水稻种子内生泛菌促进小球藻生长和油脂积累

【目的】从水稻种子中分离出优势内生菌，研究其对小球藻生长、生化组分及油脂积累的影响，探索植物内生菌作为促进微藻生长和生化品质改良的新微生物资源的可行性。【方法】以收获自大田的粳稻品种日本晴种子为试验材料，采用传统表面消毒法分离获得内生菌。内生菌的鉴定采用形态观察和16S rDNA序列分析法。以蛋白核小球藻为研究对象，采用共培养法评估内生菌对小球藻生长及生化指标的影响，以纯藻为对照，将内生菌与蛋白核小球藻种子细胞按菌藻比（10﹕1）接种至BG11培养基进行共培养。采用血球板计数法测定藻细胞数目。同时以纯菌培养为对照，采用平板稀释菌落计数法测定菌藻共培养体系中菌浓的变化曲线。小球藻细胞中叶绿素a和类胡萝卜素的提取采用甲醇浸提法，定量测定采用紫外分光光度法。小球藻油脂提取采用氯仿甲醇浸提法，脂肪酸组分分析采用气相色谱-质谱联用法。【结果】分离获得一株在水稻种子中常见且数量较多的内生细菌，编号为REY-1。REY-1为革兰氏阴性短杆状，经16S rDNA鉴定属于泛菌属Pantoea sp.。REY-1与蛋白核小球藻共培养14 d后，菌藻共培养组中藻细胞浓度达到1.15×10⁸ cells/mL，是对照组的1.97倍。菌藻共培养组与纯菌培养对照组中水稻内生泛菌REY-1的浓度均呈现峰谷式变化曲线，但菌藻共培养组中REY-1的菌浓同比显著低于纯菌培养对照组。14 d培养结束时，菌藻共培养组的菌藻比反转为1﹕100，说明REY-1促进蛋白核小球藻的生长，其自身增殖又受到藻细胞的抑制，二者存在显著的互作效应。菌藻共培养组藻细胞叶绿素a和类胡萝卜素总浓度与对照差异不显著，但单细胞叶绿素a和类胡萝卜素含量分别比对照组下降42.58%和42.68%，二者差异均显著。菌藻共培养组藻细胞油脂含量达到细胞干重的29.90%，比对照组增加78.00%，差异显著，油脂产率（2.14 mg·L^-1·d^-1）比对照组增加了1.68倍。2种培养体系中藻细胞脂肪酸主要组分相似，均为棕榈酸（16﹕0）、亚油酸（18﹕2）和亚麻酸（18﹕3）。REY-1共培养处理使藻细胞的单不饱和脂肪酸相对含量比对照增加了32.37%，短链脂肪酸肉豆蔻酸（14﹕0）相对含量比对照增加了2.12倍，差异显著。此外共培养组藻细胞新合成一种长链脂肪酸-芥酸（22﹕1），相对含量达到藻细胞总脂肪酸的1.68%。【结论】水稻种子内生泛菌REY-1能在BG11培养液中长期存活并与蛋白核小球藻产生互作，显著提高小球藻生物量和油脂含量，大幅提高小球藻油脂产率并影响脂肪酸相对组成比例的生物学效应。水稻种子内生菌可以作为开发小球藻共生菌的优良微生物新资源。

Growth and Lipid Accumulation Promotion of Chlorella by Endophytic Pantoea sp. from Rice Seeds

【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.