自养和兼养条件下蛋白核小球藻昼夜节律的响应

【目的】研究自养和兼养两种培养方式对蛋白核小球藻(Chlorella pyrenoidosa)生长、细胞分裂和生化组分积累的影响,探讨人工培养蛋白核小球藻的昼夜节律响应机制和优化技术。【方法】小球藻自养培养采用BG11培养基,兼养培养基在BG11培养基中添加4种不同浓度(1、5、10、20 g/L)的葡萄糖,培养周期为10 d。血球板计数法测定藻细胞浓度,干重法测定藻细胞生物量。显微观察藻细胞大小和分裂情况。脂染色法测定小球藻总脂的含量,藻细胞的叶绿素、蛋白和淀粉分别采用甲醇、氢氧化钠、硝酸钙浸提后通过紫外分光光度法定量测定。【结果】葡萄糖兼养培养对蛋白核小球藻具有显著的促生长效应,最适浓度为10 g/L。10 d收获时,兼养组(10 g/L葡萄糖)藻细胞浓度和干重分别是自养组的2.57倍和6.73倍。分析一昼夜中的藻细胞增殖规律可知,第2天和第5天时自养组中增殖的新生子细胞约有76.00%在黑暗期分裂产生,而兼养组中第2天和第5天光照期的新细胞增殖量占比分别达到40.90%和67.50%。一昼夜内藻细胞大小的迁移动态监测表明,第2天自养组藻细胞的体积变化静息期为8 h,兼养组只有4 h;第5天两组藻细胞大小迁移动态的昼夜节律明显,但兼养组黑暗结束后较大细胞(D6μm)占比显著高于自养组。第8天时,兼养组藻细胞已处于稳定期,总脂和蛋白含量均显著高于自养组,藻细胞总脂和色素含量在一昼夜中相对稳定,但蛋白和淀粉含量分别在光照8 h和12 h左右达到峰值。从第2天开始,对兼养组细胞每天进行2 h光延长,收获时藻细胞浓度和干重分别比对照组提高13%和11%。【结论】葡萄糖兼养培养能大幅提高蛋白核小球藻的生物量。蛋白核小球藻生长增殖与生化组分积累均受昼夜节律调控,自养条件下藻细胞以光照期生长黑暗期增殖为主。兼养培养提高藻细胞生物量的机制在于缩短藻细胞生长静息期,在昼夜节律中加速藻细胞生长并显著提高通过细胞周期检查点的细胞比例,光照期效应尤其明显。藻细胞蛋白和淀粉含量昼夜节律明显,最佳收获时间分别在光照8 h和12 h后。

Circadian rhythm response of Chlorella pyrenoidosa under autotrophic and mixotrophic cultivation

Objective] To study the effect of autotrophic and mixotrophic cultivation on the growth, division and biochemical components accumulation of Chlorella pyrenoidosa, then to explore the response mechanism on circadian rhythm under artificial cultivation and optimize the corresponding technology. Methods] BG11 medium was used for autotrophic cultivation of Chlorella pyrenoidosa and BG11 medium supplemented with glucose of different concentrations (1, 5, 10, 20 g/L) for mixotrophic cultivation. The algae cell number was determined by a hemocytometer, biomass was measured by dry weight, and the size and division of algae cells were observed by a microscope. We used staining method for quantification the total lipids of Chlorella pyrenoidosa. Chlorophyll, protein and starch contents were determined by ultraviolet spectrophotometry. Results] Glucose had a significant growth promoting effect on Chlorella pyrenoidosa, and the optimum concentration was 10 g/L. Under a circadian rhythm, about 76.00% of new daughter cells in the autotrophic cultivation group were proliferated under dark period on the second and fifth day, whereas 67.50% of the new daughter cells were generated under light period in the mixotrophic group. The silent period of the algae in the autotrophic group was 8 hours, but that in the mixotrophic condition was only 4 hours on the second day. On the eighth day, the contents of total lipids and chlorophyll of algae cells kept relatively stable under a light-dark period, the protein and starch content of that reached their respective peak after 8 and 12 hours in light. Conclusion] Mixotrophic cultivation with glucose can increase the biomass of Chlorella pyrenoidosa notably. The growth, proliferation and the biochemical components accumulation of Chlorella pyrenoidosa were regulated by circadian rhythm, the algae in autotrophic culture mainly grow during day in light and undergo cell proliferation in dark. The machanism of mixotrophic cultivation increasing the biomass was shortening the growth silence period of algae cells, and increasing the proportion of larger cells for passing through commitment point of cell-cycle, it was especially obvious during light period. The protein and starch contents of Chlorella pyrenoidosa showed obvious circadian rhythm, their best harvest time were 8 and 12 hours after in light, respectively.