost of the feedstock is too high to yield an economically competitive biofuel. Processing cost-advantaged feedstocks creates other unique challenges in adapting HTL to upgrade biomass with higher than typical ash content and less preferred composition (low lipid). Despite the challenges, HTL of cost-advantaged algae results in economically competitive pricing scenarios and significant advantages in reducing net emissions below 70% of the petroleum baseline. The utilization of a variety of potential non-fuel products from algal HTL, such as the use of HTL solids as a cement additive, provides a significant reduction in net emissions by offsetting emissions from other carbon-intense products. This report presents an analysis of the research conducted at PNNL to develop an economically and environmentally beneficial process for algae HTL.

09 BIOMASS FUELS↗