Euglena Cells Research Articles

Dielectrophoretic characterization and selection of non-spherical flagellate algae in parallel channels with right-angle bipolar electrodes.

Non-spherical flagellate algae play an increasingly significant role in handling problematic issues as versatile biological micro/nanorobots and resources of valuable bioproducts. However, the commensalism of flagellate algae with distinct structures and constituents causes considerable difficulties in their further biological utilization. Therefore, it is imperative to develop a novel method to realize high-efficiency selection of non-spherical flagellate algae in a non-invasive manner. Enthused by these, we proposed a novel method to accomplish the selection of flagellate algae based on the numerical and experimental investigation of dielectrophoretic characterizations of flagellate algae. Firstly, an arbitrary Lagrangian-Eulerian method was utilized to study the electro-orientation and dielectrophoretic assembly process of spindle-shaped and ellipsoid-shaped cells in a uniform electric field. Secondly, we studied the equilibrium state of spherical, ellipsoid-shaped, and spindle-shaped cells under positive DEP forces actuated by right-angle bipolar electrodes. Thirdly, we investigated the dielectrophoretic assembly and escape processes of the non-spherical flagellate algae in continuous flow to explore their influences on the selection. Fourthly, freshwater flagellate algae (Euglena, H. pluvialis, and C. reinhardtii) and marine ones (Euglena, Dunaliella salina, and Platymonas) were separated to validate the feasibility and adaptability of this method. Finally, this approach was engineered in the selection of Euglena cells with high viability and motility. This method presents immense prospects in the selection of pure non-spherical flagellate algae with high motility for chronic wound healing, bio-micromotor construction, and decontamination with advantages of no sheath, strong reliability, and shape-insensitivity.

Tocopherol Content of Euglena sp. Isolated from Yogyakarta under Glucose and Ethanol Mixture Treatment

Euglena sp. is a microalgae with significant potential for utilization as a high-value product because of the presence of protein, lipid, paramylon, and other compounds. Even though these microalgae may be found in freshwater, research on enhancing Euglena sp. cultivation is still limited in Indonesia. Tocopherols are antioxidants that can effectively protect against diseases caused by oxidative stress. The isomer of tocopherol with the highest biological activity is α-tocopherol. Euglena sp. cells had the highest levels of α-tocopherol compared to other microorganisms. Scientists are continuously trying to determine how to obtain a high α-tocopherol concentration and a significant Euglena cell biomass. Photosynthetic organisms culture has been found to boost α-tocopherol content in Euglena sp., although heterotrophic culture can potentially increase biomass. This study used photoheterotrophic culture with a mixture of glucose and ethanol to increase the α-tocopherol and biomass concentration inside the culture of the local strain of Euglena sp. The addition of treatments in a glucose and ethanol combination with levels of 3:2; 2.5: 2.5; 2: 2; and 0:0 (control) g L-1 was used in this study to assess the impact of Euglena sp. culture on growth, biomass, and α-tocopherol concentration. According to the findings of this study, the 3:2 treatment produced the most significant specific growth rate and biomass, including 0.992 (OD680/OD680/day) and 8.480 (g L-1). In contrast, the 2.5:2.5 treatment produced the highest α-tocopherol content, specifically 7.09±0.096 mg L-1.

Fast and efficient molecule delivery into Euglena gracilis mediated by cell-penetrating peptide or dimethyl sulfoxide.

This study describes the development of two methods for delivering exogenous materials into Euglena gracilis, a unicellular flagellate organism. We report that the use of Pep-1, a short cell-penetrating peptide (CPP), or dimethyl sulfoxide (DMSO) can mediate fast and efficient intracellular delivery of exogenous materials into E.gracilis, achieving cellular entry efficiency as high as 70-80%. However, compared with human cells, the penetration of this algal cell with CPP requires a much higher concentration of purified proteins. In addition, upon convenient treatment with DMSO, E.gracilis cells can efficiently adsorb exogenous proteins and DNA with 10% DMSO as the optimal concentration for Euglena cells. Our results provide more options for the E.gracilis transformation 'toolkit box' and will facilitate future molecular manipulations of this microalgal organism.

Biochemical composition of <i>Euglena gracilis</i> cells during mixotrophic growth in the presence of various organic substrates

The general biochemical composition and profile of low molecular weight metabolites of euglena (Euglena gracilis Klebs.) cells grown in a mixotrophic culture supplemented with 0.5% ethanol, glucose, butanol, glycine, or glycerol was studied. Ethanol and glucose significantly stimulated the growth of the Euglena culture and the accumulation of storage compounds (paramylon and wax esters) in the cells. Butanol also contributed to the division of euglena cells and increased biosynthesis of protein, mono- and disaccharides, carotenoids, and tocopherols. Glycine and glycerol were metabolized more slowly than other substrates, and they accumulated in E. gracilis cells along with their direct derivatives. Glycine did not stimulate the growth of the culture, but it caused the accumulation of paramylon, organic acids of the Krebs cycle, and nitrogen-containing metabolites (chlorophyll, free amino acids, and nitrogenous bases) in the cells. Apparently, glycine is equally effectively used by Euglena as an additional source of both carbon and nitrogen under the conditions of mixotrophy. Assimilation of glycerol was accompanied by the accumulation of wax esters and the amino acids proline and ornithine in Euglena cells. In general, the results obtained demonstrate the ability of Euglena to significantly rearrange its metabolism during the assimilation of organic substrates of various chemical nature. Our data can be used in the context of E. gracilis practical applications.

Heterotrophic Cultivation of Euglena gracilis in Stirred Tank Bioreactor: A Promising Bioprocess for Sustainable Paramylon Production.

Paramylon is a valuable intracellular product of the microalgae Euglena gracilis, and it can accumulate in Euglena cells according to the cultivation conditions. For the sustainable production of paramylon and appropriate cell growth, different bioreactor processes and industrial byproducts can be considered as substrates. In this study, a complex medium with corn steep solid (CSS) was used, and various bioreactor processes (batch, fed batch, semicontinuous and continuous) were performed in order to maximize paramylon production in the microalgae Euglena gracilis. Compared to the batch, fed batch and repeated batch bioprocesses, during the continuous bioprocess in a stirred tank bioreactor (STR) with a complex medium containing 20 g/L of glucose and 25 g/L of CSS, E. gracilis accumulated a competitive paramylon content (67.0%), and the highest paramylon productivity of 0.189 g/Lh was observed. This demonstrated that the application of a continuous bioprocess, with corn steep solid as an industrial byproduct, can be a successful strategy for efficient and economical paramylon production.

Fatty Acid Accumulations and Transcriptome Analyses Under Different Treatments in a Model Microalga Euglena gracilis

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.

High-light modification of excitation-energy-relaxation processes in the green flagellate Euglena gracilis.

Photosynthetic organisms finely tune their photosynthetic machinery including pigment compositions and antenna systems to adapt to various light environments. However, it is poorly understood how the photosynthetic machinery in the green flagellate Euglena gracilis is modified under high-light conditions. In this study, we examined high-light modification of excitation-energy-relaxation processes in Euglena cells. Oxygen-evolving activity in the cells incubated at 300µmol photons m-2s-1 (HL cells) cannot be detected, reflecting severe photodamage to photosystem II (PSII) in vivo. Pigment compositions in the HL cells showed relative increases in 9'-cis-neoxanthin, diadinoxanthin, and chlorophyll b compared with the cells incubated at 30µmol photons m-2s-1 (LL cells). Absolute fluorescence spectra at 77K exhibit smaller intensities of the PSII and photosystem I (PSI) fluorescence in the HL cells than in the LL cells. Absolute fluorescence decay-associated spectra at 77K of the HL cells indicate suppression of excitation-energy transfer from light-harvesting complexes (LHCs) to both PSI and PSII with the time constant of 40ps. Rapid energy quenching in LHCs and PSII in the HL cells is distinctly observed by averaged Chl-fluorescence lifetimes. These findings suggest that Euglena modifies excitation-energy-relaxation processes in addition to pigment compositions to deal with excess energy. These results provide insights into the photoprotection strategies of this alga under high-light conditions.

Nanofibers made from acetylparamylons by a soaking method

A novel method was developed for preparing nanofibers from acetylparamylons, which are acetylated derivatives of a β-1,3-glucan (paramylon) produced by Euglena cells. This method was primarily based on the self-assembling tendency of β-1,3-glucans. First, a solid consisting of aggregated nanofibers (~100 nm in diameter) was precipitated by pouring a homogeneous reaction mixture containing acetylparamylons into water. Second, the solid precipitate was soaked in a halogenated solvent. Scanning probe microscopic observations confirmed that soaking in chloroform liberated nanofibers (105.0 ± 8.9 nm in diameter and over ~7.0 μm in length) from the solid precipitates of acetylparamylon with an acetylation degree of ~1.4.

How Euglena gracilis swims: Flow field reconstruction and analysis.

Euglena gracilis is a unicellular organism that swims by beating a single anterior flagellum. We study the nonplanar waveforms spanned by the flagellum during a swimming stroke and the three-dimensional flows that they generate in the surrounding fluid. Starting from a small set of time-indexed images obtained by optical microscopy on a swimming Euglena cell, we construct a numerical interpolation of the stroke. We define an optimal interpolation (which we call synthetic stroke) by minimizing the discrepancy between experimentally measured velocities (of the swimmer) and those computed by solving numerically the equations of motion of the swimmer driven by the trial interpolated stroke. The good match we obtain between experimentally measured and numerically computed trajectories provides a first validation of our synthetic stroke. We further validate the procedure by studying the flow velocities induced in the surrounding fluid. We compare the experimentally measured flow fields with the corresponding quantities computed by solving numerically the Stokes equations for the fluid flow, in which the forcing is provided by the synthetic stroke, and find good matching. Finally, we use the synthetic stroke to derive a coarse-grained model of the flow field resolved in terms of a few dominant singularities. The far field is well approximated by a time-varying Stresslet, and we show that the average behavior of Euglena during one stroke is that of an off-axis puller. The reconstruction of the flow field closer to the swimmer body requires a more complex system of singularities. A system of two Stokeslets and one Rotlet, that can be loosely associated with the force exerted by the flagellum, the drag of the body, and a torque to guarantee rotational equilibrium, provides a good approximation.

The dialogue between protozoa and bacteria in a microfluidic device.

In nature, protozoa play a major role in controlling bacterial populations. This paper proposes a microfluidic device for the study of protozoa behaviors change due to their chemotactic response in the presence of bacterial cells. A three-channel microfluidic device was designed using a nitrocellulose membrane into which channels were cut using a laser cutter. The membrane was sandwiched between two glass slides; a Euglena suspension was then allowed to flow through the central channel. The two side channels were filled with either, 0.1% peptone as a negative control, or a Listeria suspension respectively. The membrane design prevented direct interaction but allowed Euglena cells to detect Listeria cells as secretions diffused through the nitrocellulose membrane. A significant number of Euglena cells migrated toward the chambers near the bacterial cells, indicating a positive chemotactic response of Euglena toward chemical cues released from Listeria cells. Filtrates collected from Listeria suspension with a series of molecular weight cutoffs (3k, 10k and 100k) were examined in Euglena chemotaxis tests. Euglena cells were attracted to all filtrates collected from the membrane filtration with different molecular weight cutoffs, suggesting small molecules from Listeria might be the chemical cues to attract protozoa. Headspace volatile organic compounds (VOC) released from Listeria were collected, spiked to 0.1% peptone and tested as the chemotactic effectors. It was discovered that the Euglena cells responded quickly to Listeria VOCs including decanal, 3,5- dimethylbenzaldehyde, ethyl acetate, indicating bacterial VOCs were used by Euglena to track the location of bacteria.

Interactive programming paradigm for real-time experimentation with remote living matter

Recent advancements in life-science instrumentation and automation enable entirely new modes of human interaction with microbiological processes and corresponding applications for science and education through biology cloud laboratories. A critical barrier for remote and on-site life-science experimentation (for both experts and nonexperts alike) is the absence of suitable abstractions and interfaces for programming living matter. To this end we conceptualize a programming paradigm that provides stimulus and sensor control functions for real-time manipulation of physical biological matter. Additionally, a simulation mode facilitates higher user throughput, program debugging, and biophysical modeling. To evaluate this paradigm, we implemented a JavaScript-based web toolkit, "Bioty," that supports real-time interaction with swarms of phototactic Euglena cells hosted on a cloud laboratory. Studies with remote and on-site users demonstrate that individuals with little to no biology knowledge and intermediate programming knowledge were able to successfully create and use scientific applications and games. This work informs the design of programming environments for controlling living matter in general, for living material microfabrication and swarm robotics applications, and for lowering the access barriers to the life sciences for professional and citizen scientists, learners, and the lay public.

Swimming Euglena respond to confinement with a behavioral change enabling effective crawling.

Some euglenids, a family of aquatic unicellular organisms, can develop highly concerted, large amplitude peristaltic body deformations. This remarkable behavior has been known for centuries. Yet, its function remains controversial, and is even viewed as a functionless ancestral vestige. Here, by examining swimming Euglena gracilis in environments of controlled crowding and geometry, we show that this behavior is triggered by confinement. Under these conditions, it allows cells to switch from unviable flagellar swimming to a new and highly robust mode of fast crawling, which can deal with extreme geometric confinement and turn both frictional and hydraulic resistance into propulsive forces. To understand how a single cell can control such an adaptable and robust mode of locomotion, we developed a computational model of the motile apparatus of Euglena cells consisting of an active striated cell envelope. Our modeling shows that gait adaptability does not require specific mechanosensitive feedback but instead can be explained by the mechanical self-regulation of an elastic and extended motor system. Our study thus identifies a locomotory function and the operating principles of the adaptable peristaltic body deformation of Euglena cells.

A major isoform of mitochondrial trans-2-enoyl-CoA reductase is dispensable for wax ester production in Euglena gracilis under anaerobic conditions.

Under anaerobic conditions, Euglena gracilis produces a large amount of wax ester through mitochondrial fatty acid synthesis from storage polysaccharides termed paramylon, to generate ATP. Trans-2-enoyl-CoA reductases (TERs) in mitochondria have been considered to play a key role in this process, because the enzymes catalyze the reduction of short chain length CoA-substrates (such as crotonyl-CoA). A TER enzyme (EgTER1) has been previously identified and enzymologically characterized; however, its physiological significance remained to be evaluated by genetic analysis. We herein generated EgTER1-knockdown Euglena cells, in which total crotonyl-CoA reductase activity was decreased to 10% of control value. Notably, the knockdown cells showed a severe bleaching phenotype with deficiencies in chlorophylls and glycolipids, but grew normally under heterotrophic conditions (with glucose supplementation). Moreover, the knockdown cells accumulated much greater quantities of wax ester than control cells before and after transfer to anaerobic conditions, which was accompanied by a large metabolomic change. Furthermore, we failed to find any contribution of other potential TER genes in wax ester production. Our findings propose a novel role of EgTER1 in the greening process and demonstrate that this enzyme is dispensable for wax ester production under anaerobic conditions.

Bio-inspired neurocomputing with 256 noise oscillators simulating photo response of Euglena cells

We developed a bio-inspired neurocomputing approach based on our earlier biological neurocomputer, which leverages the survival strategies of living micro-algae cells (Euglena gracilis) to soft computing. Instead of using the real living cells, the bio-inspired neurocomputing in this study (namely, Euglena-inspired neurocomputing) mimics the photophobic responses of the cells using photo-responsive (PR) noise oscillators. The PR noise oscillators play the role of neurons during computation and their output signals are autonomously changed both by noise generation and firing of the neuron. The Euglena-inspired neurocomputing achieved a high performance in searching for multiple solutions continuously and autonomously for a combinatorial optimization problem, 16-city TSP as instance. We analyzed the temporal evolution of the computation and its dependence on the parameter set of the PR noise oscillators and identified the source of the high performance as the trade-off between noise amplitude and the reduction ratio of the oscillators. We next introduced two specific survival strategies observed in the real Euglena cells to PR noise oscillators, and elucidated their positive effects on the performance. The Euglena-inspired neurocomputing examined in this study can be used to address dynamically changing optimization problems, since the computation tracks changes in the imposed conditions by autonomous and non-converged searching for the solutions.

Controllable alignment of elongated microorganisms in 3D microspace using electrofluidic devices manufactured by hybrid femtosecond laser microfabrication

This paper presents a simple technique to fabricate new electrofluidic devices for the three-dimensional (3D) manipulation of microorganisms by hybrid subtractive and additive femtosecond (fs) laser microfabrication (fs laser-assisted wet etching of glass followed by water-assisted fs laser modification combined with electroless metal plating). The technique enables the formation of patterned metal electrodes in arbitrary regions in closed glass microfluidic channels, which can spatially and temporally control the direction of electric fields in 3D microfluidic environments. The fabricated electrofluidic devices were applied to nanoaquariums to demonstrate the 3D electro-orientation of Euglena gracilis (an elongated unicellular microorganism) in microfluidics with high controllability and reliability. In particular, swimming Euglena cells can be oriented along the z-direction (perpendicular to the device surface) using electrodes with square outlines formed at the top and bottom of the channel, which is quite useful for observing the motions of cells parallel to their swimming directions. Specifically, z-directional electric field control ensured efficient observation of manipulated cells on the front side (45 cells were captured in a minute in an imaging area of ~160×120 μm), resulting in a reduction of the average time required to capture the images of five Euglena cells swimming continuously along the z-direction by a factor of ~43 compared with the case of no electric field. In addition, the combination of the electrofluidic devices and dynamic imaging enabled observation of the flagella of Euglena cells, revealing that the swimming direction of each Euglena cell under the electric field application was determined by the initial body angle.

Wax Ester Fermentation and Its Application for Biofuel Production.

In Euglena cells under anaerobic conditions, paramylon, the storage polysaccharide, is promptly degraded and converted to wax esters. The wax esters synthesized are composed of saturated fatty acids and alcohols with chain lengths of 10-18, and the major constituents are myristic acid and myristyl alcohol. Since the anaerobic cells gain ATP through the conversion of paramylon to wax esters, the phenomenon is named "wax ester fermentation". The wax ester fermentation is quite unique in that the end products, i.e. wax esters, have relatively high molecular weights, are insoluble in water, and accumulate in the cells, in contrast to the common fermentation end products such as lactic acid and ethanol.A unique metabolic pathway involved in the wax ester fermentation is the mitochondrial fatty acid synthetic system. In this system, fatty acid are synthesized by the reversal of β-oxidation with an exception that trans-2-enoyl-CoA reductase functions instead of acyl-CoA dehydrogenase. Therefore, acetyl-CoA is directly used as a C2 donor in this fatty acid synthesis, and the conversion of acetyl-CoA to malonyl-CoA, which requires ATP, is not necessary. Consequently, the mitochondrial fatty acid synthetic system makes possible the net gain of ATP through the synthesis of wax esters from paramylon. In addition, acetyl-CoA is provided in the anaerobic cells from pyruvate by the action of a unique enzyme, oxygen sensitive pyruvate:NADP+ oxidoreductase, instead of the common pyruvate dehydrogenase multienzyme complex.Wax esters produced by anaerobic Euglena are promising biofuels because myristic acid (C14:0) in contrast to other algal produced fatty acids, such as palmitic acid (C16:0) and stearic acid (C18:0), has a low freezing point making it suitable as a drop-in jet fuel. To improve wax ester production, the molecular mechanisms by which wax ester fermentation is regulated in response to aerobic and anaerobic conditions have been gradually elucidated by identifying individual genes related to the wax ester fermentation metabolic pathway and by comprehensive gene/protein expression analysis. In addition, expression of the cyanobacterial Calvin cycle fructose-1,6-bisphosphatase/sedohepturose-1,7-bisphosphatase, in Euglena provided photosynthesis resulting in increased paramylon accumulation enhancing wax ester production. This chapter will discuss the biochemistry of the wax ester fermentation, recent advances in our understanding of the regulation of the wax ester fermentation and genetic engineering approaches to increase production of wax esters for biofuels.

Biochemistry and Physiology of Heavy Metal Resistance and Accumulation in Euglena.

Free-living microorganisms may become suitable models for removal of heavy metals from polluted water bodies, sediments, and soils by using and enhancing their metal accumulating abilities. The available research data indicate that protists of the genus Euglena are a highly promising group of microorganisms to be used in bio-remediation of heavy metal-polluted aerobic and anaerobic acidic aquatic environments. This chapter analyzes the variety of biochemical mechanisms evolved in E. gracilis to resist, accumulate and remove heavy metals from the environment, being the most relevant those involving (1) adsorption to the external cell pellicle; (2) intracellular binding by glutathione and glutathione polymers, and their further compartmentalization as heavy metal-complexes into chloroplasts and mitochondria; (3) polyphosphate biosynthesis; and (4) secretion of organic acids. The available data at the transcriptional, kinetic and metabolic levels on these metabolic/cellular processes are herein reviewed and analyzed to provide mechanistic basis for developing genetically engineered Euglena cells that may have a greater removal and accumulating capacity for bioremediation and recycling of heavy metals.

Large-Scale Cultivation of Euglena.

From the middle of the twentieth century, microalgae have been exploited as a candidate biomass source of food and other products. One such candidate source is the fast-proliferating microalga Euglena gracilis. The commercial cultivation of E. gracilis began in 2007, after the success of its outdoor mass cultivation and improvement of the harvesting and drying methods suitable for Euglena cells. The commercialization of Euglena production is based on the strategy of "5Fs of Biomass," which refers to the development and production of commercial products including food, fiber, feed, fertilizer, and fuel from biomass." Although room for improvement remains in the productivity of Euglena biomass, the product with the highest value-food-is already profitable. By enhancing the productivity of its biomass, other Euglena products, including fiber, feed, fertilizer, and fuel, can be commercialized. Breeding and recombinant DNA technology studies are being conducted to accomplish more extensive application of Euglena. In addition, the search for a better place for outdoor mass cultivation of Euglena is ongoing.

Equilibrium and kinetic studies of Cu(II) and Ni(II) sorption on living Euglena gracilis

Biosorption and bioaccumulation involve the decrease in concentration or removal of metals from aqueous solution through the sequestering of ions by active or metabolically inert biomass. In this study, the potential use of Euglena gracilis, a free-floating, flagellated unicellular species of protist, to remove Cu and Ni ions at environmentally relevant levels from aqueous solutions was investigated. Adsorption isotherms were used in a batch system to describe the kinetic and equilibrium characteristics of metal removal. The effects of pH and initial concentration of metal ions on the adsorption of Cu and Ni ions were examined. Results indicate that the sorption reaction occurred quickly (<30 min) in both Cu and Ni monometallic systems, and adsorption followed a pseudo-second-order kinetics model for both metals. Comparable metal sorption was found at pH 5 and 7.5, suggesting that metal sorption onto live Euglena cells is not pH dependent. No significant differences in metal sorption were found at pH 5 and 7.5. Removal efficiencies for Cu decreased with higher initial concentrations (3–30 μg L−1) and conformed to the Freundlich sorption model. Ni removal was found to increase with greater initial concentration values (5–110 mg L−1) and conformed to the Freundlich isotherm. The removal efficiency for Cu and Ni was 58 ± 3 and 44 ± 6%, respectively. In bimetallic systems, sorption of both metals were equivalent, suggesting that E. gracilis may be appropriate for mining effluent metal removal due to their ability to simultaneously tolerate, sorb, and accumulate multiple metals in solution in a range of environmental conditions.

배양방법에 따른 Euglena gracilis의 성장 및 지방산 조성

The quality and quantity of live food sources strongly influence the success of fish production in farming operations. Thus, critical studies of live forage species are a crucial element for progress in fish aquaculture. The fat content of food is an especially important determinant of growth in marine fish. Omega-3 highly unsaturated fatty acids (HUFA) are essential components of diet that determine the nutritional value of larval fish. Euglena is a protist that has potential as a forage species. These single-celled organisms have plant and animal characteristics they are motile, elliptical in shape and 15&#x2013;500 &#x3BC;m in diameter. Their nutritional content is excellent, but most studies have focused on cells raised in autotrophic culture. We therefore examined differences in the lipid and fatty acid contents, and the growth of Euglena cells grown under autotrophic, heterotrophic, and mixotrophic conditions. Biomass production reached 15.03 g/L, 12.28 g/L, and 3.66 g/L under mixotrophy, heterotrophy, and autotrophy, respectively. The proportional n-3 HUFA content differed among culture methods: 10.04%, 5.80% and 10.01% in mixotrophic, heterotrophic and autotrophic cultures, respectively. Mixotrophy was to be the best form of cultivation for improving the growth and nutritional content of Euglena.