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Isolation and Characterization of Euglena gracilis-Associated Bacteria, Enterobacter sp. CA3 and Emticicia sp. CN5, Capable of Promoting the Growth and Paramylon Production of E. gracilis under Mixotrophic Cultivation.

ABSTRACT: Euglena gracilis produces paramylon, which is a feedstock for high-value functional foods and nutritional supplements. The enhancement of paramylon productivity is a critical challenge. Microalgae growth-promoting bacteria (MGPB) can improve microalgal productivity; however, the MGPB for E. gracilis remain unclear. This study isolated bacteria capable of enhancing E. gracilis growth and paramylon production under mixotrophic conditions. Enterobacter sp. CA3 and Emticicia sp. CN5 were isolated from E. gracilis grown with sewage-effluent bacteria under mixotrophic conditions at pH 4.5 or 7.5, respectively. In a 7-day E. gracilis mixotrophic culture with glucose, CA3 increased E. gracilis biomass and paramylon production 1.8-fold and 3.5-fold, respectively (at pH 4.5), or 1.9-fold and 3.5-fold, respectively (at pH 7.5). CN5 increased E. gracilis biomass and paramylon production 2.0-fold and 4.1-fold, respectively (at pH 7.5). However, the strains did not show such effects on E. gracilis under autotrophic conditions without glucose. The results suggest that CA3 and CN5 promoted both E. gracilis growth and paramylon production under mixotrophic conditions with glucose at pH 4.5 and 7.5 (CA3) or pH 7.5 (CN5). This study also provides an isolation method for E. gracilis MGPB that enables the construction of an effective E. gracilis-MGPB-association system for increasing the paramylon yield of E. gracilis.

SUBMITTER: Rubiyatno

PROVIDER: S-EPMC8303684 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMicroalgae 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.ResultsIn 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).ConclusionsThese 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.

Project description: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.

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Isolation and Characterization of Euglena gracilis-Associated Bacteria, Enterobacter sp. CA3 and Emticicia sp. CN5, Capable of Promoting the Growth and Paramylon Production of E. gracilis under Mixotrophic Cultivation.

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