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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 55 records · Page 3

Systems-wide analysis revealed shared and unique responses to moderate and acute high temperatures in the green alga Chlamydomonas reinhardtii

Different intensities of high temperatures affect the growth of photosynthetic cells in nature. To elucidate the underlying mechanisms, we cultivated the unicellular green alga Chlamydomonas reinhardtii under highly controlled photobioreactor conditions and revealed systems-wide shared and unique responses to 24-hour moderate (35°C) and acute (40°C) high temperatures and subsequent recovery at 25°C. We identified previously overlooked unique elements in response to moderate high temperature. Heat at 35°C transiently arrested the cell cycle followed by partial synchronization, up-regulated transcripts/proteins involved in gluconeogenesis/glyoxylate-cycle for carbon uptake and promoted growth. But 40°C disrupted cell division and growth. Both high temperatures induced photoprotection, while 40°C distorted thylakoid/pyrenoid ultrastructure, affected the carbon concentrating mechanism, and decreased photosynthetic efficiency. We demonstrated increased transcript/protein correlation during both heat treatments and hypothesize reduced post-transcriptional regulation during heat may help efficiently coordinate thermotolerance mechanisms. During recovery after both heat treatments, especially 40°C, transcripts/proteins related to DNA synthesis increased while those involved in photosynthetic light reactions decreased. We propose down-regulating photosynthetic light reactions during DNA replication benefits cell cycle resumption by reducing ROS production. Our results provide potential targets to increase thermotolerance in algae and crops.

59 BASIC BIOLOGICAL SCIENCES↗

A phytophotonic approach to enhanced photosynthesis

Photosynthesis is the dominant biotic carbon sink on earth and hence presents an opportunity for enhanced sequestration of CO 2 . If the average net carbon fixation efficiency of terrestrial plants could be increased by 3.3%, all anthropogenic CO 2 accumulating in the atmosphere could instead be reduced and incorporated into terrestrial biomass. Plants make inefficient use of the overly abundant sunlight available to them, a result of having evolved to be competitive and survive highly dynamic environmental conditions rather than maximize photosynthetic productivity. We explore herein a phytophotonic approach to enhanced photosynthesis, whereby sunlight is redistributed by means of luminescent or persistent luminescent (PersL) materials. Phytophotonics has potential at varied scales, ranging from photobioreactors to greenhouses all the way to crops in the field, the latter having the potential to impact planetary CO 2 levels. The approach is three-fold: a spectral redistribution to relieve high-light-stress at the top surface of leaves and increasingly drive photosynthesis deeper in leaves and canopies; a minute-scale temporal redistribution to bridge periods of intermittent shade and reduce shock associated with variable light conditions; and a multiple-hour temporal redistribution to shift a fraction of high-intensity midday lighting to evening hours. Based on simulations of photoluminescent materials and light quality experiments with a model algal system, it is shown that while lengthening daylight hours will require significant improvements in PersL materials, the other two approaches show more immediate promise. We demonstrate a means of concentrating PersL light from SrAl 2 O 4 :Eu,Dy, approaching levels needed to effectively bridge periods of natural shade, and outline the scientific questions and technical hurdles remaining to realize the benefits of the proposed spectral shift.

59 BASIC BIOLOGICAL SCIENCES↗

Productivity and bioproduct formation in phototropic knock/out mutants in micro algae

Phototropin is a blue light receptor, which mediates a variety of blue-light elicited physiological processes in plants and algae. In higher plants these processes include phototropism, chloroplast movement and stomatal opening. In the green alga Chlamydomonas reinhardtii, phototropin plays a vital role in progression of the sexual life cycle and in the control of the eye spot size and light sensitivity. Phototropin is also involved in blue-light mediated changes in the synthesis of chlorophylls, carotenoids, chlorophyll binding proteins. We compared the transcriptome of phototropin knock out (PHOT KO) mutant and wild-type parent to analyze differences in gene expression in high light grown cultures (500 μmol photons m⁻²s⁻¹). Our results indicate the up-regulation of genes involved in photosynthetic electron transport chain, carbon fixation pathway, starch, lipid, and cell cycle control genes. With respect to photosynthetic electron transport genes, genes encoding proteins of the cytochrome b6f and ATP synthase complex were up regulated potentially facilitating proton-coupled electron transfer. In addition genes involved in limiting steps in the Calvin cycle Ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO), Sidoheptulose 1,7 biophosphatase (SBPase), Glyceraldehyde-3-phosphate dehydrogenase (3PGDH) and that mediate cell-cycle control (CDK) were also up regulated along with starch synthase and fatty acid biosynthesis genes involved in starch and lipid synthesis. In addition, transmission electron micrographs show increased accumulation of starch granules in PHOT mutant compared to wild type, which is consistent with the higher expression of starch synthase genes. Collectively, the altered patterns of gene expression in the PHOT mutants were associated with a two-fold increase in growth and biomass accumulation compared to wild type when grown in environmental photobioreactors (Phenometrics) that simulate a pond environment. In conclusion, our studies suggest that phototropin may be a master gene regulator that suppresses rapid cell growth and promotes gametogenesis and sexual recombination in wild type strains.

59 BASIC BIOLOGICAL SCIENCES↗

CO 2 to Bioplastics: Beneficial Re-use of Carbon Emissions from Coal-fired Power Plants using Microalgae

This project sought to address the technical and economic barriers to carbon dioxide (CO 2 ) capture and utilization using microalgae. Specifically, a dual photobioreactor (PBR)/open raceway pond (ORP) cultivation system was evaluated with respect to capital and operational costs, productivity, and culture health, and compared to an ORP-only cultivation system. In the dual (or “hybrid”) system, a cyclic flow PBR was used to provide inoculum for two open raceway ponds after each harvest, with the hypothesis that this PBR + pond system should result in improved performance compared to traditional raceway ponds. Two other raceway ponds were operated conventionally as a control, the experiments being performed at Duke Energy’s East Bend Station in northern KY using coal-derived flue gas as the CO 2 source. Although algae productivity achieved was mediocre due to the fact that the experiments had to be performed late in the growing season when climatic conditions were not optimal for high growth, the hybrid cultivation system showed a higher level of algae productivity (statistically significant) compared to the traditional ponds. In order to realize the maximum value of the algal biomass produced, fractionation of the biomass was examined. Bioplastic compounding and material characterization was subsequently performed using three feed stocks: whole biomass, lipid-extracted biomass and the proteinaceous residue from full fractionation. In general, the whole and lipid-extracted biomass gave similar results in terms of the properties of the resulting bioplastics, while the proteinaceous residue showed promise for the production of a polybutylene adipate terephthalate (PBAT) blend. Finally, sustainability assessment of a putative biorefinery system was conducted, including techno-economic and life cycle impact assessment. Nine different production scenarios were considered, comprising combinations of the three different biomass growth architectures (ORP, PBR and dual PBR-ORP systems) coupled with three different algae biomass processing pathways (drying only, lipid extraction and fractionation). Results show that the minimum selling price of the bioplastic feed stock (BPFS) is within the realm of economic competition with prices as low as $970 USD tonne -1 . Additionally, life cycle impact assessment results indicate drastic improvements in performance of the produced BPFS, with reductions in greenhouse gas emissions ranging between 67 and 116% compared to a petroleum based plastic feedstock.

01 COAL, LIGNITE, AND PEAT↗

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↗

A Novel Platform for Algal Biomass Production Using Cellulosic Mixotrophy (CeMix) (Final Technical Report)

This novel project, branded CeMIX for cellulosic mixotrophy, targets solutions for several technological barriers limiting the deployment of algae cultivation in sunny, arid regions (specifically the southwest United States). Opening up the region for algae cultivation could increase DOE’s resource assessments and biofuel potential for the U.S. Mixotrophic metabolism in support of liquid fuel production is completely dependent on the availability of waste organic carbon in order to avoid diversion of food resources for fuel production. The use of cellulosic sugar hydrolysate (CSH) produced via the NREL process provided a standardized carbon source for study of the underlying biochemistry and metabolic adjustments to mixotrophy. Mixotrophic metabolism uses both photosynthetic CO 2 fixation and sugar oxidation to improve biomass productivity and harvest yield. The project was high-risk, high-reward in the sense that adding organic carbon to algal cultivation is an invitation for rapid-growth, heterotrophic contaminants to overtake the culture and consume mineral nutrients needed for algal growth. To help evade this outcome the project focused on acidophilic red algae that require low pH conditions with 40-48°C optimal temperature profiles that define them as low-range thermophiles. This is the only project in the BETO portfolio to utilize extreme conditions of low pH and high operating temperatures to control pathogens and competitors in mass culture. The project yielded five major outcomes. 1) Harvest densities of 5-10 g ash-free dry weight are easily achieved, providing a 10-fold reduction in water required and dewatering demand. 2) Catabolic repression of photosynthesis in G. sulphuraria is conditionally repressed by low oxygen. 3) Mixotrophic growth on cellulosic hydrolysate consumes all C6 and C5 sugars concomitant with increases in floridean starch with little change in lipid content. Protein content can be manipulated between 35 and 54 wt% by varying the C:N molar ratio from 10:1 to 20:1, respectively. 4) Mixotrophic cultures with up to 50 mM total sugar are remarkably stable at pH 2.5 and daily average reactor temperatures of 40°C. 5) Capital costs for glass tubular photobioreactors are prohibitive for liquid fuel production using a hydrothermal liquefaction processing pathway. The economic outlook for covered raceway ponds is better with a project minimum fuel selling price at $3.32 dm 3 GE -1 .

09 BIOMASS FUELS↗

Solar Spectrum Conversion for an Algae Bioreactor (CRADA Final Report)

This project focused on developing advanced optical coatings to improve solar energy utilization. The research aimed to create lanthanide-doped upconversion nanoparticles (UCNPs) capable of capturing unused near-infrared (NIR) light from the sun and converting it into visible light (blue and red photons) that can be used for photosynthesis. The primary goal was to identify, synthesize, and integrate highly efficient UCNPs into a transparent thin-film device. Through a comprehensive workflow involving computer simulations, high-throughput robotic synthesis, and detailed optical characterization, the project successfully developed a high-performance material. The key technical achievement was the creation of a core-shell UCNP (NaYF₄:20%Yb³⁺, 2%Er³⁺ coated with a 10 nm NaYF₄ shell) that demonstrated a quantum yield of 3.2% for converting 980 nm NIR light into visible light. Transparent thin films fabricated from these nanoparticles showed excellent optical properties, confirming their potential for practical applications. This research adds to the scientific understanding of energy transfer in lanthanide materials and demonstrates a technically effective method for creating efficient light-converting coatings. The primary benefit to the public lies in the potential for these coatings to enhance the efficiency of solar-driven processes, such as boosting the growth of algae in photobioreactors for biofuel production.

14 SOLAR ENERGY↗

Cas9 deletion of lutein biosynthesis in the marine alga Picochlorum celeri reduces photosynthetic pigments while sustaining high biomass productivity

Domestication of algae for food and renewable biofuels remains limited by the low photosynthetic efficiencies of processes that have evolved to be competitive for optimal light capture, incentivizing the development of large antennas in light-limiting conditions, thus decreasing efficient light utilization in cultivated ponds or photobioreactors. Reducing the pigment content to improve biomass productivity has been a strategy discussed for several decades and the ability to reduce pigment significantly is now fully at hand thanks to the widespread use of genome editing tools. Picochlorum celeri is one of the fastest growing marine algae identified and holds particular promise for outdoor cultivation, especially in saline water and warm climates. We show that while chlorophyll b is essential to sustain high biomass productivities under dense cultivation, removing Picochlorum celeri’s main carotenoid, lutein, leads to a decreased total chlorophyll content, higher a/ b ratio, reduced functional LHCII cross section and higher maximum quantum efficiencies at lower light intensities, resulting in an incremental increase in biomass productivity and increased PAR-to-biomass conversion efficiency. These findings further strengthen the existing strategies to improve photosynthetic efficiency and biomass production in algae.

59 BASIC BIOLOGICAL SCIENCES↗

Manipulation of glycogen and sucrose synthesis increases photosynthetic productivity in cyanobacteria

Photosynthetic productivity is limited by low energy conversion efficiency in naturally evolved photosynthetic organisms, via multiple mechanisms that are not fully understood. Here we show evidence that extends recent findings that cyanobacteria use “futile” cycles in the synthesis and degradation of carbon compounds to dissipate ATP. Reduction of the glycogen cycle or the sucrose cycle in the model cyanobacterium Synechocystis 6803 led to redirection of cellular energy toward faster growth under simulated outdoor light conditions in photobioreactors that was accompanied by higher energy charge [concentration ratio of ATP/(ATP + ADP)]. Such manipulation of energy metabolism may have potential in engineering microalgal chassis cells to increase productivity of biomass or target metabolites.

09 BIOMASS FUELS↗

Leveraging CRISPR Cas9 RNPs and Cre- loxP in Picochlorum celeri for generation of field deployable strains and selection marker recycling

As new highly productive strains of algae are discovered and developed to meet the energy, chemical, and food requirements of the future, genetic engineering of those strains in a manner that yields deployable transformants is paramount. This study introduces the novel CRoxP ($\underline{\textrm{C}}$$\textrm{as9}$ $\underline{\textrm{R}}$$\textrm{NPs}$ coupled with an inducible $\underline{\textrm{CR}}$$\textrm{e}$-$\textrm{l}\underline{\textrm{oxP}}$) system for rapid generation of marker- and transgene-free strains of Picochlorum celeri. The CRoxP system allows reuse of selection markers without Cas9 expression in vivo, eliminating many of the bottlenecks associated with conventional CRISPR Cas9 use for precise genome editing. In P. celeri, transformants were generated with a turnaround time as short as 21 days between transformation and being ready for another round of transformation with the same selection marker by using the CRoxP system. As a use-case for CRoxP, depigmented strains of P. celeri were generated by multiplexed Cas9 disruption of major LHCII genes followed by either a second round of LHCII targeting, or knockout of an LHCI gene. One transformant tested in flask culture (R6) exhibited similar biomass production to the wild type with 46% less Chl a + b on a biomass basis. In photobioreactors and under diel light simulating a solar day, a transformant (LhcBM31) exhibited 34 g AFDW m –2 d –1 with 54% less Chl a + b on a biomass basis vs. wild type.

09 BIOMASS FUELS↗

Biocontainment of Genetically Engineered Algae

Algae (including eukaryotic microalgae and cyanobacteria) have been genetically engineered to convert light and carbon dioxide to many industrially and commercially relevant chemicals including biofuels, materials, and nutritional products. At industrial scale, genetically engineered algae may be cultivated outdoors in open ponds or in closed photobioreactors. In either case, industry would need to address a potential risk of the release of the engineered algae into the natural environment, resulting in potential negative impacts to the environment. Genetic biocontainment strategies are therefore under development to reduce the probability that these engineered bacteria can survive outside of the laboratory or industrial setting. These include active strategies that aim to kill the escaped cells by expression of toxic proteins, and passive strategies that use knockouts of native genes to reduce fitness outside of the controlled environment of labs and industrial cultivation systems. Several biocontainment strategies have demonstrated escape frequencies below detection limits. However, they have typically done so in carefully controlled experiments which may fail to capture mechanisms of escape that may arise in the more complex natural environment. The selection of biocontainment strategies that can effectively kill cells outside the lab, while maintaining maximum productivity inside the lab and without the need for relatively expensive chemicals will benefit from further attention.

59 BASIC BIOLOGICAL SCIENCES↗

Novel Concept for LSS Based on Advanced Microalgal Biotechnologies

One of the key issues for successful human space exploration is biomedical life support in hostile space and planetary environments that otherwise cannot sustain life. Bioregenerative life support systems (LSS) are one of the options for atmospheric regeneration. To date, no bioregenerative LSS has shown capability for 100% air regeneration. Nor have these LSS been robust enough to simultaneously provide a regenerable complete food source. In contrast to microalgae, traditional plant approaches, e.g. wheat and lettuce, are lacking essential amino acids, vitamins, and micronutrients. Moreover, the rate of photosynthesis by microalgae significantly exceeds that of high plants. Nevertheless, the employment of microalgae in LSS technology was restricted, until recently, due to high water demands. Also the per person requirement of a 40L volume of microalgae in a photobioreactor, to provide daily O2 production, made an algae-based approach less attractive. By employing a vertically stacked membrane bioreactor, coupled with a solar tracker and photon-delivery system, a lightweight air revitalization system for space based applications, with minimal water requirements, can be developed. Our preliminary estimations suggest that a membrane bioreactor, 8m3 in volume, comprised of 80m2 (twenty 2m x 2m membranes, each spaced 10 cm apart), and a total of 70L of water could produce 2.7 kg of dried microalgal biomass that would supply the energy and essential amino acid requirements, as well as producing sufficient O2 for the daily needs of a 15 member crew. Research on the biochemical content of edible blue-green alga Spirulina (Arthrospira) platensis shows a wide spectrum of stable Spirulina mutants with an enhanced content of amino acids, -carotene, and phycobilliprotein c-phycocyanin. Feeding animals suffering from radiation-induced lesions, c-phycocyanin, extracted from strain 27G, led to a correction in the decrement of dehydrogenase activity and energy-rich phosphate levels, as well as improved antioxidant defense and pyruvate levels, compared to untreated animals. Experimental anemia in rats was corrected by feeding Spirulina platensis strains 198B and 27G, (with an enhanced content of methionine, phycobiliproteins and carotenoids). Spirulina was recently shown by Ananyev et al, 2005, to be an oxygenic organism with the highest level of photosystem II activity (O2 production). We propose therefore to develop a design for membrane-based photoreactors for Lunar and Mars exploration habitat LSS, for the cultivation of genetically modified strains of Spirulina to scrub CO2 and supply astronauts with O2, protein, vitamins, and immunostimulators.

Brown, I.↗

OMEGA for the Future of Biofuels

OMEGA: Offshore Membrane Enclosure for Growing Algae. To develop a photobioreactor (PBR) for growing algae (Oil, food, fertilizer) that does not compete with agriculture for land (deployed offshore), water or fertilizer (uses/treats wastewater).

Trent, Jonathan↗

OMEGA (Offshore Membrane for Enclosing Algae). NASA-NAVY: A Strategic Planning Discussion

This briefing packet provides a short introduction to OMEGA and a truncated version of our project approach, with an example of the kind of work break down structure (WBS) used to guide our Phase I activities. It is meant to give you an impression of how we are approaching the challenge of creating the world's first marine photobioreactor (PBR) that will scale to address the strategic energy problems confronting the United States and the world. Some of our conceptual PBR designs and plans for logistics are included to communicate the path we have taken. We have also included an aerial photograph of the experimental tanks we are using at the Cal Fish and Game, followed by concluding remarks. The overarching purpose of the strategic planning discussion in Norfolk is to establish the relationship between the NASA OMEGA Team and the Navy, to unite the strengths of both agencies, and to map a mutual way forward along the project's established critical path.

Trent, Jonathan↗

Development of a Prototype Algal Reactor for Removing CO2 from Cabin Air

Controlling carbon dioxide in spacecraft cabin air may be accomplished using algal photobioreactors (PBRs). The purpose of this project was to evaluate the use of a commercial microcontroller, the Arduino Mega 2560, for measuring key phot~ioreactor variables: dissolved oxygen, pH, temperature, light, and carbon dioxide. The Arduino platform is an opensource physical computing platform composed of a compact microcontroller board and a C++/C computer language (Arduino 1.0.5). The functionality of the Arduino platform can be expanded by the use of numerous add-ons or 'shields'. The Arduino Mega 2560 was equipped with the following shields: datalogger, BNC shield for reading pH sensor, a Mega Moto shield for controlling CO2 addition, as well as multiple sensors. The dissolved oxygen (DO) probe was calibrated using a nitrogen bubbling technique and the pH probe was calibrated via an Omega pH simulator. The PBR was constructed using a 2 L beaker, a 66 L box for addition of CO2, a micro porous membrane, a diaphragm pump, four 25 watt light bulbs, a MasterFiex speed controller, and a fan. The algae (wild type Synechocystis PCC6803) was grown in an aerated flask until the algae was dense enough to used in the main reactor. After the algae was grown, it was transferred to the 2 L beaker where CO2 consumption and O2 production was measured using the microcontroller sensor suite. The data was recorded via the datalogger and transferred to a computer for analysis.

Patel, Vrajen↗

Algae to Enhance Water Recovery for Future Space Habitats

Bioregenerative technologies have been suggested for human life support in space for decades. Such technologies have not yet been incorporated due to 1) assumed unreliability and 2) bioregeneration rates for given processes are slower when compared to equivalent physical/chemical treatment technologies. Slower treatment capacity for biological system result in larger technology infrastructure (i.e., higher mass, power, and volume requirements), and thus less attractive. Current ISS (International Space Station) life support systems are strictly physical/chemical. However, bioregenerative systems are being suggested for future surface systems (on the Moon or Mars) due to the limited access to resupplied materials and continued need for resiliency and sustainability. In the realm of water reclamation, the ISS system processes only urine, metabolic condensates, and hygiene (i.e., handwash and oral, no shower or laundry) waters with 75 percent closure. For future surface habitats with 4 crew members, approximately 30 liters per day of wastewater will be generated, containing estimated 850 milligrams per liter NH4-N. Conventional algae PBRs (photobioreactors) require dilution to accommodate such high concentrations of ammonium. If dilution is required, resulting technology hardware will increase dramatically in mass. A new approach was explored, whereby high carbon dioxide (1200-2000 parts per million) and light (400-600 micromoles per square meter per second) conditions were provided to treat the undiluted wastewater stream. Daily treatment capacity for Chlorella sorokiniana and Chlorella vulgaris was observed to be 85 and 107 milligrams NH4-N per gram of biomass, respectively. This preliminary study shows that there is capacity to increase ammonium removal rates by algal species, and thus reactor size (mass and volume) for future surface systems. Smaller reactor volumes will help bioregenerative treatment technologies compete with the presently accepted physical/chemical treatment technologies.

Water Purification↗

Combined Techno-Economic Analysis and Life Cycle Assessment of an Integrated Direct Air Capture System with Advanced Algal Biofuel Production

The continuous increase in carbon dioxide (CO2) concentration in the atmosphere since the First Industrial Revolution correlates convincingly with the ongoing rise of the Earth's global average temperature contributing to climate change. As a response, carbon capture and sequestration (CCS) technologies are being implemented to mitigate anthropogenic CO2 emissions by capturing CO2 from high emitting point sources, such as power plants, refineries, and cement factories, and subsequently buried in geological formations underground for long term storage. Recently direct air capture (DAC) has emerged as a promising alternative technology that captures CO2 directly from the atmosphere for use or sequestration. This study investigates the potential synergistic benefits of integrating a solid amine-based DAC system with advanced algal biofuel production in photobioreactors (PBRs). DAC utilization allows the removal of atmospheric CO2 while also decoupling algae production facilities from anthropogenic point CO2 sources and avoiding the cost and logistics challenges of transporting CO2 long distances to remote facilities. Techno-economic analysis and life cycle assessment are performed to assess the economic and environmental benefits of heat and mass integration between the DAC and the PBR for biofuel production. The DAC-PBR system integration also considers on-site flue gas handling options, DAC capital utilization, the tradeoff between centralization or decentralization of key unit operations, and the PBR array. This presentation will discuss how optimizing DAC-PBR process integration can enhance the algal biofuel's economic and environmental sustainability and the prospect of DAC enabling the circular carbon economy.

algal biofuel production↗