Extracellular vesicles (EVs) are nanoscale particles with membrane structures secreted by cells, facilitating the transfer of proteins, lipids, small RNAs, lncRNAs, and DNAs. These vesicles have been extensively studied for their role in mediating cell-to-cell communication in mammalian systems and their potential applications in disease diagnosis and treatment. While extensively studied in mammalian systems, the functions of microalgal EVs, particularly their roles in environmental adaptation, remain underexplored. In this study, we identified EVs in four selected microalgal cultures, including Chlamydomonas, and we found EVs can transfer miRNAs and proteins between Chlamydomonas cells. Notably, miRNAs in EVs were selectively packaged and distinct from those in host cells, suggesting their role in stress signaling and cellular adaptation. Proteomic analyses identified stress-responsive proteins and potential biomarkers for nutrient depletion. These findings suggest that Chlamydomonas EVs play a significant role in nutrient sensing and information transfer between cells, especially under nutrient stress conditions. Our research highlights the critical role of EVs in nutrient sensing and intercellular communication in microalgae, offering insights into their adaptive strategies in dynamic aquatic ecosystems.

IntroductionMicroalgae, known for their adaptability to extreme environments, are important for basic research and industrial applications. Euglena, unique for its lack of a cell wall, has garnered attention due to its versatility and the presence of bioactive compounds. Despite its potential, few studies have focused on Euglena’s cold adaptation mechanisms.MethodsThis study investigates the cold adaptation mechanisms of Euglena gracilis, a microalga found in highly diverse environmental habitats, by comparing its growth, photosynthetic performance, and physiological and biochemical responses under two low-temperature cultivation modes: pre-adaptation to 16°C followed by exposure to 4°C (PreC) and direct exposure to 4°C (DirC).Results and discussionIn this study, the PreC group exhibited superior growth rates, higher photosynthetic efficiency, and more excellent antioxidant activity compared to the DirC group. These advantages were attributed to higher levels of protective compounds, enhanced membrane stability, and increased unsaturated fatty acid content. The PreC group’s ability to maintain higher cell vitality under cold stress conditions underscores the significance of pre-adaptation in enhancing cold tolerance. The findings from this research provide valuable insights into the mechanisms underlying cold adaptation in E. gracilis, emphasizing the benefits of pre-adaptation. These insights are crucial for optimizing the cultivation of algal species under cold stress conditions, which is essential for both biotechnological applications and ecological studies. This study not only advances our understanding of Euglena’s adaptive responses to low temperatures but also contributes to the broader field of algal research and its industrial exploitation.

Euglena gracilis (E. gracilis), pivotal in the study of photosynthesis, endosymbiosis, and chloroplast development, is also an industrial microalga for paramylon production. Despite its importance, E. gracilis genome exploration faces challenges due to its intricate nature. In this study, we achieved a chromosome-level de novo assembly (2.37 Gb) using Illumina, PacBio, Bionano, and Hi-C data. The assembly exhibited a contig N50 of 619 Kb and scaffold N50 of 1.12 Mb, indicating superior continuity. Approximately 99.83% of the genome was anchored to 46 chromosomes, revealing structural insights. Repetitive elements constituted 58.84% of the sequences. Functional annotations were assigned to 39,362 proteins, enhancing interpretative power. BUSCO analysis confirmed assembly completeness at 80.39%. This first high-quality E. gracilis genome offers insights for genetics and genomics studies, overcoming previous limitations. The impact extends to academic and industrial research, providing a foundational resource.

In recent years, the proliferation of industrial effluents has posed significant challenges. In comparison to traditional physical and chemical methods, biological approaches have gained prominence among researchers due to their cost-effectiveness, abundant sources, ease of operation, and minimal secondary pollution. Moreover, employing microalgae for bioremediation offers the added advantage of concurrent production of diverse sustainable resources and biofuels. Nevertheless, challenges pertaining to recovery and reaction conditions continue to impede its large-scale implementation. Thus, exploration of improved solutions, such as enhanced reaction conditions, is imperative to maximize efficiency. In this review, we elucidate the mechanisms and pivotal factors involved in the removal of heavy metals by microalgae. Notably, we highlight innovative bioreactor technologies, including immobilization carriers, modified microalgae, and symbiotic systems, as focal points of this discussion. This comprehensive overview underscores the potential of microalgae-based bioremediation and paves the way for future advancements in this critical field.

IntroductionThe microalga Chlorella sp. NeZha, recently isolated from a balcony environment, shows significant adaptability across various salinity conditions, including seawater (SeaW), freshwater (FreshW), and high salinity levels (45‰). This study investigates its potential for sustainable aquaculture and biotechnological applications.MethodsMorphological and genetic identification were conducted using optical microscopy and DNA sequencing. The microalga was cultivated in a 400 L outdoor photobioreactor, and its biochemical composition, including chlorophyll a, carbohydrate, protein, and lipid content, was analyzed. Its compatibility with zooplankton and growth in aquaculture wastewater were also evaluated.ResultsChlorella sp. NeZha produced chlorophyll a at concentrations exceeding seaweed and Spirulina by 10- and 5-fold, respectively, with a dry weight chlorophyll a content of 34.25 mg/g and 25 pg./cell. The microalga also contained carbohydrate (~33%), protein (~20%), and lipids (~14%). It was compatible with zooplankton species, such as rotifers and brine shrimp, and showed promising growth in aquaculture wastewater.DiscussionThe findings suggest that Chlorella sp. NeZha is a viable candidate for sustainable aquaculture and biotechnological applications, offering high nutritional value and environmental resilience. Its adaptability to diverse salinity conditions and ability to thrive in wastewater highlight its potential for bioremediation and use as feedstock for zooplankton. Further research is recommended to optimize its cultivation and explore broader applications.

Life cycle assessment (LCA) is a powerful tool to evaluate environmentally sustainable production or consumption of various goods or services. Microalgae are single-celled green factories and good resources of biofuels, bioactive products, food ingredients, and degradable biomaterials. Currently, microalgae are also valuable for mitigating elevated greenhouse gases like CO 2 levels and treatment of wastewater. LCA evaluation was limited and separated, majorly in microalgal biofuels and heterotrophic cultivation. Comparative LCA for different final algal products such as algal powder, bio-oil, total fatty acid, and residue recycling is still limited, especially autotrophic algal cultivation for products other than bio-oils and biofuels. Thus, we chose several autotrophic cultivated microalgae and made a comparative LCA among these selected species and a detailed step-by-step production in Chlorella sp. Results indicated that we could significantly reduce the production cost and lower environmental impacts by selecting algal species and final products, optimizing methods for algal cultivation, biomass separation, and drying process, and land selection plus electricity renewable energy, together with thermal power plants nearby for CO 2 or flu gas. It shed light on the insight of microalgal consumption selection under current international requirements and challenges for carbon sequestration.

Euglena gracilis , a single-celled microalga with various trophic growth styles under different cultivation conditions, contains nutrients, such as ß-1,3-glucans, essential amino acids, fatty acids, vitamins, and minerals. It has recently attracted attention as a new health food. Among them, ß-1,3-glucans, paramylon of Euglena , is an insoluble dietary fiber and is well known as an immune booster, attenuator of obesity and diabetes, reducer of acute liver injury, and suppressor of atopic dermatitis, and other chronic inflammatory disorders. Recently, evidence has appeared for the positive health effects of foods, food ingredients, or biochemical compounds derived from several other microalgae, such as Chlorella , Spirulina , Dunaliella , Phaeodactylum , and Pavlova . Until most recently, the prebiotic activity of Euglena and paramylon was reported. Emerging prospects of microalgae as prebiotics were well summarized, but the mechanisms behind the bacterial growth promotion by microalgae are not elucidated yet. Thus, we evaluated the prebiotic prospects of both autotrophic and heterotrophic Euglena on six different Lactobacillus . What’s more, the stimulated mechanism was revealed by bacterial culture medium metabolomic analysis. This study could widen the knowledge about the prebiotic activity of Euglena as a next-generation prebiotic and other microalgae-derived compounds as potential health foods.

With the continuous growth of the world’s population and the increasing development of industrialization, the demand for energy by human beings has been expanding, resulting in an increasingly severe energy crisis. Microalgae are considered the most potential alternatives to traditional fossil fuels due to their many advantages, like fast growth rate, strong carbon sequestration capacity, and low growth environment requirements. Euglena can use carbon sources such as glucose, ethanol, and others for heterotrophic growth. Moreover, Euglena is highly adaptable to the environment and has a high tolerance to various environmental stresses, such as salinity, heavy metals, antibiotics, etc. Different treatments of Euglena cells could affect their growth and the accumulation of bioactive substances, especially fatty acids. To expand the industrial application of Euglena as a potential biodiesel candidate, we determine the physiological responses of Euglena against environmental stresses (antibiotics, heavy metals, salinity) or carbon resources (glucose and ethanol), and evaluate the potential for higher quality and yield of fatty acid with a high growth rate. Adding glucose into the culture media increases cell biomass and fatty acid production with high-quality biodiesel characters. The transcriptome analysis helped explore the possible regulation and biosynthesis of fatty acids under different treatments and exploited in the improvement of biodiesel production. This study provides insights for further improvement and various culture treatments for Euglena-based biodiesel and jet fuels.

The carotenoids, including lycopene, lutein, astaxanthin, and zeaxanthin belong to the isoprenoids, whose basic structure is made up of eight isoprene units, resulting in a C40 backbone, though some of them are only trace components in Euglena. They are essential to all photosynthetic organisms due to their superior photoprotective and antioxidant properties. Their dietary functions decrease the risk of breast, cervical, vaginal, and colorectal cancers and cardiovascular and eye diseases. Antioxidant functions of carotenoids are based on mechanisms such as quenching free radicals, mitigating damage from reactive oxidant species, and hindering lipid peroxidation. With the development of carotenoid studies, their distribution, functions, and composition have been identified in microalgae and higher plants. Although bleached or achlorophyllous mutants of Euglena were among the earliest carotenoid-related microalgae under investigation, current knowledge on the composition and biosynthesis of these compounds in Euglena is still elusive. This review aims to overview what is known about carotenoid metabolism in Euglena, focusing on the carotenoid distribution and structure, biosynthesis pathway, and accumulation in Euglena strains and mutants under environmental stresses and different culture conditions. Moreover, we also summarize the potential applications in therapy preventing carcinogenesis, cosmetic industries, food industries, and animal feed.

The genus Euglena contains more than 1000 species of single-celled flagellated microorganisms with both plant and animal characteristics. As a model organism, E. gracilis has been studied well to address fundamental questions in chloroplast development and photosynthesis, with implications in physiology, biochemistry and cell biology (Schwartzbach and Shigeoka, 2017). However, research in bioengineering and biotechnology in Euglena, especially using genome editing, remains insufficient (Figure 1a). Nomura et al. (2019) reported successful CRISPR/Cas9-mediated genome editing in Euglena using electroporation of Cas9 ribonucleoproteins (RNPs) targeting EgGSL2 and obtained mutant rates of approximately 70–90% based on morphology and amplicon-sequencing (Nomura et al. 2019). The protocol for implementing the E. gracilis CRISPR experiments was also described in further detail and published (Nomura et al., 2020). To date, these are the only research articles related to CRISPR genome editing of E. gracilis. Since their publication, no other successful case or report has been published by other groups, including ours. In this study, we repeated the CRISPR genome editing experiments on E. gracilis (Nomura et al., 2019, 2020) and assessed their efficiency by high-throughput sequencing. Further, sgRNA targeting the E. gracilis crtP1 gene for phytoene desaturase (PDS) was designed as a reference (Method S1). The EG300 experimental procedures and parameters strictly followed the protocol described by Nomura et al. (2020) (Figure 1c). Moreover, E. gracilis protoplast-like cells (Plas) treated with proteinase K were also used with extended voltage parameters from 150 to 600 V using two types of electroporation devices, NEPA21 and Bio-Rad Xcell (Figure 1c). Additional details of the process are described in Method S1. The result showed that the CRISPR-RNPs system worked efficiently to induce enzymatic cleavage of the partial EgGSL2 gene in vitro (Figure 1b). T7 Endonuclease I (T7E I) assays of three EG300 replications indicated their low editing efficiency from 'target1' on the partial EgGSL2 by electroporation (Figure 1d). Further, high-throughput sequencing of the 'target1' amplicons was performed using the Illumina NovaSeq 6000 platform. After data control, sequences were aligned and operational taxonomic units (OTUs) were clustered (FLASH v1.2.11 and USEARCH v10.0.240); three OTUs were found to be clustered in most high-quality sequences, which represented wild type, mutant1 and mutant2 (Figure 1e) (accessible data set in China National GeneBank (CNGB); accession no.: CNP0002995). The available sequences were obtained ranging from 29,541 to 353,307 in eight samples. However, only one sample, EG450, revealed mutant sequences with the mutant1 (0.81%) and mutant2 (0.17%), respectively (Figure 1f). The results showed that CRISPR genome editing of E. gracilis targeting the EgGSL2 by electroporation was successful but had very low efficiency. The efficiency of EG300 and other samples except EG450 was 0% (Figure 1f). With the crtP1 gene, eight mutant OTUs were obtained with an efficiency rate of approximately 3% in total (accessible data set in CNGB; accession no.: CNP0003142) (Figure S1). These results indicate that the delivery of RNPs into E. gracilis cells by electroporation is difficult. Microinjection is a physical method to deliver a small volume of substances into cells at the appropriate location, such as the cytoplasm or nucleus (Zhang and Yu, 2008). It is a visible and real-time traceable method that has been widely used in zebrafish embryos and mouse zygotes because of its high efficiency and low lethality (Gordon et al., 1980; Yuan and Sun, 2009). However, the application of microinjection to microalgae, although reported, is very rare (Nichols and Rikmenspoel, 1978), especially if the cell size is less than 100 μm. Here, we present a microinjection method to deliver exogenous materials into E. gracilis cells. For example, the dihydrochloride (DAPI) stain was successfully injected into E. gracilis cells using TransferMan 4r and FemtoJet 4i (Eppendorf, Germany), and blue fluorescence was observed in the nuclear region at the appropriate excitation/emission wavelengths (364 nm/454 nm) under a DMi3000B epifluorescence microscope (Leica, Germany) (Figure 1g). For genome editing E. gracilis, the sgRNA targeting the crtP1 gene for PDS and sgRNA of target1 on the EgGSL2 gene were used. In the crtP1 gene group, six cells among 100 injected cells survived, and each clone was sequenced to verify specific mutation. According to the DNA sequencing results, one clone showed precise genome editing on the crtP1 gene. The cytosine base was deleted at 295 in the partial DNA sequence of the crtP1 gene. The colour differences between the WT and crtP1-mutant were evident under the same cell density at 2.4 × 106 cells/ml (Figure 1h). The efficiency of genome editing was as high as 16.7% based on the surviving cells, whereas 1.0% was calculated based on the number of processed cells. The crtP1 mutant obtained using CRISPR technology coupled with microinjection was maintained even after repeated cultivation for a year. However, no mutant could be obtained in the EgGSL2 gene group, even though twelve clones survived. Microinjection is not a newly emerging technique but rather an advanced and sophisticated technique, primarily when performed on cells smaller than 20 μm (Chen et al., 2022). E. gracilis presents sphere-shaped cells smaller than 20 μm with a flexible pellicle, which hinders manipulation by microinjection. The size of the open tip of the injection pipette should be no more than 500 nm, and tips ranging from 50 to 100 nm are suitable to reduce cell mortality. The injection pressure varies between 100 and 2500 hPa depending on the specific circumstances. Several industrial microalgae cells are tiny with thick cell walls and movability. This may explain why the application of microinjection to microalgae compared with mammalian cells or zygotes is rare. This study represents the first successful report of a microinjection method for delivering CRISPR/Cas9 RNPs into microalgal cells. In summary, we repeated the experiment parameters from Nomura et al. (2020), and obtained suboptimal results, with a maximum editing efficiency of 0.98% by electroporation. Moreover, we conducted CRISPR/Cas9-mediated genome editing of E. gracilis and successfully knocked out the crtP1 gene by microinjection with relatively high efficiency (16.7%). The generated stable crtP1 mutant can be a good candidate for studying carotenoid metabolism in E. gracilis. To the best of our knowledge, this is the first application of microinjection to genome editing in microalgae. Overall, we demonstrate that microinjection-based single-celled manipulation has potential exogenous material delivery to facilitate the bioengineering and biotechnology of microalgae. We thank the Instrument Analysis Center of Shenzhen University. This work was partially supported by China's National Key R&D Programs (2018YFA0902500; 2020YFA0908703; and 2021YFA0910800) and the National Natural Science Foundation of China (41876188). The authors declare no conflicts of interest. ZC and JW conceived and designed the experiments. JZ, ZC, MD, RY and WF helped to perform experiments. AL and JW helped revise the manuscript. All authors read and approved the final manuscript. Figure S1 Mutant types of the sequence targeted on the crtP1 gene after electroporation, determined by high-throughput sequencing Method S1. Methods for Cas9 RNPs electroporation and single-celled microinjection on E. gracilis Movie S1 Short movie of single-celled microinjection on E. gracilis Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Naturally occurring compounds isolated from the microalga Euglena gracilis, such as polysaccharide paramylon, exhibit antimicrobial, anti-viral, antitumor, and anti-inflammatory activities. Whether live E. gracilis cells and its aqueous extract accelerate burn wound healing remains to be investigated. In this study, live E. gracilis cells and its aqueous extract were mixed with chitosan-hyaluronic acid hydrogel (CS/HA) to form cell + CS/HA and extract + CS/HA, which were then smeared onto the deeply burned skin of mice. The efficacy of these mixtures in accelerating wound healing was assessed through wound size reduction measurement, histological and immunofluorescence analyses, and serum pro-inflammatory cytokine level (INF-γ, IL-1β, and IL-6) determination. The live E. gracilis cells and its aqueous extract were found to facilitate wound healing by enhancing re-epithelization and reducing fibroplasia without stimulating excessive inflammatory response. In conclusion, live E. gracilis cells and its aqueous extract can be potentially used to treat cutaneous wounds.

Background Microalgae can contribute to more than 40% of global primary biomass production and are suitable candidates for various biotechnology applications such as food, feed products, drugs, fuels, and wastewater treatment. However, the primary limitation for large-scale algae production is the fact that algae requires large amounts of fresh water for cultivation. To address this issue, scientists around the world are working on ways to reuse the water to grow microalgae so that it can be grown in successive cycles without the need for fresh water. Results In this study, we present the results when we cultivate microalgae with cultivation water that is purified and reused. Specifically, we purify the cultivation water using an ultrafiltration membrane (UFM) treatment and investigate how this treatment affects: the biomass and biochemical components of the microalgae; characteristics of microalgae growth inhibitors; the mechanism whereby potential growth inhibitors are secreted (followed using metabolomics analysis); the effect of activated carbon (AC) treatment and advanced oxidation processes (AOPs) on the removal of growth inhibitors of Euglena gracilis . Firstly, the results show that E. gracilis can be only cultivated through two growth cycles with water that has been filtered and reused, and the growth of E. gracilis is significantly inhibited when the water is used a third time. Secondly, as the number of reused water cycles increases, the Cl − concentration gradually increases in the cultivation water. When the Cl − concentration accumulates to a level of fivefold higher than that of the control, growth of E. gracilis is inhibited as the osmolality tolerance range is exceeded. Interestingly, the osmolality of the reused water can be reduced by replacing NH 4 Cl with urea as the source of nitrogen in the cultivation water. Thirdly, E. gracilis secretes humic acid (HA)—which is produced by the metabolic pathways for valine, leucine, and isoleucine biosynthesis and by linoleic acid metabolism—into the cultivation water. Because HA contains large fluorescent functional groups, specifically extended π(pi)-systems containing C=C and C=O groups and aromatic rings, we were able to observe a positive correlation between HA concentration and the rate of inhibition of E. gracilis growth using fluorescence spectroscopy. Moreover, photosynthetic efficiency is adversely interfered by HA, thereby reductions in the synthetic efficiency of paramylon and lipid in E. gracilis . In this way, we are able to confirm that HA is the main growth inhibitor of E. gracilis . Finally, we verify that all the HA is removed or converted into nutrients efficiently by AC or UV/H 2 O 2 /O 3 treatments, respectively. As a result of these treatments, growth of E. gracilis is restored (AC treatment) and the amount of biomass is promoted (UV/H 2 O 2 /O 3 treatment). Conclusions These studies have important practical and theoretical significance for the cyclic cultivation of E. gracilis and for saving water resources. Our work may also provide a useful reference for other microalgae cultivation.