The impact of elevated sulfur and nitrogen levels on cadmium tolerance in Euglena species.

Heavy metal (HM) pollution threatens human and ecosystem health. Current methods for remediating water contaminated with HMs are expensive and have limited effect. Therefore, bioremediation is being investigated as an environmentally and economically viable alternative. Freshwater protists Euglena gracilis and Euglena mutabilis were investigated for their tolerance to cadmium (Cd). A greater increase in cell numbers under Cd stress was noted for E. mutabilis but only E. gracilis showed an increase in Cd tolerance following pre-treatment with elevated concentrations of S or N. To gain insight regarding the nature of the increased tolerance RNA-sequencing was carried out on E. gracilis. This revealed transcript level changes among pretreated cells, and additional differences among cells exposed to CdCl2. Gene ontology (GO) enrichment analysis reflected changes in S and N metabolism, transmembrane transport, stress response, and physiological processes related to metal binding. Identifying these changes enhances our understanding of how these organisms adapt to HM polluted environments and allows us to target development of future pre-treatments to enhance the use of E. gracilis in bioremediation relating to heavy metals.

Metabolic responses of Euglena gracilis under photoheterotrophic and heterotrophic conditions.

The protist Euglena gracilis has various trophic modes including heterotrophy and photoheterotrophy. To investigate how cultivation mode influences metabolic regulation, the chemical composition of cellular metabolites of Euglena gracilis grown under heterotrophic and photoheterotrophic conditions was monitored from the early exponential phase to the mid-stationary phase using two different techniques, i.e, nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HRMS). The combined metabolomics approach allowed an in-depth understanding of the mechanism of photoheterotrophic and heterotrophic growth for biomolecule production. Heterotrophic conditions promoted the production of polar amino and oxygenated compounds such as proteins and polyphenol compounds, especially at the end of the exponential phase while photoheterotrophic cells enhanced the production of organoheterocyclic compounds, carbohydrates, and alkaloids.

Metabolomic analysis and pathway profiling of paramylon production in Euglena gracilis grown on different carbon sources.

Paramylon (ß-1,3-glucan) produced by Euglena gracilis displays antioxidant, antitumor, and hypolipidaemic functions. The biological properties of paramylon production by E. gracilis can be understood by elucidating the metabolic changes within the algae. In this study, the carbon sources in AF-6 medium were replaced with glucose, sodium acetate, glycerol, or ethanol, and the paramylon yield was measured. Adding 0.1260 g/L glucose to the culture medium resulted in the highest paramylon yield of 70.48 %. The changes in metabolic pathways in E. gracilis grown on glucose were assessed via non-targeted metabolomics analysis using ultra-high-performance liquid chromatography coupled to high-resolution quadrupole-Orbitrap mass spectrometry. We found that glucose, as a carbon source, regulated some differentially expressed metabolites, including l-glutamic acid, γ-aminobutyric acid (GABA), and l-aspartic acid. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes further showed that glucose regulated the carbon and nitrogen balance through the GABA shunt, which enhanced photosynthesis, regulated the flux of carbon and nitrogen into the tricarboxylic acid cycle, promoted glucose uptake, and increased the accumulation of paramylon. This study provides new insights into E. gracilis metabolism during paramylon synthesis.

Evaluation of the Effects of Euglena gracilis on Enhancing Immune Responses in RAW264.7 Cells and a Cyclophosphamide-Induced Mouse Model.

In this study we evaluated the immune-enhancing effects of ß-glucan, the main component of Euglena gracilis (Euglena), and Euglena on inflammatory factor expression in RAW264.7 macrophages and ICR mice with cyclophosphamide-induced immunosuppression. Macrophages were treated with ß-glucan or Euglena for 48 h. The ß-glucan and Euglena groups exhibited higher levels of inducible nitric oxide synthase, nitric oxide, and tumor necrosis factor (TNF)-α than the control (vehicle alone) group. Animals were fed saline and ß-glucan (400 mg/kg body weight (B.W.)) or Euglena (400 or 800 mg/kg B.W.) for 19 days, and on days 17-19, cyclophosphamide (CCP, 80 mg/kg B.W.) was administered to induce immunosuppression in the ICR mouse model. CCP reduced the body weight, spleen index, and cytokine expression of the mice. To measure cytokine and receptor expression, splenocytes were treated with concanavalin A (ConA) or lipopolysaccharide (LPS) as a mitogen for 24 h. In vivo, ConA stimulation significantly upregulated the expression of interferon (IFN)-γ, interleukin (IL)-10, IL-12 receptor ß1, IL-1ß, and IL-2 in splenocytes from the ß-glucan- or Euglena-treated groups compared with those in the splenocytes from the CCP-treated group; LPS stimulation increased the levels of the cytokines TNF-α, IL-1ß, and IL-6 in splenocytes from the ß-glucan- or Euglena-treated groups compared with those from the CCP-treated group, but most of these differences were not significant. These results demonstrate the effect of Euglena in ameliorating macrophages and immunosuppression in CCP-treated mice. Thus, Euglena has the potential to enhance macrophage- and splenocyte-mediated immune-stimulating responses.

Influence of Carbon Sources on the Phenolic Compound Production by Euglena gracilis Using an Untargeted Metabolomic Approach.

Industrial development and urbanization has led to the diverse presence of metals in wastewater that are often improperly treated. The microalgae Euglena gracilis can tolerate high concentrations of metal via the excretion of organic metabolites, including phenolics. This study aims to evaluate how carbon amendment stimulates phenolic compound production by E. gracilis. The number, relative intensity and molecular composition of the phenolic compounds were significantly different between each of four carbon amended cultures (i.e., glutamic acid, malic acid, glucose, reduced glutathione) during the log phase. Phenolic compounds were mainly produced during the minimum growth rate, likely a response to stressful conditions. A better understanding of phenolic compounds production by E. gracilis and the impact of growth conditions will help identify conditions that favor certain phenolic compounds for dietary and metal chelation applications.

Oral administration of Euglena gracilis paramylon ameliorates chemotherapy-induced leukocytopenia and gut dysbiosis in mice.

Euglena gracilis (EUG) is a food supplement rich in beta-glucans, which are stored in the form of granules called paramylon. We determined whether EUG improved chemotherapy-induced leukocytopenia and dysbiosis. Mice were orally administered EUG prior to gemcitabine treatment. Analyses of the blood cell count, leukocyte population in the spleen, granulocyte/macrophage-colony-stimulating factor (GM-CSF) production by splenocytes, and fecal microbiome were conducted. The recovery of total leukocytes, neutrophils, and monocytes was accelerated after a single gemcitabine treatment. A more rapid lymphocyte recovery rate was observed after four gemcitabine treatments. No difference was observed in the percentage of T, B, or myeloid cells or in the expression of Dectin-1 in the spleens of the gemcitabine and EUG/gemcitabine groups. The EUG/gemcitabine group showed an enhanced GM-CSF production by lipopolysaccharides-stimulated splenocytes. Next-generation sequencing revealed that gemcitabine-induced dysbiosis was alleviated. This study demonstrated that EUG-derived beta-glucans could act as a biological response modifier as well as prebiotics for ameliorating chemotherapy-induced adverse effects.

Euglenatides, Potent Antiproliferative Cyclic Peptides Isolated from the Freshwater Photosynthetic Microalga Euglena gracilis.

By limiting the nitrogen source to glutamic acid, we isolated cyclic peptides from Euglena gracilis containing asparagine and non-proteinogenic amino acids. Structure elucidation was accomplished through spectroscopic methods, mass spectrometry and chemical degradation. The euglenatides potently inhibit pathogenic fungi and cancer cell lines e.g., euglenatide B exhibiting IC50 values of 4.3 µM in Aspergillus fumigatus and 0.29 µM in MCF-7 breast cancer cells. In an unprecedented convergence of non-ribosomal peptide synthetase and polyketide synthase assembly-line biosynthesis between unicellular species and the metazoan kingdom, euglenatides bear resemblance to nemamides from Caenorhabditis elegans and inhibited both producing organisms E. gracilis and C. elegans. By molecular network analysis, we detected over forty euglenatide-like metabolites in E. gracilis, E. sanguinea and E. mutabilis, suggesting an important biological role for these natural products.

Simultaneous probing of dual intracellular metabolites (ATP and paramylon) in live microalgae using graphene oxide/aptamer nanocomplex.

The development of an intracellular metabolite imaging platform for live microorganisms has been a challenge in the study of microbes. Herein, we performed metabolite imaging in live microalgal cells using a graphene oxide (GO)/aptamer complex. The properties of the GO were characterized using dynamic light scattering (DLS) and atomic force microscopy (AFM), which were determined to have 140 ± 3 nm in mean diameter. An ATP-specific aptamer was mixed with GO to form a GO/aptamer complex, and the feasibility of the complex was tested in vitro. The high correlation between the fluorescence intensity and concentration of ATP was observed in the range 0-10 mM. Next, the feasibility of the complex was confirmed in vivo. Under both phototrophic and heterotrophic culture conditions, Euglena gracilis internalized the complex, and bright fluorescence was observed as the aptamer was bound to the target metabolite (ATP). The fluorescence intensity of cells was correlated to the ATP concentration in the cells. Imaging of dual intracellular metabolites (ATP and paramylon) was achieved by simply using two different aptamers (ATP-specific aptamer and paramylon-specific aptamer) together, showing the great potential of the complex as a dual-sensing/imaging platform. In addition, the GO/aptamer complex exhibited low cytotoxicity; the proliferation and viability of E. gracilis cells were not significantly affected by the complex. Our results suggested that this new imaging platform can be efficiently used for detecting dual intracellular metabolites in live microalgal cells.

Immunoenhancing Effects of Euglena gracilis on a Cyclophosphamide-Induced Immunosuppressive Mouse Model.

In this study, the effects of the immune stimulator Euglena gracilis (Euglena) in cyclophosphamide (CCP)-induced immunocompromised mice were assessed. The key component ß-1,3-glucan (paramylon) constitutes 50% of E. gracilis. Mice were orally administered Euglena powder (250 and 500 mg/kg body weight (B.W.)) or ß-glucan powder (250 mg/kg B.W.) for 19 days. In a preliminary immunology experiment, ICR mice were intraperitoneally injected with 80 mg of CCP/kg B.W. during the final 3 consecutive days. In the main experiment, BALB/c mice were treated with CCP for the final 5 days. To evaluate the enhancing effects of Euglena on the immune system, mouse B.W., the spleen index, natural killer (NK) cell activity and mRNA expression in splenocytes lungs and livers were determined. To detect cytokine and receptor expression, splenocytes were treated with 5 µg/ml concanavalin A or 1 µg/ml lipopolysaccharide. The B.W. and spleen index were significantly increased and NK cell activity was slightly enhanced in all the experimental groups compared to the CCP group. In splenocytes, the gene expression levels of tumor necrosis factor-α, interferon-γ, interleukin (IL)-10, IL-6, and IL-12 receptor were increased in the E. gracilis and ß-glucan groups compared to the CCP group, but there was no significant difference. Treatment with 500mg of Euglena/kg B.W. significantly upregulated dectin-1 mRNA expression in the lung and liver compared to the CCP group. These results suggest that Euglena may enhance the immune system by strengthening innate immunity through immunosuppression.

Paramylon from Euglena gracilis Prevents Lipopolysaccharide-Induced Acute Liver Injury.

Acute liver injury (ALI) is a life-threatening syndrome with high mortality and lacks effective therapies. Rodents under LPS (lipopolysaccharide)/D-Gal (D-galactosamine) stress mimic ALI by presenting dramatically increased inflammation and cell death in the liver. Euglena gracilis, functioning like dietary fiber, is commonly used as a paramylon (Pa)-rich nutritional supplement that has various biological effects such as regulating immune system, anti-obesity, and anti-tumor. Here, we found that Pa or sonicated and alkalized paramylon (SA-Pa) alleviated the LPS/D-Gal-induced hepatic histopathological abnormalities in mice. Compared with Pa, SA-Pa had lower molecular weights/sizes and showed better efficacy in alleviating injury-induced hepatic functions, as well as the transcriptional levels of inflammatory cytokines. Moreover, SA-Pa treatment promoted M2 macrophage activation that enhanced the anti-inflammatory function in the liver, and downregulated STAT3 target genes, such as Fos, Jun, and Socs3 upon the injury. Meanwhile, SA-Pa treatment also alleviated apoptosis and necroptosis caused by the injury. Our results demonstrated that SA-Pa efficiently protected the liver from LPS/D-Gal-induced ALI by alleviating inflammation and cell death.

Cryo-EM structure of the highly atypical cytoplasmic ribosome of Euglena gracilis.

Ribosomal RNA is the central component of the ribosome, mediating its functional and architectural properties. Here, we report the cryo-EM structure of a highly divergent cytoplasmic ribosome from the single-celled eukaryotic alga Euglena gracilis. The Euglena large ribosomal subunit is distinct in that it contains 14 discrete rRNA fragments that are assembled non-covalently into the canonical ribosome structure. The rRNA is substantially enriched in post-transcriptional modifications that are spread far beyond the catalytic RNA core, contributing to the stabilization of this highly fragmented ribosome species. A unique cluster of five adenosine base methylations is found in an expansion segment adjacent to the protein exit tunnel, such that it is positioned for interaction with the nascent peptide. As well as featuring distinctive rRNA expansion segments, the Euglena ribosome contains four novel ribosomal proteins, localized to the ribosome surface, three of which do not have orthologs in other eukaryotes.

Immune activation of murine RAW264.7 macrophages by sonicated and alkalized paramylon from Euglena gracilis.

BACKGROUND: Euglena is a new super health food resource that is rich in the natural polysaccharide paramylon, a linear ß-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.

Water extract from Euglena gracilis prevents lung carcinoma growth in mice by attenuation of the myeloid-derived cell population.

The partially purified water extract from Euglena gracilis (EWE) was evaluated for its antitumor and immunomodulatory effects in cell cultures and in a mouse orthotopic lung carcinoma allograft model. In two-dimensional cell culture, the EWE treatment inhibited cell growth of both murine Lewis lung carcinoma (LLC) and human lung carcinoma cells (A549 and H1299) in a dose- and time-dependent manner. In contrast, the growth of mouse bone marrow cells (BMCs), but not mouse splenocytes (SPLs), was stimulated by the treatment with EWE. In three-dimensional spheroid culture, spheroid growth of LLC cells was significantly attenuated by EWE treatment. In a mouse LLC orthotopic allograft model, pretreatment with EWE (150-200 mg/kg/day, via drinking water) three weeks prior to the LLC cell inoculation, but not post-treatment after LLC cell inoculation, significantly attenuated the growth of LLC tumors in immunocompetent syngeneic mouse lung. This tumor growth attenuation coincided with a significant decrease in the population of myeloid-derived cells, primarily neutrophils. Flow cytometric analysis revealed that the EWE treatment significantly attenuated growth of granulocytic myeloid-derived suppressor cells (gMDSC) in BMCs and that this decrease was due to induction of gMDSC-specific apoptosis and differentiation of monocytic MDSCs (mMDSC) to macrophages. The present study provides evidence that EWE pretreatment inhibits lung carcinoma growth mainly by stimulating host antitumor immunity through attenuation of growth of gMDSCs and decreasing the number of peripheral granulocytes. This study suggests that the partially purified extract derived from Euglena gracilis contains significant bioactive materials that prevent lung carcinoma growth.

Anaerobic respiration coupled with mitochondrial fatty acid synthesis in wax ester fermentation by Euglena gracilis.

In Euglena gracilis, wax ester fermentation produces ATP during anaerobiosis. Here, we report that anaerobic wax ester production is suppressed when the mitochondrial electron transport chain complex I is inhibited by rotenone, whereas it is increased by the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). The ADP/ATP ratio in anaerobic cells is elevated by treatment with either rotenone or CCCP. Gene silencing experiments indicate that acyl-CoA dehydrogenase, electron transfer flavoprotein (ETF), and rhodoquinone (RQ) participate in wax ester production. These results suggest that fatty acids are synthesized in mitochondria by the reversal of ß-oxidation, where trans-2-enoyl-CoA is reduced mainly by acyl-CoA dehydrogenase using the electrons provided by NADH via the electron transport chain complex I, RQ, and ETF, and that ATP production is highly supported by anaerobic respiration utilizing trans-2-enoyl-CoA as a terminal electron acceptor.

Nickel accumulation by the green algae-like Euglena gracilis.

Nickel accumulation and nickel effects on cellular growth, respiration, photosynthesis, ascorbate peroxidase (APX) activity, and levels of thiols, histidine and phosphate-molecules were determined in Euglena gracilis. Cells incubated with 0.5-1mM NiCl2 showed impairment of O2 consumption, photosynthesis, Chl a+b content and APX activity whereas cellular integrity and viability were unaltered. Nickel accumulation was depressed by Mg2+ and Cu2+, while Ca2+, Co2+, Mn2+ and Zn2+ were innocuous. The growth half-inhibitory concentrations for Ni2+ in the culture medium supplemented with 2 or 0.2mM Mg2+ were 0.43 or 0.03mM Ni2+, respectively. Maximal nickel accumulation (1362mg nickel/Kg DW) was achieved in cells exposed to 1mM Ni2+ for 24h in the absence of Mg2+ and Cu2+; accumulated nickel was partially released after 72h. GSH polymers content increased or remained unchanged in cells exposed to 0.05-1mM Ni2+; however, GSH, cysteine, γ-glutamylcysteine, and phosphate-molecules all decreased after 72h. Histidine content increased in cells stressed with 0.05 and 0.5mM Ni2+ for 24h but not at longer times. It was concluded that E. gracilis can accumulate high nickel levels depending on the external Mg2+ and Cu2+ concentrations, in a process in which thiols, histidine and phosphate-molecules have a moderate contribution.

Genotoxicity and subchronic toxicity evaluation of dried Euglena gracilis ATCC PTA-123017.

Euglena gracilis is a microalga capable of synthesizing various nutrients of interest in human and animal nutrition. When cultivated aerobically in the dark, Euglena synthesize paramylon, a storage polysaccharide comprised of high molecular weight beta-1,3-D-glucose polymers organized in cytoplasmic granules. Beta-glucans have been shown to have immune modulation effects, including anti-microbial, anti-tumor, and anti-oxidant properties, and metabolic effects, such as regulation of cholesterol and blood sugar levels. Preparations of E. gracilis and paramylon may therefore have potential utility as functional food ingredients for human and animal nutrition. A battery of toxicological studies was conducted on a dried preparation of E. gracilis and paramylon to support their safe food use. The dried alga was not genotoxic in a bacterial reverse mutation test and mammalian micronucleus test. In the subchronic toxicity study, rats were provided E. gracilis in the diet at levels of 0, 12,500, 25,000 or 50,000 ppm. Paramylon was provided at a concentration of 50,000 ppm. No effects that could be attributable to treatment were observed in clinical observations, body weight, food consumption, ophthalmology, hematology and clinical chemistry, urinalysis, and macroscopic and microscopic findings. A NOAEL of 50,000 ppm in the diet was determined for both ingredients.

Efficient selective breeding of live oil-rich Euglena gracilis with fluorescence-activated cell sorting.

Euglena gracilis, a microalgal species of unicellular flagellate protists, has attracted much attention in both the industrial and academic sectors due to recent advances in the mass cultivation of E. gracilis that have enabled the cost-effective production of nutritional food and cosmetic commodities. In addition, it is known to produce paramylon (ß-1,3-glucan in a crystalline form) as reserve polysaccharide and convert it to wax ester in hypoxic and anaerobic conditions-a promising feedstock for biodiesel and aviation biofuel. However, there remain a number of technical challenges to be solved before it can be deployed in the competitive fuel market. Here we present a method for efficient selective breeding of live oil-rich E. gracilis with fluorescence-activated cell sorting (FACS). Specifically, the selective breeding method is a repetitive procedure for one-week heterotrophic cultivation, staining intracellular lipids with BODIPY(505/515), and FACS-based isolation of top 0.5% lipid-rich E. gracilis cells with high viability, after inducing mutation with Fe-ion irradiation to the wild type (WT). Consequently, we acquire a live, stable, lipid-rich E. gracilis mutant strain, named B1ZFeL, with 40% more lipid content on average than the WT. Our method paves the way for rapid, cost-effective, energy-efficient production of biofuel.

Cadmium removal by Euglena gracilis is enhanced under anaerobic growth conditions.

The facultative protist Euglena gracilis, a heavy metal hyper-accumulator, was grown under photo-heterotrophic and extreme conditions (acidic pH, anaerobiosis and with Cd(2+)) and biochemically characterized. High biomass (8.5×10(6)cellsmL(-1)) was reached after 10 days of culture. Under anaerobiosis, photosynthetic activity built up a microaerophilic environment of 0.7% O2, which was sufficient to allow mitochondrial respiratory activity: glutamate and malate were fully consumed, whereas 25-33% of the added glucose was consumed. In anaerobic cells, photosynthesis but not respiration was activated by Cd(2+) which induced higher oxidative stress. Malondialdehyde (MDA) levels were 20 times lower in control cells under anaerobiosis than in aerobiosis, although Cd(2+) induced a higher MDA production. Cd(2+) stress induced increased contents of chelating thiols (cysteine, glutathione and phytochelatins) and polyphosphate. Biosorption (90%) and intracellular accumulation (30%) were the mechanisms by which anaerobic cells removed Cd(2+) from medium, which was 36% higher versus aerobic cells. The present study indicated that E. gracilis has the ability to remove Cd(2+) under anaerobic conditions, which might be advantageous for metal removal in sediments from polluted water bodies or bioreactors, where the O2 concentration is particularly low.

Zn-bis-glutathionate is the best co-substrate of the monomeric phytochelatin synthase from the photosynthetic heavy metal-hyperaccumulator Euglena gracilis.

The phytochelatin synthase from photosynthetic Euglena gracilis (EgPCS) was analyzed at the transcriptional, kinetic, functional, and phylogenetic levels. Recombinant EgPCS was a monomeric enzyme able to synthesize, in the presence of Zn(2+) or Cd(2+), phytochelatin2-phytochelatin4 (PC2-PC4) using GSH or S-methyl-GS (S-methyl-glutathione), but not γ-glutamylcysteine or PC2 as a substrate. Kinetic analysis of EgPCS firmly established a two-substrate reaction mechanism for PC2 synthesis with Km values of 14-22 mM for GSH and 1.6-2.5 µM for metal-bis-glutathionate (Me-GS2). EgPCS showed the highest Vmax and catalytic efficiency with Zn-(GS)2, and was inactivated by peroxides. The EgPCS N-terminal domain showed high similarity to that of other PCSases, in which the typical catalytic core (Cys-70, His-179 and Asp-197) was identified. In contrast, the C-terminal domain showed no similarity to other PCSases. An EgPCS mutant comprising only the N-terminal 235 amino acid residues was inactive, suggesting that the C-terminal domain is essential for activity/stability. EgPCS transcription in Euglena cells was not modified by Cd(2+), whereas its heterologous expression in ycf-1 yeast cells provided resistance to Cd(2+) stress. Phylogenetic analysis of the N-terminal domain showed that EgPCS is distant from plants and other photosynthetic organisms, suggesting that it evolved independently. Although EgPCS showed typical features of PCSases (constitutive expression; conserved N-terminal domain; kinetic mechanism), it also exhibited distinct characteristics such as preference for Zn-(GS)2 over Cd-(GS)2 as a co-substrate, a monomeric structure, and ability to solely synthesize short-chain PCs, which may be involved in conferring enhanced heavy-metal resistance.

Chromium induced stress conditions in heterotrophic and auxotrophic strains of Euglena gracilis.

Oxidative stress parameter and antioxidant defense compound as well as enzyme activity were studied in relation to different Cr(VI) concentrations (0, 10, 20, 40 µM) in two strains of Euglena gracilis, one isolated from a polluted river (MAT) and the other acquired from a culture collection (UTEX). Chromium toxicity was measured in the auxotrophic and obligated heterotrophic variants of the two strains. Chromium uptake was higher in auxotrophic cultures, reflected by their higher cell proliferation inhibition and lower IC50 levels compared to heterotrophic ones. In the Cr(VI) treatments a reduction of chlorophyll a and b ratio (Chl a/Chl b) was observed, the ratio of protein to paramylon content was augmented, and total lipid content increased, having the auxotrophic strains the highest values. TBARS content increased significantly only at 40 µM Cr(VI) treatment. Unsaturated fatty acids also increased in the Cr(VI) treatments, with the higher storage lipid (saturated acids) content in the heterotrophic cells. The antioxidant response, such as SOD activity and GSH content, increased with chromium concentration, showing the highest GSH values in the heterotrophic cultures and the SOD enzyme participation in chromium toxicity. The MAT strain had higher IC50 values, higher carbohydrate and saturated acid content, and better response of the antioxidant system than the UTEX one. This strain isolated from the polluted place also showed higher GSH content and SOD activity in control cells and in almost all treated cultures. SOD activity reached a 9-fold increase in both MAT strains. These results suggest that tolerance of MAT strain against Cr(VI) stress is not only related to GSH level and/or biosynthesis capacity but is also related to the participation of the SOD antioxidant enzyme.