水稻种子内生泛菌促进小球藻生长和油脂积累

doi:10.3864/j.issn.0578-1752.2016.08.001

Abstract

Abstract: 【Objective】The objective of this study is to isolate the dominant endophytic bacterial strain from rice seeds and identify its effects on the growth, biochemical composition and lipid accumulation of Chlorella, to explore the feasibility of plant endophytes as new microbial resources for growth promotion and biochemical quality improvement of microalgae. 【Method】The endophytic bacteria was isolated from rice seeds by the traditional surface sterilization method, using rice (Oryza sativa L. japonica. cv. Nipponbare) seeds which were harvested from the paddy fieldas experimental materials. Identification of the endophytic bacteria was based on morphological observation and 16S rDNA sequence analysis. Using Chlorella pyrenoidosa as the experimental subject, effects of the endophytic bacteria on the growth and biochemical composition of Chlorella were evaluated by co-cultivation method. The algal cells were co-inoculated with endophyte at the number ratio of 1:10 in BG11 medium with axenic C. pyrenoidosa as control. The algal cell number was determined by monitoring the cell count using hemocytometer. Meanwhile, the change curve of the concentration of bacteria in bacterial-algae co-cultivation was determined by the colony form counting method with flat plate dilution for single bacterial culture as the control. The algal chlorophyll a and carotenoids were extracted with methanol and the contents were determined by UV spectrophotometry. The algal total lipids were extracted with chloroform/methanol and the fatty acid composition was analyzed by gas chromatography/mass spectrometry. 【Result】A endophytic bacterial strain was isolated and named as REY-1, which is very commonly distributed in rice seeds with larger number. The REY-1 is Gram-negative, short rod in cell-shaped. The REY-1 strain belongs to Pantoea sp. according to the 16S rDNA identification. After 14 days co-cultivation of the REY-1 and C. pyrenoidosa, the algal cell concentration in bacterial-algal co-cultivation reached 1.15×10⁸ cells/mL that was 1.97 times as many as the control. The concentration of REY-1 in bacterial-algal co-cultivation and single bacterial culture both had peak valley changes curve, but the concentration of bacteria in bacterial-algal co-cultivation was significantly lower than that of single bacterial culture at the same period. The ratio of bacterial concentration to algal concentration decreased from 10:1 to 1:100 at the end of incubation. The result showed that REY-1 can promote the growth of C. pyrenoidosa, but its proliferation was inhibited by algae at the same time, and there was a significant interaction effect between the two. The total concentration of chlorophyll a and carotenoids in the co-cultivation system had no significant difference with the control, whereas the decrease of chlorophyll a and carotenoid contents in single algae cell was significant at 42.58% and 42.68% compared with the control, respectively. The total lipid content of algae in co-cultivation reached 29.90% by a significantly increase of 78.00% compared with the control, and the lipid productivity (2.14 mg·L^-1·d^-1) was 1.68 times higher than the control. Palmitic acid (16:0), linoleic acid (18:2) and linolenic acid (18:3) are major fatty acids in algae cells in both cultivations. Co-cultivation treatment with REY-1 increased the relative content of monounsaturated fatty acids and short chain myristic acid (14:0) in algae cells by 32.37% and 2.12 times, respectively, compared with the control, which reached to significant or very significant levels. In addition, a new long chain fatty acid, erucic acid was synthesized in co-cultivation algae cells, with relative content reached 1.68% of that of the algae total fatty acid. 【Conclusion】The endophytic Pantoea sp. REY-1 isolated from rice seeds can survive in long-term in BG11 liquid medium and interact with C. pyrenoidosa. It can significantly lead to a cumulative increase in biomass and lipid content of C. pyrenoidosa, and change its relative composition content of fatty acids. It can also promote the biosynthesis of short and long chain fatty acids. Rice seeds endophytic bacteria can be used as a good new microbial resource to develop beneficial symbiotic bacteria for Chlorella sp.

Key words: rice seed, endophytic Pantoea sp., Chlorella pyrenoidosa, co-cultivation method, lipid, fatty acid composition

SHI Yu-qian, ZHAO Yan. Growth and Lipid Accumulation Promotion of Chlorella by Endophytic Pantoea sp. from Rice Seeds[J].Scientia Agricultura Sinica, 2016, 49(8): 1429-1442.

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