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Euglena gracilis produces ATP in the anaerobic mitochondria with concomitant wax ester formation, and NADH is essential for ATP formation and fatty acid synthesis in the mitochondria. This study demonstrated that mitochondrial cofactor conversion by nicotinamide nucleotide transhydrogenase (NNT), converting NADPH/NAD+ to NADP+ /NADH, is indispensable for sustaining anaerobic metabolism. Silencing of NNT genes significantly decreased wax ester production and cellular viability during anaerobiosis but had no such marked effects under aerobic conditions. An analogous phenotype was observed in the silencing of the gene encoding a mitochondrial NADP+ -dependent malic enzyme. These results suggest that the reducing equivalents produced in glycolysis are shuttled to the mitochondria as malate, where cytosolic NAD+ regeneration is coupled with mitochondrial NADPH generation.

As an excellent cell factory rich in polyunsaturated fatty acids, β-1,3-glucan, amino acids, and vitamins, Euglena has gained great attention with high-value products in recent years. In order to determine the optimal cultivation method of Euglena pisciformis AEW501, the effects of phototrophic, mixotrophic, and heterotrophic mode on its growth were investigated, and humic acid (HA) as a growth stimulant was added into the mixotrophic culture system to verify the hypothesis that HA addition could accelerate the growth and lipid accumulation of E. pisciformis AEW501. The results indicated that biomass of mixotrophic mode (330 ± 0.05 mg L−1) was 4.80 times and 7.65 times higher than that of phototrophic and heterotrophic mode, respectively, and mixotrophy was proved as the optimal trophic mode. Moreover. The maximum biomass productivity (34.63 mg L−1 day−1) and the highest lipid content (59.51%) were achieved in 50 mg L−1 HA treatment though there was no significant difference between 10 mg L−1 and 50 mg L−1 treatments. While photosynthetic efficiency of photosystem II, pigments content, cell size, and yield of eicosapentaenoic (EPA) and docosahexaenoic (DHA) in 10 mg L−1 HA treatment were the highest. It could be concluded that HA showed good promotion effects on growth and high-value products accumulation of E. pisciformis AEW501. This research will be helpful for the large-scale cultivation of E. pisciformis AEW501.

The identification and pharmacological validation of plant-based lead compounds for the cure of different diseases including cancer have always been globally strived. In addition to possessing numerous medicinal properties, many of the phytochemicals display antioxidant potential activities. Reactive oxygen species (ROS) causeoxidative stress leading to several severe diseases such as cancer. The antioxidants are substances that fight against ROS to protect the cells from their damaging effects. In the present study, the effects of methanol extract of Euglena tuba(ETME) have been evaluated for its antioxidant and antitumor potential against Dalton’s lymphoma (DL) introduced in BALB/cmice. After 24 h of intraperitoneal inoculation of DL cells in mice, ETME (300 mg kg−1 body weight) was administered intraperitoneally upto18 alternative days. On the 18th day, the mice were sacrificed; the blood and tissues (liver and brain) were collected to determine the tumor growth parameters including morphological, behavioural, haematological profile, and antioxidant indices. The results indicated that ETME exhibited significant antioxidative and antitumor properties when compared with the data from DL bearing mice. The results from the present study indicated that ETME contained remarkable antitumor efficacy, which was mediated through amelioration of oxidative stress. The data suggested that ETME could be used as a potential natural anticancer agent.

Single-cell metabolite analysis plays an important role in biological study. While mass spectrometry is a powerful tool for identification and quantitation of metabolites, the low absolute analyte amounts in single cell and difficulty in sampling represent significant challenges in single cell analysis. In this study, we developed an effective method with a simple sampling procedure for analyzing single cells. A single cell was driven to a capillary tip through electro-migration, followed by releasing the cell contents through electroporation, into a sealed small volume (∼1.5 pL) to prevent dilution. Subsequent mass spectrometry analysis was performed directly with nanoelectrospray ionization. This method was applied for analyzing a variety of cells and monitoring the metabolic changes in response to perturbed cell culturing conditions. This method opens a new avenue for easy and rapid analysis of single cells with high sensitivity.

Asymmetrical flow field-flow fractionation (AF4) and high-resolution Orbitrap mass spectrometry (HRMS) were used to separate and characterize cellular fractions of the dark- and light-grown Euglena gracilis cellular material. Biological replicates analyzed by HRMS shared 21–73% of commonly detected m/z values. Greater variability in shared features was found in light-grown cellular fractions (p

It is difficult to match annual vaccines against the exact influenza strain that is spreading in any given flu season. Owing to the emergence of drug-resistant viral strains, new approaches for treating influenza are needed. Euglena gracilis (hereinafter Euglena), microalga, used as functional foods and supplements, have been shown to alleviate symptoms of influenza virus infection in mice. However, the mechanism underlying the inhibitory action of microalgae against the influenza virus is unknown. Here, we aimed to study the antiviral activity of Euglena extract against the influenza virus and the underlying action mechanism using Madin–Darby canine kidney (MDCK) cells. Euglena extract strongly inhibited infection by all influenza virus strains examined, including those resistant to the anti-influenza drugs oseltamivir and amantadine. A time-of-addition assay revealed that Euglena extract did not affect the cycle of virus replication, and cell pretreatment or prolonged treatment of infected cells reduced the virus titer. Thus, Euglena extract may activate the host cell defense mechanisms, rather than directly acting on the influenza virus. Moreover, various minerals, mainly zinc, in Euglena extract were found to be involved in the antiviral activity of the extract. In conclusion, Euglena extract could be a potent agent for preventing and treating influenza.

Еffects of β-1,3-glucan from euglena (Euglena gracilis) were studied in vivo by evaluating pH changes outside and inside the cell in the tissues of the 7-day leaves of barley seedlings with the use of pH-sensitive probes of FITC-dextran and pyranine. It was found that the incubation of barley leaves separated from the roots in the solution of β-1,3-glucan (0.01 %) for 40 min did not cause acidification of cytoplasm as a typical nonspecific plant response to wound stress. The inoculation of intact seedlings with Bipolaris sorokiniana (Sacc.) Shoem. spores resulted in alkalization of apoplast by 1.7 pH units, and pretreatment of seedlings with β-1,3-glucan a day before the fungal infection promoted its acidification (1.04 pH units) compared to the infected variant, indicating an increased performance of ATPase, which pumped protons from cytoplasm into apoplast. The conducted studies contribute to the selection of optimal concentrations of β-1,3-glucan for immunomodulatory mixtures.

Enzymatic degradation of the β-1,3-glucan paramylon could enable the production of bioactive compounds for healthcare and renewable substrates for biofuels. However, few enzymes have been found to degrade paramylon efficiently and their enzymatic mechanisms remain poorly understood. Thus, the aim of this work was to find paramylon-degrading enzymes and ways to facilitate their identification. Towards this end, a Euglena gracilis-derived cDNA expression library was generated and introduced into Escherichia coli. A flow cytometry-based screening assay was developed to identify E. gracilis enzymes that could hydrolyse the fluorogenic substrate fluorescein di-β-d-glucopyranoside in combination with time-saving auto-induction medium. In parallel, four amino acid sequences of potential E. gracilis β-1,3-glucanases were identified from proteomic data. The open reading frame encoding one of these candidate sequences (light_m.20624) was heterologously expressed in E. coli. Finally, a Congo Red dye plate assay was developed for the screening of enzyme preparations potentially able to degrade paramylon. This assay was validated with enzymes assumed to have paramylon-degrading activity and then used to identify four commercial preparations with previously unknown paramylon degradation ability.

Paramylon is a β-1,3-glucan photosynthesized by Euglena. In this study, the four paramylon ester derivatives, paramylon propionate (PaPr), paramylon butyrate (PaBu), paramylon valerate (PaVa), paramylon hexanoate (PaHe), were prepared and their crystal structures were determined by wide-angle X-ray diffraction. All of the reflections are indexed to a hexagonal crystal system and all of the molecular chains have five-fold screw symmetry along the molecular axes, despite of the different side-chain lengths of the ester groups. The crystal lattice parameters increase with increasing of side-chain length of the ester groups. The crystalline elastic modulus of PaPr, PaBu, and PaVa parallel to the fiber axis (El) were determined by time-resolved X-ray diffraction at SPring-8, which is a large synchrotron radiation facility. The crystalline elastic modulus of PaPr, PaBu, and PaVa are 2.5, 1.9, and 1.0 GPa, respectively. The decreasing El values are mainly caused by increasing of cross-sectional area of the molecular chains, which is affected by the side-chain length. The El values of the paramylon esters are one-tenth those of previously reported cellulose esters. This seems to be because of the different helical structure of the backbone chain.

This short communication investigated biodiesel production from Euglena Sanguineamicroalgaeand custard appleusing nano CaO as a heterogeneous catalyst. Different solvents were used to extract the oil at a fixed speed, time, and temperature for the samples to estimate the optimized oil yield%. The catalyst was synthesized by sol gel method in nano-scale. It was further characterized by FTIR spectroscopy, SEM, and XRD. The algal oil was pre-treated and trans-esterified with a catalyst to produce alkyl esters. The optimized process variables were determined using response surface methodology by varying parameters such as methanol to oil ratio and catalyst weight% for algal bio-oil and MeOH to oil ratio, time, and catalyst weight% for seed oil. The GC-MS was done to characterize the presence of biodiesel. Kinetic studies were done for the optimized condition for the algal oil and seed oil and it follows the pseudo-first order reaction.

Paramylon from Euglena gracilis is an insoluble crystalline β-1,3-glucan which have pharmaceutical and nutraceuticals applications. The present study aims to check the prebiotic potential of paramylon derived from heterotrophically grown E. gracilis in bioreactor. The Paramylon was extracted using sodium dodecyl sulfate from E. gracilis biomass. The Fourier Transform-Infra Red spectroscopy and scanning electron microscopy demonstrated the isolated paramylon to be equivalent to that of analytical standard. The prebiotic activity of E. gracilis cell extract and isolated paramylon was studied. E. gracilis cell extract as well as isolated paramylon led to cell number enhancement of Lacfid (Lactobacillus) strain exhibiting the prebiotic activities., Euglena, Paramylon, Prebiotic, Soluble.

Euglena gracilis is widely utilized as food or supplement to promote human and animal health, as it contains rich nutrients. In this study, we administered spray-dried powder of E. gracilis and paramylon, β-glucan stored in E. gracilis cells, to A4gnt knockout (KO) mice. A4gnt KO mice are a mutant mouse model that spontaneously develops gastric cancer through hyperplasia-dysplasia-adenocarcinoma sequence in the antrum of the stomach, and we observed the effects of E. gracilis and paramylon on the early involvements of A4gnt KO mice. Male and female 10-week-old A4gnt KO mice and their age-matched wildtype C57BL/6J mice were orally administered with 50 mg of E. gracilis or paramylon suspended in saline or saline as a control. After 3-week administration, animals were euthanatized and the stomach was examined histopathologically and immunohistochemically. Gene expression patterns of the stomach, which have been reported to be altered with A4gnt KO, and IgA concentration in small intestine were also analyzed with real-time PCR and ELISA, respectively. Administration of Euglena significantly reduced the number of stimulated CD3-positive T-lymphocytes in pyloric mucosa of A4gnt KO mice and tend to reduce polymorphonuclear leukocytes infiltration. Euglena administration further downregulated the expression of Il11 and Cxcl1 of A4gnt KO mice. Euglena administration also affected IgA concentration in small intestinal contents of A4gnt KO mice. Paramylon administration reduced the number of CD3-positive lymphocytes in pyloric mucosa of A4gnt KO mice, and downregulated the expressions of Il11 and Ccl2 of A4gnt KO mice. Although we found no significant effects on gross and microscopic signs of gastric dysplasia and cell proliferation, the present study suggests that the administration of Euglena and paramylon may ameliorate the early involvements of A4gnt mice through the effects on inflammatory reactions in the gastric mucosa. The cancer-preventing effects should be studied with long-term experiments until actual gastric cancer formation.

Euglena gracilis, a green microalga known as a potential candidate for jet fuel producers and new functional food resources, is highly tolerant to antibiotics, heavy metals, and other environmental stresses. Its cells contain many high-value products, including vitamins, amino acids, pigments, unsaturated fatty acids, and carbohydrate paramylon as metabolites, which change contents in response to various extracellular environments. However, mechanism insights into the cellular metabolic response of Euglena to different toxic chemicals and adverse environmental stresses were very limited. We extensively investigated the changes of cell biomass, pigments, lipids, and paramylon of E. gracilis under several environmental stresses, such as heavy metal CdCl2, antibiotics paromomycin, and nutrient deprivation. In addition, global metabolomics by Ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) was applied to study other metabolites and potential regulatory mechanisms behind the differential accumulation of major high-valued metabolites. This study collects a comprehensive update on the biology of E. gracilis for various metabolic responses to stress conditions, and it will be of great value for Euglena cultivation and high-value [154mm][10mm]Q7metabolite production.

9 pags., 7 figs., 3 tabs., A variety of natural biofilms were collected from an extremely acidic environment at Rı´o Tinto (Spain). In order to provide insights into the structure–function relationship, the microstructure of the biofilms was explored using low temperature scanning electron microscopy (LTSEM) in combination with rheological analysis. The creep-recovery experiment results have demonstrated the typical behaviour of viscoelastic materials that combine both elastic and viscous characters. The LTSEM visualization and rheological characterization of biofilms revealed that the network density increased in bacterial biofilms and was the lowest in protist Euglena biofilms. This means that, in the latter biofilms, a lower density of interactions exist, suggesting that the whole system experiences enhanced mobility under external mechanical stress. The samples with the highest dynamic moduli (Leptospirillum–Acidiphilium, Zygnemopsis, Chlorella and Cyanidium) have shown the typical strain thinning behaviour, whereas the Pinnularia and Euglena biofilms exhibited a viscous thickening reaction. The Zygnemopsis filamentous floating structure has the highest cohesive energy and has shown distinctive enhanced resilience and connectivity. This suggests that biofilms should be viewed as soft viscoelastic systems the properties of which are determined by the main organisms and their extracellular polymeric substances. The fractional Maxwell model has been found to explain the rheological behaviour of the observed complex quite well, particularly the power-law behaviour and the characteristic broad relaxation response of these systems., The TEM-BIOPHYM Service at the IEM-CSIC is acknowledged for granting the use of the facilities. This work was funded by the Spanish Science and Innovation Ministry (grants CGL2017-92086-EXP, RTI2018- 094867-BI00 and PID2019-104205GB-C22).

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 300 µmol photons m−2 s−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 30 µmol photons m−2 s−1 (LL cells). Absolute fluorescence spectra at 77 K 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 77 K 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 40 ps. 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.

Activation of microglia results in the increased production and release of a series of inflammatory and neurotoxic mediators, which play essential roles in structural and functional neuronal damage and in the development and progression of a number of neurodegenerative diseases. The microalga Euglena gracilis (Euglena), rich in vitamins, minerals, and other nutrients, has gained increasing attention due to its antimicrobial, anti-viral, antitumor, and anti-inflammatory activities. In particular, anti-inflammatory properties of Euglena could exert neuroprotective functions in different neurodegenerative diseases related to inflammation. However, the mechanisms underlying the anti-inflammatory effect of Euglena are not fully understood. In this study, we investigated whether Euglena could attenuate microglia activation and we also studied the mechanism of its anti-inflammatory activity. Our results showed that non-cytotoxic concentrations of a Euglena acetone extract (EAE) downregulated the mRNA expression levels and release of pro-inflammatory mediators, including NO, IL-1β, and TNF-α in LPS-stimulated microglia. EAE also significantly blocked the LPS-induced nuclear translocation of NF-κB p65 subunit and increased the mRNA expression of nuclear factor erythroid 2–related factor (Nrf2) and heme oxygenase-1 (HO-1). Furthermore, the release of pro-inflammatory mediators and NF-κB activation were also blocked by EAE in the presence of ML385, a specific Nrf2 inhibitor. Together, these results show that EAE overcomes LPS-induced microglia pro-inflammatory responses through downregulation of NF-κB and activation of Nrf2 signaling pathways, although the two pathways seem to get involved in an independent manner.

Photoactivated adenylyl cyclase (PAC) was first discovered to be a sensor for photoavoidance in the flagellate Euglena gracilis. PAC is a flavoprotein that catalyzes the production of cAMP upon illumination with blue light, which enables us to optogenetically manipulate intracellular cAMP levels in various biological systems. Recent progress in genome sequencing has revealed several related proteins in bacteria and ameboflagellates. Among them, the PACs from sulfur bacterium Beggiatoa sp. and cyanobacterium Oscillatoria acuminata have been well characterized, including their crystalline structure. Although there have not been many reported optogenetic applications of PACs so far, they have the potential to be used in various fields within bioscience.

Recent experiments and thermodynamic arguments suggest that mitochondrial temperatures are higher than those of the cytoplasm. A "hot mitochondrion" calls for a closer examination of the energy balance that endows it with these claimed elevated temperatures. As a first step in this effort, we present here a semi-quantitative bookkeeping whereby, in one stroke, a formula is proposed that yields the rate of heat production in a typical mitochondrion and a formula for estimating the number of active ATP synthase molecules per mitochondrion. Scaling laws are shown to determine the number of active ATP synthase molecules in a mitochondrion and mitochondrial rate of heat production. Mitochondrial population of active ATP synthases and mitochondrial rate of heat production appear, both, to scale with cell volume. Four heterotrophic protozoa cell types are considered in this study. The studied cells, selected to cover a wide range of sizes (volumes) from ca. 100 μm3 to 1 million μm3, are estimated to exhibit a power per mitochondrion ranging from ca. 1 pW to 0.03 pW. The corresponding number of active ATP synthases per mitochondrion in these cells ranges from 5,000 to just about a hundred.

Worldwide increasing levels of lead in water systems require the search for efficient ecologically friendly strategies to remove it. Hence, lead accumulation by the free-living algae-like Euglena gracilis and its effects on cellular growth, respiration, photosynthesis, chlorophyll, calcium, and levels of thiol- and phosphate-molecules were analyzed. Photosynthetic cells were able to accumulate 4627 mg lead/kgDW after 5 days of culture with 200 μM Pb2+. Nevertheless, exposure to 50, 100 and 200 μM Pb2+ for up to 8 days did not modify growth, viability, chlorophyll content and oxygen consumption/production. Enhanced biosynthesis of thiol molecules and polyphosphates, i.e. the two canonical metal ion chelation mechanisms in E. gracilis, was not induced under such conditions. However, in cells cultured in the absence of phosphate, lead accumulation and polyphosphate content markedly decreased, while culturing in the absence of sulfate did not modify the accumulation of this metal. In turn, the total amount of intracellular calcium slightly increased as the amount of intracellular lead increased, whereas under Ca2+ deficiency lead accumulation doubled. Therefore, the results indicated that E. gracilis is highly resistant to lead through mechanisms mediated by polyphosphates and Ca2+ and can in fact be classified as a lead hyperaccumulator microorganism.

The intestinal tract contains over half of all immune cells and peripheral nerves and manages the beneficial interactions between food compounds and the host. Paramylon is a &beta, 1,3-glucan storage polysaccharide from Euglena gracilis (Euglena) that exerts immunostimulatory activities by affecting cytokine production. This study investigated the signaling mechanisms that regulate the beneficial interactions between food compounds and the intestinal tract using cell type-specific calcium (Ca2+) imaging in vivo and in vitro. We successfully visualized Euglena- and paramylon-mediated Ca2+ signaling in vivo in intestinal epithelial cells from mice ubiquitously expressing the Yellow Cameleon 3.60 (YC3.60) Ca2+ biosensor. Moreover, in vivo Ca2+ imaging demonstrated that the intraperitoneal injection of both Euglena and paramylon stimulated dendritic cells (DCs) in Peyer&rsquo, s patches, indicating that paramylon is an active component of Euglena that affects the immune system. In addition, in vitro Ca2+ imaging in dorsal root ganglia indicated that Euglena, but not paramylon, triggers Ca2+ signaling in the sensory nervous system innervating the intestine. Thus, this study is the first to successfully visualize the direct effect of &beta, 1,3-glucan on DCs in vivo and will help elucidate the mechanisms via which Euglena and paramylon exert various effects in the intestinal tract.

Background Euglena is a new super health food resource that is rich in the natural polysaccharide paramylon, a linear β-1,3-glucan with various biological activities including activity on the immune system in different cell lines and animals. Despite these reports, the immune regulation mechanism of paramylon is still unclear. Results We investigate the signaling pathways paramylon impacts in immune macrophages. In RAW264.7 macrophages, sonicated and alkalized paramylon oligomers up-regulated inducible nitric oxide synthase (iNOS) and increased secretion of nitric oxide (NO), interleukin (IL)-6 and tumor necrosis factor (TNF)-α, in a concentration-dependent manner. In addition, paramylon activated the nuclear factor-κB(NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways and inhibiting these pathways attenuated the paramylon-induced secretion of the above immune-mediators. Conclusions These results demonstrate that Euglena gracilis paramylon modulates the immune system via activation of the NF-κB and MAPK signaling pathways and thus has potential therapeutic benefits.

In the present study, the chemical composition of the microalga Euglena cantabrica was investigated. The extraction of bioactive compounds was done using pressurized liquid extraction (PLE) at different temperatures (40&ndash, 180 &deg, C) and using green solvents (ethanol-water mixtures). A statistical design of experiments was used to optimize the maximum antioxidant capacity of the extracts by response surface methodology. The antioxidant capacity was determined through the inhibition of 2,2&rsquo, azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, while the chemical analyses of the extracts were carried out using different chromatographic techniques. Chlorophylls and carotenoids were analyzed by high-performance liquid chromatography coupled to a diode array detector and mass spectrometry (HPLC-DAD-MS/MS) and carbohydrates by gas chromatography with flame ionization detection (GC-FID) and high-pressure size-exclusion chromatography coupled to an evaporative light-scattering detector (HPSEC-ELSD). The results showed different possibilities for the extraction conditions, depending on the desired bioactivity or chemical composition. Briefly, (i) mixtures of ethanol-water containing around 40% ethanol at 180 &deg, C gave the best antioxidant capacity, (ii) mixtures containing around 50% ethanol at 110 &deg, C gave the best yield of &beta, glucan paramylon, and (iii) the use of pure ethanol at a low temperature (40 &deg, C) is the best choice for the recovery of carotenoids such as diatoxanthin. Summing up, E. cantabrica seems to be a good candidate to be used in biorefinery to obtain different bioactive compounds.

Eukaryotes arose about 1.6 billion years ago, at a time when oxygen levels were still very low on Earth, both in the atmosphere and in the ocean. According to newer geochemical data, oxygen rose to approximately its present atmospheric levels very late in evolution, perhaps as late as the origin of land plants (only about 450 million years ago). It is therefore natural that many lineages of eukaryotes harbor, and use, enzymes for oxygen-independent energy metabolism. This paper provides a concise overview of anaerobic energy metabolism in eukaryotes with a focus on anaerobic energy metabolism in mitochondria. We also address the widespread assumption that oxygen improves the overall energetic state of a cell. While it is true that ATP yield from glucose or amino acids is increased in the presence of oxygen, it is also true that the synthesis of biomass costs thirteen times more energy per cell in the presence of oxygen than in anoxic conditions. This is because in the reaction of cellular biomass with O2, the equilibrium lies very far on the side of CO2. The absence of oxygen offers energetic benefits of the same magnitude as the presence of oxygen. Anaerobic and low oxygen environments are ancient. During evolution, some eukaryotes have specialized to life in permanently oxic environments (life on land), other eukaryotes have remained specialized to low oxygen habitats. We suggest that the Km of mitochondrial cytochrome c oxidase of 0.1–10 μM for O2, which corresponds to about 0.04%–4% (avg. 0.4%) of present atmospheric O2 levels, reflects environmental O2 concentrations that existed at the time that the eukaryotes arose., Graphical abstract Image 1, Highlights • The first 1.5 billion years of life history took place without molecular oxygen. • The first eukaryotes appeared ca. 1.6 billion years ago, oxygen rose with land plants. • Eukaryotes arose and diversified with low oxygen, anaerobic ATP synthesis is ancient. • Anaerobic energy metabolism in mitochondria is common among eukaryotic lineages. • The Km of cytochrome c oxidase might reflect low environmental O2 at eukaryote origin.

Microalgae-derived functional materials, such as food supplements, drugs, and biofuels have gained a great deal of attention in recent times. Among the several microalgae investigated for biomaterials production, Euglena spp. are particularly attractive due to their easiness to grow in cultures and the ability to produce numerous bioactive compounds through extremely complex metabolic pathways. The genus Euglena comprises more than 300 species of unicellular, predominantly freshwater flagellates of worldwide distribution. The plastids in Euglena cells have been recognized as a site of the production of proteins, fatty acids, and many other value-added metabolites. Euglena is one of the few microorganisms that simultaneously produces antioxidants, such as β-carotene, l -ascorbic acid, and α-tocopherol, along with wax esters, phytotoxins and polyunsaturated fatty acids (PUFAs), that are useful in the manufacture of pharmaceuticals, cosmeceuticals, and nutraceuticals. Nevertheless, the biotechnological potency of Euglena for industrial production has been exploited limitedly. This review summarizes the major value-added compounds obtained from the protist genus Euglena and their potential and prospects for commercial production.

© 2018 Elsevier B.V. Euglena gracilis is a unicellular microalga studied for the production of nutraceuticals, cosmeceuticals and biofuel. Full exploitation of the organism requires the development of genetic engineering tools including a method for obtaining genetically stable transformants. In this work, Agrobacterium mediated transformation, biolistic bombardment and electroporation were explored to obtain stable nuclear E. gracilis transformants. Two 3′UTR fragments of the E. gracilis gapC gene were added at the 5′and 3′ ends of the pCambia1302 T-DNA to promote homologous integration of the transforming DNA into the genome. E. gracilis transformants growing on hygromycin plates and expressing the mgfp5 gene coding for green fluorescent protein were obtained from all three approaches. Maintenance of the transforming DNA in the nucleus was confirmed by PCR. Agrobacterium-mediated transformation yielded 10 transformants, biolistic bombardment seven and electroporation one transformant per 10,000 cells plated. Transformants from biolistic bombardment and electroporation were able to transiently express the hptII gene based on their growth on hygromycin containing plates, but this property was lost during repeated rounds of cultivation suggesting lack of (stable) integration of the transforming DNA into the Euglena genome. In contrast, Agrobacterium-mediated transformation produced stable nuclear transformants growing on hygromycin plates even after 12 rounds of cultivation. This work will pave the way for further improvement of E. gracilis strains for the production of valuable compounds.

Euglena gracilis is a promising source of commercially important metabolites such as vitamins, wax esters, paramylon, and amino acids. However, the molecular tools available to create improved Euglena strains are limited compared to other microorganisms that are currently exploited in the biotechnology industry. The complex poly-endosymbiotic nature of the Euglena genome is a major bottleneck for obtaining a complete genome sequence and thus represents a notable shortcoming in gaining molecular information of this organism. Therefore, the studies and applications have been more focused on using the wild-type strain or its variants and optimizing the nutrient composition and cultivation conditions to enhance the production of biomass and valuable metabolites. In addition to producing metabolites, the E. gracilis biorefinery concept also provides means for the production of biofuels and biogas as well as residual biomass for the remediation of industrial and municipal wastewater. Using Euglena for bioremediation of environments contaminated with heavy metals is of special interest due to the strong ability of the organism to accumulate and sequester these compounds. The published draft genome and transcriptome will serve as a basis for further molecular studies of Euglena and provide a guide for the engineering of metabolic pathways of relevance for the already established as well as novel applications.

This study evaluated methane (CH 4) emission, intake, digestibility, and nitrogen efficiency in sheep fed diets containing replacement levels (0%, 33%, 50%, and 67% of soybean meal with euglena). In this experiment, four Corriedale wether sheep with an initial body weight of 53.8 ± 4.6 were arranged in a 4 × 4 Latin square design. This experiment lasted 84 days, divided into four experimental periods. Each period lasted 21 days, which consists of 14 days of adaptation to the diets, 5 days to collect samples, and 2 days to collect gas emission from sheep. Methane emission expressed as L/kg DM intake or g/kg DM intake reduced by up to 37% and the energy loss via CH 4 (% of GE intake) reduced by up to 34%. No differences (p > 0.05) were observed in DM and OM intake and whole tract apparent DM digestibility due to substitution of soybean meal with euglena. The total CP loss reduced significantly (linear, p

In the present study, five different DNA extraction procedures were examined to determine their effectiveness for extracting DNA suitable for NGS applications. This included two silica-membrane spin column kits, phenol:chloroform, and two CTAB-based methods. Spectrophotometric and fluorimetric measurements as well as standard gel electrophoresis were used as criteria for evaluating the quantity and quality of the isolated DNA prior to the sequencing. Herein, the method of establishing and maintaining axenic Euglena cultures is also presented. The modified CTAB-based method proved to be highly efficient. In terms of DNA quantity and purity (according to the absorbance ratios), the chosen method resulted in DNA of high molecular weight and quality, which fulfills the library construction requirements. Genomic DNA of Euglena hiemalis (CCAP 1224/35) and E. longa (CCAP 1204-17a) isolated using the suggested protocol had been successfully sequenced on the Illumina HiSeq platform. A modified, rapid CTAB-based method of total DNA isolation from Euglena has been described. In terms of the DNA quantity and quality, the protocol devised involving the washing step with DMSO:acetonitrile proved superior to the commonly used, commercially manufactured kits and isolation with phenol:chloroform. The method is also less labor-intensive and time-consuming than the traditional CTAB-based protocol.

Wax esters (WEs) synthesized by Euglena gracilis are potential sources for alternative fuels because of their high productivity, recent success in mass cultivation, and low energy consumption in extraction. In this study, deoxygenation of Euglena WE and conversion to hydrocarbons in a catalytic cracking process under a hydrogen-free atmosphere was investigated using a residue fluid catalytic cracking equilibrium catalyst with enhanced hydrogen-transfer activity. The deoxygenation of Euglena WE proceeded more rapidly with higher H2O selectivity than that of saturated triglycerides. This is because initial β-elimination of WEs produces saturated fatty acids and higher olefins; the higher olefins rapidly release hydrogen species during cracking, cyclization and aromatization, and the hydrogen species accelerate hydrodeoxygenation of the saturated fatty acids. Furthermore, the cracking of Euglena WE produced large amounts of paraffins and olefins instead of aromatics. Therefore, Euglena WE was confirmed to be a preferable feedstock for the catalytic cracking process for hydrocarbon fuel production.

Paramylon, which is a β-(1,3)-D-glucan photosynthesized by Euglena, was chemically modified by esterification. Various paramylon triesters with different alkyl chain lengths (carbon numbers 2–12) were successfully prepared. All of the paramylon triesters have higher thermal degradation temperatures than that of neat paramylon. Moreover, it was found that the paramylon triesters with C2–C6 alkyl chains are crystalline polymers with melting temperatures from 281 °C to 114 °C, and those with C8–C12 alkyl chains are amorphous polymers, confirmed by both DSC and X-ray diffraction analysis. Paramylon triesters with C3–C12 alkyl chains could shape self-sustaining films by both solvent-casting and melt-quench methods with high optical transmittance and sufficient tensile strength or elongation at break. Thermal and mechanical properties of paramylon triesters can be controlled freely from hard to soft by substituted acyl length. In the cases of the crystalline paramylon triesters, highly oriented and crystallized films could be fabricated by the thermally stretched method, and their tensile strengths have been obviously improved. Well-oriented X-ray fiber diagrams of the stretched and crystallized films suggest that all of the paramylon triesters have rare 5-fold helix conformation of molecular chains in crystal.

Euglena gracilis produces several important health-enhancing metabolites including ascorbate, α-tocopherol and free amino acids (faa). The yield of metabolites is dependent on the strain of E. gracilis and the metabolic growth condition. Here we investigated the effects of photoautotrophic (PT), mixotrophic (MT) and heterotrophic (HT) cultivation on the synthesis of ascorbate, α-tocopherol and faa in E. gracilis var. saccharophila, using label-free shotgun proteomics, and metabolite analysis using colourimetric assay, high-performance and ultra-performance liquid chromatography (HPLC/UPLC). PT cultivation resulted in the production of more antioxidants (up to 4.13 mg g−1 ascorbate and 2.52 mg g−1 α-tocopherol) than the MT and HT growth conditions (up to 0.97 and 0.50 mg g−1 ascorbate, and 1.40 and 0.21 mg g−1 α-tocopherol, respectively). The relative abundance of several faa varied between mid-log and initial stationary growth phases, but the total amount of faa remained about the same, with arginine as the most abundant amino acid. Proteomic analysis revealed a total of 3843 non-redundant proteins in E. gracilis var. saccharophila, of which 1890 were common among all cultivations. Gene ontology annotations suggested derivatisation of metabolic pathways from different organisms, such as lysine biosynthesis from fungi and serine biosynthesis from plants, while a few pathways were unique to Euglena, such as those of ascorbate and arginine. Some enzymes exhibited several isoforms that were influenced by the metabolic growth condition. For example, one of the isozymes of threonine aldolase was expressed in HT/MT cultures only, and one of the isozymes of phosphoglycerate dehydrogenase was expressed in PT cultures only. This is the first proteomic study of E. gracilis var. saccharophila, which provides a mechanistic insight into the biosynthetic pathway dynamics of primary metabolites (antioxidants and faa). This new information can serve as a framework for further development of Euglena as a producer of nutraceuticals.

Paramylon, a storage polysaccharide of Euglena gracilis, is a linear β-1,3-glucan with a weight-average molecular weight of ∼2.0×105. Sequential long-chain acylation and succinylation of paramylon yielded amphiphilic paramylon acylate succinates. Owing to their amphiphilicity, these paramylon derivatives showed higher viscosity than paramylon succinate when dispersed in an aqueous solution. Examination of the viscosity of aqueous solutions containing paramylon acylate succinates differing in chain length and degree of substitution of long-chain acyl groups (DSlca) revealed that the longer the acyl chain and the higher the DSlca, the higher the viscosity of the aqueous solution. Solution casting yielded transparent and mechanically tough films from paramylon acylate succinates. These films had high water absorbability, up to ∼1000 times their weight.

In the microalga Euglena gracilis, the storage polysaccharide paramylon (β-1,3-D-glucan) is degraded to glucose, and finally converted to wax esters under anaerobic conditions. The wax esters and paramylon are now considered to be valuable materials for applications in biofuel production and in medicine. Genetic improvements of wax esters and paramylon accumulation in Euglena would facilitate their large-scale industrial applications; however, such improvements have thus far been difficult to realize because the regulatory factors involved in the wax ester fermentation pathway remain mostly unknown. Recently, two of dual-specificity Tyr phosphorylation-regulated kinases, starch degradation 1 (STD1) and triacylglycerol accumulation regulator 1 (TAR1), have been reported to regulate triacylglycerol metabolism in Chlamydomonas reinhardtii. In this study, we identified the Euglena DYRK orthologous sequences, and gene-silencing of EgSTD1 and EgSTD2 showed an increase in accumulation of paramylon and the following anaerobic wax ester production. This result indicated that EgSTD1 and EgSTD2 play a significant role in regulation of the wax ester fermentation pathway.

Purpose To test the effects of short-term exposure of aquatic organisms to electric field (EF) with negligible magnetic component. Materials and methods We built a plate capacitor that served as a source of EF of strengths that can be found in nature near transmission lines. We exposed two cultured protist species Euglena viridis and Paramecium caudatum to EFs for 24 hours and monitored their abundance, morphology, intracellular superoxide anion (by dihydroethidium [DHE]), hydrogen peroxide by (H2DCF) and lipid peroxidation (MDA) contents, catalase (CAT) and superoxide dismutase (SOD) activity. Results We found that even short-term exposure to low strength EF causes changes in population abundance, morphology and oxidative stress response in both species. As the EF strength increased, abundance of both species decreased. However, at weaker EFs, fission rates were seemingly promoted. We noted a decrease in size in both organisms in directions perpendicular to their fission planes correlated with EF strength. DHE and H2DCF fluorescence intensity and SOD activity were higher in organisms exposed to the stronger EFs. Conclusions We suggest that the electric component of the field, rather than the magnetic, is the main cause of all the noted effects. As a result, aquatic organisms should be given greater importance in studies assessing the effects of EMFs in spite of the attenuating effects of water to EF strengths.

The phytoflagellate Euglena gracilis produces a large amount of paramylon (PM), a conglomerate of liner β-1,3-glucan chains, as a storage polysaccharide. PM is synthesized from uridine diphosphate-glucose, but its mechanism of formation is largely unknown. Two enzymes, glucan synthase-like (EgGSL) 1 and EgGSL2 were previously identified as candidates for PM synthesis in a Euglena transcriptome analysis. Here, we performed a reverse genetic analysis on these enzymes. Knockdown of EgGSL2, but not EgGSL1, significantly inhibits PM accumulation in Euglena cells. Additionally, β-1,3-glucan synthesis is detected in a PM-associated membrane fraction extracted from Euglena cells. Our findings indicate that EgGSL2 is the predominant enzyme for PM biosynthesis.

application/pdf, This in vivo study was conducted to evaluate the effect of supplementation with different rates of euglena (Euglena gracilis) on nutrient intake, digestibility, nitrogen balance and rumen fermentation. Four rumen cannulated Corriedale wethers sheep with an average body weight of 44.25 +/- 3.86 kg were arranged in a 4 x 4 Latin square design and fed a basal diet of Guinea grass (Panicum maximum) hay and concentrate mixture at the maintenance level with four different rates of euglena (0, 50, 100 and 150 g/kg DM intake). The experiment was conducted over 80 days in four 20 day periods that consisted of 14 days of acclimatization, 5 days of measurement and 1 more day for rumen liquor sample collection. The data were subjected to polynomial regression analysis. Dry matter (DM), organic matter (OM), acid detergent fibre (ADF) and gross energy (GE) intake increased linearly (P< 0.001) and quadratically (P=0,002) with increasing concentrations of euglena. Similarly, crude protein (CP) intake was increased linearly (P< 0.001). Dry matter, OM, NDF, ADF and GE digestibility were not affected by supplementation of euglena (P> 0.11) while apparent CP digestibility increased linearly (P= 0.009). As a result, protein retention (g/d) was increased linearly (P 0.23) the total volatile fatty acid (VFA) concentration and the molar proportions of acetate, propionate, butyrate and the acetate: propionate ratio. The finding of this study indicated that the addition of euglena increased nutrient intake without affecting total tract digestibility. It has been also demonstrated that addition of euglena at a dose of 150 g/kg DM improved CP retention by 31%, which may be associated with increased CP digestibility and efficiency of utilization. Thus, euglena supplementation up to 150 g/kg DM of the diet could be a possible option for substitution of protein and energy sources. (C) 2017 Elsevier B.V. All rights reserved.

Euglena sanguinea is known to produce the alkaloid toxin euglenophycin and is known to cause fish kills and inhibit mammalian tissue and microalgal culture growth. An analysis of over 30 species of euglenoids for accumulation of euglenophycin identified six additional species producing the toxin; and six of the seven E. sanguinea strains produced the toxin. A phylogenetic assessment of these species confirmed most taxa were in the Euglenaceae, whereas synthesis capability apparently has been lost in the Phacus, Eutreptiella, and Discoplastis branches.

In this review we will describe the structure of the photosynthetic unicellular alga Euglena gracilis (Euglenozoa, Euglenophyceae). The description of the cell will proceed from the outside structures to the inside components. Details will be given at both microscopic and ultra-microscopic levels citing the most recent data, only for those structures that are not comparable with analogue structures found in most algae. Special attention will be given to the peculiar features of Euglena such as pellicle, photoreceptive system, storage products, and chloroplasts. We will also analyze the swimming behavior of the cell highlighting the role of photoreception in guiding the cell movement. The importance of Euglena as a promising source of immunopotentiating compounds will be discussed.