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DOE OSTI2025

Knocking out the carboxyltransferase interactor 1 (CTI1) in Chlamydomonas boosted oil content by fivefold without affecting cell growth

Summary The first step in chloroplast de novo fatty acid synthesis is catalysed by acetyl‐CoA carboxylase (ACCase). As the rate‐limiting step for this pathway, ACCase is subject to both positive and negative regulation. In this study, we identify a Chlamydomonas homologue of the plant carboxyltransferase interactor 1 (CrCTI1) and show that this protein interacts with the Chlamydomonas α‐carboxyltransferase (Crα‐CT) subunit of the ACCase by yeast two‐hybrid protein–protein interaction assay. Three independent CRISPR‐Cas9 mediated knockout mutants for CrCTI1 each produced an ‘enhanced oil’ phenotype, accumulating 25% more total fatty acids and storing up to fivefold more triacylglycerols (TAGs) in lipid droplets. The TAG phenotype of the crcti1 mutants was not influenced by light but was affected by trophic growth conditions. By growing cells under heterotrophic conditions, we observed a crucial function of CrCTI1 in balancing lipid accumulation and cell growth. Mutating a previously mapped in vivo phosphorylation site (CrCTI1 Ser108 to either Ala or to Asp), did not affect the interaction with Crα‐CT. However, mutating all six predicted phosphorylation sites within Crα‐CT to create a phosphomimetic mutant reduced this pairwise interaction significantly. Comparative proteomic analyses of the crcti1 mutants and WT suggested a role for CrCTI1 in regulating carbon flux by coordinating carbon metabolism, antioxidant and fatty acid β‐oxidation pathways, to enable cells to adapt to carbon availability. Taken together, this study identifies CrCTI1 as a negative regulator of fatty acid synthesis in algae and provides a new molecular brick for the genetic engineering of microalgae for biotechnology purposes.

Li, Zhongze [Aix‐Marseille Université, CEA, CNRS, ↗

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 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 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↗

DOE OSTI2024

Algae, Wastewater Treatment and SAF

Microalgal biomass production has been proposed for many years for the low-cost production of foods, feeds, fuels and other bio-products, and to treat wastewaters, while reducing greenhouse gas emissions, with raceway ponds the most economical technology for cultivating microalgae.

09 BIOMASS FUELS↗

DOE OSTI2024

Algae, Wastewater Treatment and SAF

09 BIOMASS FUELS↗

DOE OSTI2024

Algae, Wastewater Treatment and SAF

09 BIOMASS FUELS↗

DOE OSTI2025

Evaluation of DNA Extraction Efficiency in Diverse Algae Strains Using Commercial Kits and Lysis Approaches

Efficient DNA extraction is essential for accurately monitoring microalgae communities in large-scale cultivation systems such as raceway ponds and wastewater ponds. Traditional phenol chloroform extracts are a staple in microbiology but are obsolete for routine sampling due to its high toxicity reagents and time intensive setups. Commercial DNA extraction kits are more favorable for the microbes found in these ponds, but lack specific kits made for these communities. Little is known about which kits perform the best, leading researchers to use a variety of different kits with inconsistent results. This project compared one precipitation based commercial kit (Lucigen Masterpure) and five wash based kits (Monarch, Zymo Quick-DNA, and three Qiagen DNeasy kits) using four brackish algae strains to determine which methods yield the greatest quantity and quality of genomic DNA. Extractions were evaluated using the manufacturers protocol, and additional pretreatment options were administered before a single kit to compare its potential in being added routinely before extractions. Pretreatment options included both cryogenic freeze-thawing and heat incubation using enzymes. DNA was quantified using Qubit fluorometry and NanoDrop purity ratios. Overall, the Qiagen PowerWater kit provided the highest DNA yield and purity, but at a significantly higher cost then the precipitation-based kit (MasterPure). It was also noted that while the precipitation-based kit was significantly cheaper, provided similar results, it took significantly more time to complete a single run. Cryogenic pretreatment (6x cycles) increased average DNA yields by up to 80%, whereas enzymatic pretreatment most improved purity ratios without substantially improving quantity. The results suggest that it may be more cost and time efficient to use Qiagen kits with the addition of lysis pretreatments to procure better results. Future works includes developing a better system to efficiently collect multi variable data, and to upscale to artificial polycultures using similar methodologies alongside sequencing to confirm kit results.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Enhanced Production of Algae Lipids and Carbohydrates for Fuel and Polyurethane Precursors

This Final Technical Report summarizes the goals, approach, and outcomes of the project “Enhanced Production of Algae Lipids and Carbohydrates for Fuel and Polyurethane Precursors”. The project addressed the challenge of improving microalgae biomass productivity and simultaneously producing valuable polyurethane precursors (PUPs), from both lipids and carbohydrates, that can be converted into renewable biofuels and bio-based polyurethane (PU) products. By integrating advanced genetic engineering, traditional breeding, high-throughput screening, pilot cultivation, and chemical conversion technologies, the team achieved significant advancements in algae biotechnology.

09 BIOMASS FUELS↗

DOE OSTI2025

Combined Algae Processing (CAP): Progress, Challenges, Opportunities, and Future Directions

This white paper summarizes the historical progress and perspectives on future opportunities for Combined Algae Processing (CAP), a conversion approach centered around fractionating microalgae biomass and producing a slate of fuels and products. We discuss the critical challenges associated with the commercialization of algal biofuels and demonstrate how the CAP approach is uniquely suited to overcome these barriers. The versatility of applying CAP to various sources of algae is highlighted along with conversion to various fuels and product options.

09 BIOMASS FUELS↗

DOE OSTI2026

Improved Microalgal Carbon Utilization Efficiency via Integrated CO 2 Electro-Conversion to Formate and Microalgal Sequestration

This project developed a process to convert industrial carbon dioxide (CO 2 ) emissions into high-value, sustainable products through genetically engineered algae cultivation. While traditional microalgae cultivation depends on sparging CO 2 gas through water, this method is often inefficient because much of the gas escapes into the atmosphere before the algae can consume it. To overcome this challenge, the project designed an integrated system that first uses a CO 2 to formic acid electrolyzer to convert CO 2 into water-soluble formic acid/formate, then introduces formic acid/formate into the algae pond for cultivation, which allows the algae to access and utilize nearly all of the provided carbon, greatly increasing the efficiency of carbon utilization. The project team has successfully scaled up the CO 2 to formic acid electrolyzer from lab-scale to 1000 cm² and demonstrated industrially relevant current densities with the scaled-up electrolyzers using a CO 2 source that simulates industrial CO 2 waste.

42 ENGINEERING↗

DOE OSTI2025

AlgaeOrtho, a bioinformatics tool for processing ortholog inference results in algae

Introduction: Microalgae constitute a prominent feedstock for producing biofuels and biochemicals by virtue of their prolific reproduction, high bioproduct accumulation, and the ability to grow in brackish and saline water. However, naturally occurring wild type algal strains are rarely optimal for industrial use; therefore, bioengineering of algae is necessary to generate superior performing strains that can address production challenges in industrial settings, particularly the bioenergy and bioproduct sectors. One of the crucial steps in this process is deciding on a bioengineering target: namely, which gene/protein to differentially express. These targets are often orthologs which are defined as genes/proteins originating from a common ancestor in divergent species. Although bioinformatics tools for the identification of protein orthologs already exist, processing the output from such tools is nontrivial, especially for a researcher with little or no bioinformatics experience. Methods: The present study introduces AlgaeOrtho, a user-friendly tool that builds upon the SonicParanoid orthology inference tool (based on an algorithm that identifies potential protein orthologs based on amino acid sequences) and the PhycoCosm database from JGI (Joint Genome Institute) to help researchers identify orthologs of their proteins of interest in multiple diverse algal species. Results: The output of this application includes a table of the putative orthologs of their protein of interest, a heatmap showing sequence similarity (%), and an unrooted tree of the putative protein orthologs. Notably, the tool would be instrumental in identifying novel bioengineering targets in different algal strains, including targets in not-fully annotated algal species, since it does not depend on existing protein annotations. We tested AlgaeOrtho using three case studies, for which orthologs of proteins relevant to bioengineering targets, were identified from diverse algal species, demonstrating its ease of use and utility for bioengineering researchers. Discussion: This tool is unique in the protein ortholog identification space as it can visualize putative orthologs, as desired by the user, across several algal species.

09 BIOMASS FUELS↗

DOE OSTI2024

Environmental DNA Detection in Marine Macrophyte Ecosystems as a Potential Blue Carbon Source in Sediments

“Blue carbon” refers to the carbon sequestered by the world’s oceanic and coastal ecosystems, particularly through coastal vegetation such as mangroves, salt marshes, seagrasses, and marine macroalgae. These ecosystems play a crucial role in the global carbon cycle by serving as significant carbon sinks, absorbing carbon dioxide from the atmosphere and storing it in biomass and sediments over long periods. This study explores the use of environmental DNA (eDNA) to detect marine macrophytes and microalgae assemblages contributing to blue carbon in sediments across various coastal ecosystems. The research addresses the challenges of traditional monitoring methods by utilizing high-throughput sequencing of the 18S-V9 region amplified using eDNA from sediment samples collected at eight locations in the United States and South Korea. The results reveal a diverse array of taxa, underscoring the variability in community composition across different conditions. Notably, sites with seagrass beds and Ulva blooms showed distinct patterns in microalgal community structure. This study underscores the potential of eDNA analysis in providing comprehensive insights into the biodiversity of marine macrophyte ecosystems, thus informing conservation efforts and enhancing the understanding of marine ecological dynamics.

Xing, Qikun (ORCID:0000000206179027)↗

DOE OSTI2025

Persistence of bacterial-mediated anti-rotifer protection in preliminary outdoor cultivation trial for Microchloropsis salina

Outdoor algal cultivation systems are susceptible to a wide variety of deleterious species. In previously published studies, we observed protection using microbial consortia at laboratory scale cultures; Microchloropsis salina in the presence of microbial consortia were protected from grazing from the marine rotifer, Brachionus plicatilis. Our objective for the present work was to determine if this protection conferred by microbial consortia in controlled laboratory experiments would persist in an open, outdoor multi-liter cultivation system. We found that algal protection did persist as evidenced by the presence of fewer motile rotifers and decreased rotifer-associated egg counts for the consortia-treated outdoor cultures. Due to the low temperature and light conditions that reduced growth of the algae outdoors, we performed an indoor laboratory assay which also confirmed the persistence of algal protection. Lastly, the lower numbers of motile rotifers and fewer rotifer-associated eggs in the consortia-treated algal cultures suggests a possible protective mechanism by the consortia through interfering with the rotifer lifecycle or reproduction. Finally, these initial results support the possibility that low cost, prophylactic treatments with microbial consortia can protect algae from deleterious species in outdoor cultivation systems.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Identification and overexpression of endogenous transcription factors to enhance lipid accumulation in the biotechnologically relevant species Chlamydomonas pacifica

Sustainable low-carbon energy solutions are critical to mitigating global carbon emissions. Algae-based platforms offer potential by converting carbon dioxide into valuable products while aiding carbon sequestration. However, scaling algae cultivation faces challenges like contamination in outdoor systems. Previously, our lab evolved Chlamydomonas pacifica, an extremophile green alga, which tolerates high temperature, pH, salinity, and light, making it ideal for large-scale bioproduct production, including biodiesel. Here, we enhanced lipid accumulation in evolved C. pacifica by identifying and overexpressing key endogenous transcription factors through genome-wide in-silico analysis and in-vivo testing. These factors include Lipid Remodeling Regulator 1 (CpaLRL1), Nitrogen Response Regulator 1 (CpaNRR1), Compromised Hydrolysis of Triacylglycerols 7 (CpaCHT7), and Phosphorus Starvation Response 1 (CpaPSR1). Under nitrogen deprivation, CpaLRL1, CpaNRR1, and CpaCHT7 overexpression enhanced lipid accumulation compared to wild-type. However, CpaPSR1 increased lipid accumulation compared to wild-type in normal media and did not increase further under nitrogen deprivation, highlighting the difference in function based on media conditions. Notably, lipid analysis of CpaPSR1 under normal media conditions revealed a 2.4-fold increase in triglycerides (TAGs) compared to the wild-type, highlighting its potential for biodiesel production. This approach provides a framework for transcription factor-focused metabolic engineering in algae, advancing bioenergy and biomaterial production.

Biofuels↗

DOE OSTI2025

Integrated thermal and biological conversion of microalgal proteins to lipids

Microalgal composition varies with cultivation strategy, and low-cost approaches often produce high-protein biomass. This presents challenges for biorefineries designed around static, lipid-rich feedstocks. In particular, hydrolysates from high-protein algae are nitrogen-rich and sugar-poor, limiting microbial conversion and reducing product yields. This study develops a sequential thermal conditioning and biological upgrading strategy to integrate high-protein hydrolysate processing within conventional lipid extraction and upgrading designs. Oxidative deconstruction was used to break down proteins into ammonium and short-chain carboxylates. Ammonium was subsequently removed to yield a nitrogen-depleted, carboxylate-rich medium suitable for microbial lipid production. Bioconversion trials with Cutaneotrichosporon oleaginosum showed lipid accumulation only from hydrolysates treated with both oxidative deconstruction and nitrogen removal, reaching 1.2 g/L lipids at 30 % intracellular content. This integrated approach enables protein-to-lipid conversion and improves flexibility to process variable algal feedstocks, advancing fuel-oriented microalgal biorefineries.

09 BIOMASS FUELS↗

DOE OSTI2025

Hydrothermal liquefaction of wastewater-grown algae to produce synthetic aviation fuel: A combined experimental study and techno-economic assessment

Large-scale algae farms may someday become a consistent source of biomass feedstock for biofuels. Near-term supplies of algal biomass are available at certain water resource recovery facilities as algae cultivation is used as a method for nutrient recovery from specific effluent streams. Algae grown as a service shifts the value to the service rather than its sole use as a feedstock, which could enable the provision of algal biomass at low to no cost to biofuel producers. Hydrothermal liquefaction (HTL) can readily upgrade wet feedstock slurries, such as algae, to produce a carbon-enriched biocrude. The HTL biocrude can be hydrotreated and distilled, producing a variety of distillate fuels, including synthetic aviation fuel (SAF). We present a pathway, showing the experimental production of SAF from wastewater-grown algae via HTL, along with a techno-economic assessment to identify opportunities for process improvements. Critical quality attributes of the SAF, such as density, viscosity, surface tension, and freeze point, were estimated within the expected fuel experience ranges when compared against petroleum jet fuel. The average minimum fuel selling price of fuels from wastewater-grown algae for breakeven economics was $\$9.04$ per gasoline gallon equivalent (GGE). The sale of co-products such as struvite fertilizers and cement additives can add revenue to reduce the net cost. Ultimately, the selling price is influenced by the scale of the HTL processing facility. Adjusting estimations in the process scale, algae yield, and capital cost estimation can lower the price to $\$6.51$/GGE or raise it to $13.07/GGE.

Biofuels↗