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

Relying on the Beijing-Shijiazhuang Expressway widening project near the impervious wall of a reservoir, this paper uses FLAC3D two-dimensional and three-dimensional numerical simulation methods to establish the whole process model of the impervious wall of the reservoir affected by the construction load of the high-way reconstruction section. The stress and strain state of the cut-off wall in the high-way reconstruction section and the nearby reservoir is simulated in detail, the overall deformation of the cut-off wall in the reservoir is directly reflected, and the interaction and differential deformation between the wall structures are reflected. The safety and stability of the cutoff wall of the reservoir affected by the construction load are evaluated so that various advanced mechanical behaviors of the cutoff wall can be predicted. Research results show that the horizontal displacement value of the wall gradually increases from bottom to top, and the maximum value appears at the top of the wall. The horizontal displacement value of the 1–3 walls is relatively large, with the maximum value of 22.368 mm, and the horizontal displacement value of the 4–10 walls shows little difference. This is on account of the gravity of the backfill, the strata in the whole project area having settled, and the settlement at the bottom of the cut-off wall being 2.542 mm. At the root of the rigid cut-off wall, the compressive stress concentration occurs, with the maximum value between 1.75 MPa and 2.15 MPa. Due to the size of the structure, the maximum tensile stress of 0.237 MPa appears in the local area near the guide wall of the rigid cut-off wall, which will not endanger the rigid cut-off wall because of its small value. The maximum stress in the rigid impervious wall and the plastic impervious wall are 1.90–2.15 MPa and 1.00–1.12 MPa, respectively. Apart from the small tensile stress at the connecting guide wall between the rigid cut-off wall and the plastic concrete cut-off wall, the cut-off wall is under pressure, especially the plastic cut-off wall. Combined with the analysis of the stress state of the wall, it can be determined that the anti-seepage wall (rigid cut-off wall and plastic concrete cut-off wall) is stable and safe during the construction period. [ABSTRACT FROM AUTHOR]

Bentonite, a supplementary cementitious material for Portland cement, has greatly contributed to environmental sustainability. However, few studies have investigated mortar samples produced by substituting bentonite for cement, and cement strength may be adversely affected when cement is replaced with bentonite in larger proportions. Therefore, this paper investigates and discusses the effect of microbially induced calcium carbonate precipitation (MICP) on improving the strength of bentonite-amended cement. The bio-mineralization process of MICP was characterized by SEM-EDS, while the biominerals formed in bentonite-amended mortar were identified by FIIR and XRD analysis. The results showed that: at bentonite concentrations of 0%, 10%, 20%, 30%, and 40% in cement, the bacterial suspension and reaction solution enhanced the compressive strength of bentonite-amended cement by 17%, 20%, 79%, 78%, and 38%, respectively, after 28 days, compared to control specimens; With the increased bacterial concentration in the presence of the reaction solution, the strength of the bentonite-amended cement (20% bentonite) increased remarkably compared to the control specimen (without bacteria). When the bacterial concentration was OD600 2.0, the compressive strength of bentonite-amended cement (20% bentonite) increased by 80% after 28 days; MICP process has a great effect on improving the strength of bentonite-amended cement. It is a green and economical choice to use MICP to improve the strength of bentonite-amended cement. [ABSTRACT FROM AUTHOR]

GLO:F16/01nov22:pbi13915-fig-0001.jpg PHOTO (COLOR): 1 CRISPR genome editing to target the EgGSL2 gene by electroporation and the crtP1 gene using microinjection to deliver Cas9-RNPs into E. gracilis. Keywords: Euglena gracilis; CRISPR; Gene editing; Electroporation; Microinjection EN Euglena gracilis CRISPR Gene editing Electroporation Microinjection 2048 2050 3 11/01/22 20221101 NES 221101 The genus I Euglena i contains more than 1000 species of single-celled flagellated microorganisms with both plant and animal characteristics. High-throughput sequencing revealed low-efficacy genome editing using Cas9 RNPs electroporation and single-celled microinjection provided an alternative to deliver CRISPR reagents into Euglena gracilis. [Extracted from the article]

Background: Microalgae can contribute to more than 40% of global primary biomass production and are suitable candidates for various biotechnology applications such as food, feed products, drugs, fuels, and wastewater treatment. However, the primary limitation for large-scale algae production is the fact that algae requires large amounts of fresh water for cultivation. To address this issue, scientists around the world are working on ways to reuse the water to grow microalgae so that it can be grown in successive cycles without the need for fresh water. Results: In this study, we present the results when we cultivate microalgae with cultivation water that is purified and reused. Specifically, we purify the cultivation water using an ultrafiltration membrane (UFM) treatment and investigate how this treatment affects: the biomass and biochemical components of the microalgae; characteristics of microalgae growth inhibitors; the mechanism whereby potential growth inhibitors are secreted (followed using metabolomics analysis); the effect of activated carbon (AC) treatment and advanced oxidation processes (AOPs) on the removal of growth inhibitors of Euglena gracilis. Firstly, the results show that E. gracilis can be only cultivated through two growth cycles with water that has been filtered and reused, and the growth of E. gracilis is significantly inhibited when the water is used a third time. Secondly, as the number of reused water cycles increases, the Cl− concentration gradually increases in the cultivation water. When the Cl− concentration accumulates to a level of fivefold higher than that of the control, growth of E. gracilis is inhibited as the osmolality tolerance range is exceeded. Interestingly, the osmolality of the reused water can be reduced by replacing NH4Cl with urea as the source of nitrogen in the cultivation water. Thirdly, E. gracilis secretes humic acid (HA)—which is produced by the metabolic pathways for valine, leucine, and isoleucine biosynthesis and by linoleic acid metabolism—into the cultivation water. Because HA contains large fluorescent functional groups, specifically extended π(pi)-systems containing C=C and C=O groups and aromatic rings, we were able to observe a positive correlation between HA concentration and the rate of inhibition of E. gracilis growth using fluorescence spectroscopy. Moreover, photosynthetic efficiency is adversely interfered by HA, thereby reductions in the synthetic efficiency of paramylon and lipid in E. gracilis. In this way, we are able to confirm that HA is the main growth inhibitor of E. gracilis. Finally, we verify that all the HA is removed or converted into nutrients efficiently by AC or UV/H2O2/O3 treatments, respectively. As a result of these treatments, growth of E. gracilis is restored (AC treatment) and the amount of biomass is promoted (UV/H2O2/O3 treatment). Conclusions: These studies have important practical and theoretical significance for the cyclic cultivation of E. gracilis and for saving water resources. Our work may also provide a useful reference for other microalgae cultivation. [ABSTRACT FROM AUTHOR]

Euglena gracilis is a unicellular green eukaryotic microalga that features characteristics of both plants and animals. The photosynthetic function of its chloroplast is easily lost under stress resulting in bleached mutants, while the physiological role of their residual plastid DNAs remains unclear. In this study, we obtained five bleached mutants by ofloxacin (Ofl) treatment, identified 12 residual plastid genes in five bleached mutants, and determined the mRNA levels in the wild type E. gracilis (WT) and one bleached mutant (OflB2) under dark and light stimulation conditions by quantitative reverse transcribed PCR (qRT-PCR). Results show that the expression of all selected plastid genes in both WT and OflB2 mutant did not change significantly in darkness, while their responses to light stimulation were different. Under the light stimulation conditions, half of the genes did not change significantly, while most of the other genes were down-regulated in OflB2 mutant and up-regulated in WT. Therefore, the bleached mutant retains part of the plastid genome and the plastid relic is responsive to light. Our research will help to understand the functions of residual plastid DNA and evolution of chloroplasts. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Microalgae are widely be used in carbon sequestration, food supplements, natural pigments, polyunsaturated fatty acids, biofuel applications, and wastewater treatment. However, the difficulties incurred in algae cell separation and harvesting, and the exorbitant cost required to overcome these challenges, are the primary limitations to large-scale industrial application of microalgae technology. Results: Herein, we explore the potential of inducing flocculation by adjusting the pH for pre-concentrating Euglena gracilis. Our results demonstrate that flocculation can be induced by increasing the medium pH to 8.5; however, most of the algae cells were broken by increasing the pH > 10. Magnesium phosphate, calcium phosphate, and their derivatives precipitation jointly led to flocculation, although calcium phosphate and its derivatives precipitation had a greater effect. Conclusions: This study demonstrates that pH treatment-induced flocculation is efficient and feasible for the pre-concentration of E. gracilis under a pilot-scale culture system. Moreover, it also maintained the microalgae cells' integrity, chlorophyll production, and increased paramylon production. These findings provide a theoretical basis for reducing the cost of large-scale E. gracilis harvesting; as well as provide a reference for harvesting other microalgae. [ABSTRACT FROM AUTHOR]

Purpose: Under different abiotic-stress conditions, the unicellular green microalga Dunaliella salina accumulates large amounts of carotenoids which are accompanied by fatty acid biosynthesis. Carotenoids and fatty acids both possess long carbon backbones; however, the relationship between carotenoid and fatty acid metabolism is controversial and remains poorly understood in microalgae. Methods: In this study, we investigated the growth curves and the β-carotene, lutein, lipid, and fatty acid contents of D. salina Y6 grown under different abiotic-stress conditions, including high light, nitrogen depletion, and high salinity. Results: Both high-salinity and nitrogen-depleted conditions significantly inhibited cell growth. Nitrogen depletion significantly induced β-carotene accumulation, whereas lutein production was promoted by high light. The accumulation of lipids did not directly positive correlate with β-carotene and lutein accumulation under the three tested abiotic-stress conditions, and levels of only a few fatty acids were increased under specific conditions. Conclusion: Our data indicate that cellular β-carotene accumulation in D. salina Y6 positive correlates with accumulation of specific fatty acids (C16:0, C18:3n3, C14:0, and C15:0) rather than with total fatty acid content under different abiotic stress conditions. [ABSTRACT FROM AUTHOR]

Euglena, a new superfood on the market, is a nutrient-rich, green single-celled microorganism that features the characteristics of both plant and animal. When cultivated under different conditions, Euglena produces different bioactive nutrients. Interestingly, Euglena is the only known microorganism whose chloroplasts are easy to lose under stress and become permanently bleached. We applied gas chromatography-mass spectrometry (GC-MS) to determine the metabolomes of wild-type (WT) Euglena gracilis and its bleached mutant OflB2 under light stimulation. We found a significant metabolic difference between WT and OflB2 cells in response to light. An increase of membrane components (phospholipids and acylamides) was observed in WT, while a decrease of some stimulant metabolites was detected in OflB2. These metabolomic changes after light stimulation are of great significance to the development of Euglena chloroplasts and their communications with the nucleus. [ABSTRACT FROM AUTHOR]

Epigenetic modifications such as DNA methylation are well known as connected with many important biological processes. Rapid accumulating evidence shows environmental stress can generate particular defense epigenetic changes across generations in eukaryotes. This transgenerational epigenetic inheritance in animals and plants has gained interest over the last years. Cyanobacteria play very crucial role in the earth, and as the primary producer they can adapt to nearly all diverse environments. However, few knowledge about the genome wide epigenetic information such as methylome information in cyanobacteria, especially under any environment stress, was reported so far. In this study we profiled the genome-wide cytosine methylation from a model cyanobacterium Synechocystis sp. PCC 6803, and explored the possibility of transgenerational epigenetic process in this ancient single-celled prokaryote by comparing the DNA methylomes among normal nitrogen medium cultivation, nitrogen starvation for 72 h and nitrogen recovery for about 12 generations. Our results shows that DNA methylation patterns in nitrogen starvation and nitrogen recovery are much more similar with each other, significantly different from that of the normal nitrogen. This study reveals the difference in global DNA methylation pattern of cyanobacteria between normal and nutrient stress conditions and reports the evidence of transgenerational epigenetic process in cyanobacteria. The results of this study may contribute to a better understanding of epigenetic regulation in prokaryotic adaptation to and survive in the ever changing environment. [ABSTRACT FROM AUTHOR]

Toxic effects of copper on aquatic organisms in polluted water bodies have garnered particular attention in recent years. Microalgae play an important role in aquatic ecosystems, and they are sensitive to heavy metal pollution. Thus, it is important to clarify the mechanism of copper toxicity first for ecotoxicology studies. In this study, the physiological, biochemical and gene expression characteristics of a model green microalga, Chlamydomonas reinhardtii, with 0, 50, 150 and 250 μM copper treatments were investigated. The response of C. reinhardtii to copper stress was significantly shown at a dose dependent manner. Inhibition of cell growth and variation of total chlorophyll content were observed with copper treatments. The maximum photochemical efficiency of PSII, actual photochemical efficiency of PSII and photochemical quenching value decreased in the 250 μM copper treatment with minimum values equal to 28, 24 and 60 % of the control values respectively. The content of lipid peroxidation biomarker malondialdehyde with copper treatments increased with a maximum value sevenfold higher than the control value. Inhibition of cell growth and photosynthesis was ascribed to peroxidation of membrane lipids. The glutathione content and activities of antioxidant enzymes, glutathione S-transferase, glutathione peroxidase, superoxide dismutase and peroxidase were induced by copper. Interestingly, the expression of antioxidant genes and the photosynthetic gene decreased in most copper treatments. In conclusion, oxidative stress caused by production of excess reactive oxidative species might be the major mechanism of copper toxicity on C. reinhardtii. [ABSTRACT FROM AUTHOR]

Macroalgae has bloomed in the brackish lake of Shenzhen Bay, China continuously from 2010 to 2014. Gracilaria tenuistipitata was identified as the causative macroalgal species. The aim of this study was to explore the outbreak mechanism of G. tenuistipitata, by studying the effects of salinity and nitrogen sources on growth, and the different nitrogen sources uptake characteristic. Our experimental design was based on environmental conditions observed in the bloom areas, and these main factors were simulated in the laboratory. Results showed that salinity 12 to 20 ‰ was suitable for G. tenuistipitata growth. When the nitrogen sources' (NH4+, NO3−) concentrations reached 40 µM or above, the growth rate of G. tenuistipitata was significantly higher. Algal biomass was higher (approximately 1.4 times) when cultured with NH4+ than that with NO3− addition. Coincidentally, macroalgal bloom formed during times of moderate salinity (∼12 ‰) and high nitrogen conditions. The NH4+ and NO3− uptake characteristic was studied to understand the potential mechanism of G. tenuistipitata bloom. NH4+ uptake was best described by a linear, rate-unsaturated response, with the slope decreasing with time intervals. In contrast, NO3− uptake followed a rate-saturating mechanism best described by the Michaelis-Menten model, with kinetic parameters Vmax = 37.2 µM g−1 DM h−1 and Ks = 61.5 µM. Further, based on the isotope 15N tracer method, we found that 15N from NH4+ accumulated faster and reached an atom% twice than that of 15N from NO3−, suggesting when both NH4+ and NO3− were available, NH4+ was assimilated more rapidly. The results of the present study indicate that in the estuarine environment, the combination of moderate salinity with high ammonium may stimulate bloom formation. [ABSTRACT FROM AUTHOR]

We used standard periphyton samplers to examine the colonization pattern of periphytic algae on artificial substrates (glass slides) in Lam Tsuen River, Hong Kong, in dry (winter) and wet (summer) seasons. In each season, six replicated slides were retrieved randomly and replaced by new slides at weekly intervals over a period of 6 weeks. We thus obtained two batches of slides, both with a series of different exposure times (1 to 6 weeks): one batch was set up at the same time (start of the sampling) and the other was harvested at the same time (end of the sampling). Changes in taxonomic composition, species diversity (Shannon-Wiener diversity index), standing crop (in terms of cell density and cell biovolume), and abundance of the abundant algal species were monitored and compared between the two batches of slides. The succession patterns of the periphytic algae were similar between the two batches in each season, while more remarkable differences were observed between the two seasons, suggesting that either batch would be suitable for a colonization study of periphytic algae. The cell density was dominated by diatoms in both seasons, while the cell biovolume was dominated by diatoms in winter and by green algae in summer. The cell density and biovolume attributed to blue green algae was relatively small. Most of the diatom species exhibited similar colonization patterns throughout the experiment, while green algae showed different succession patterns in different seasons or sampling methods, indicating that diatoms are better bio-indicators than green algae for a periphyton colonization study. In general, the diversity indices and the standing crops reached their maximums at around week 4, and they were higher in summer than in winter. [ABSTRACT FROM AUTHOR]

A cell-wall deficient strain of Chlamydomonas reinhardtii P. A Dang. CC-849 was cotransformed with two expression vectors, p105B124 and pH105C124, containing phbB and phbC genes, respectively, from Ralstonia eutropha. The transformants were selected on Tris-acetate-phosphate media containing 10 μg · mL−1 Zeomycin. Upon further screening, the transgenic algae were subcloned and maintained in culture. PCR analysis demonstrated that both phbB and phbC genes were successfully integrated into the algal nuclear genome. Poly-3-hydroxybutyrate (PHB) synthase activity in these transgenic algae ranged from 5.4 nmol · min−1 · mg protein−1 to 126 nmol · min−1 · mg protein−1. The amount of PHB in double transgenic algae was determined by gas chromatography–mass spectrometry (GC–MS) when comparing with PHB standard. In addition, PHB granules were observed in the cytoplasm of transgenic algal cells using TEM, which indicated that PHB was synthesized in transgenic C. reinhardtii. Hence, results clearly showed that producing PHB in C. reinhardtii was feasible. Further studies would focus on enhancing PHB production in the transgenic algae and targeting the chloroplast for PHB accumulation. [ABSTRACT FROM AUTHOR]

The cDNA of the metallothionein-like (MT-like) gene from Festuca rubra cv. Merlin was optimized with bias codon of Chlamydomonous reinhardtii chloroplast genome. The optimized MT-like gene was delivered into C. reinhardtii chloroplast and the transgenic strains expressing MT-like gene was obtained. PCR-Southern blot and RT-PCR-Southern blot analysis demonstrated that the MT-like gene was integrated into chloroplast genome of C. reinhardtii and expressed at the transcriptional level. The cadmium binding capacity of the transgenic C. reinhardtii was determined by hydride generation-atomic fluorescence spectrometry (HG-AFS) and the binding properties were analyzed. Results showed that the transgenic C. reinhardtii expressing the MT-like gene exhibited remarkably higher Cd2+ binding capacity and grew to higher densities at toxic Cd2+ concentrations (40–100 μmol/L) than the wild type strain, and that the IC50 of Cd2+ (3-d treating) to algal cell growth of transgenic strain was 55.43% higher than that of the wild type strain, indicating that the Cd2+ binding capacity and Cd2+ tolerance of C. reinhardtii was enhanced through the expression of the foreign MT-like gene in chloroplast. [ABSTRACT FROM AUTHOR]

The expression vector containing phbB and ble genes was constructed and transformed into cell-wall-deficient strain Chlamydomonas reinhardtii CC-849 by the glass-bead method. The transgenic alga was selected and maintained in the TAP agar plates containing 10 μg/mL Zeomycin. Transgenic alga, which could express phbB at the transcriptional level, was obtained and further confirmed with PCR, Southern blot and RT-PCR-DNA hybridization analysis. [ABSTRACT FROM AUTHOR]