• ISSN 1008-505X
  • CN 11-3996/S
赵艳, 汪成. 低氮胁迫对蛋白核小球藻生化组分和絮凝性能的影响[J]. 植物营养与肥料学报, 2019, 25(3): 489-497. DOI: 10.11674/zwyf.18085
引用本文: 赵艳, 汪成. 低氮胁迫对蛋白核小球藻生化组分和絮凝性能的影响[J]. 植物营养与肥料学报, 2019, 25(3): 489-497. DOI: 10.11674/zwyf.18085
ZHAO Yan, WANG Cheng. Effects of low nitrogen stress on the biochemical components and flocculation property of Chlorella pyrenoidosa[J]. Journal of Plant Nutrition and Fertilizers, 2019, 25(3): 489-497. DOI: 10.11674/zwyf.18085
Citation: ZHAO Yan, WANG Cheng. Effects of low nitrogen stress on the biochemical components and flocculation property of Chlorella pyrenoidosa[J]. Journal of Plant Nutrition and Fertilizers, 2019, 25(3): 489-497. DOI: 10.11674/zwyf.18085

低氮胁迫对蛋白核小球藻生化组分和絮凝性能的影响

Effects of low nitrogen stress on the biochemical components and flocculation property of Chlorella pyrenoidosa

  • 摘要:
    目的 蛋白核小球藻 (Chlorella pyrenoidosa) 蛋白含量高,富含各种必需氨基酸和多种营养保健因子,2012年被我国增列为新资源食品。本文以蛋白核小球藻 (Chlorella pyrenoidosa) 为材料,研究低氮胁迫对自养和兼养来源的藻细胞生化组分和絮凝性能的影响,为开发应用提供技术支撑。
    方法 分别以BG11基本培养基和BG11添加10 g/L葡萄糖接种培养蛋白核小球藻,获得自养和兼养来源的种子藻细胞。以BG11基本培养基中18 mmol/L硝酸钠为正常供氮对照,设置硝酸钠水平为3、6、9 mmol/L的低氮胁迫培养基。种子藻细胞培养14天后,分别采用干重法、脂染色法测定藻细胞干重生物量和总脂含量;蛋白和淀粉含量的测定采用紫外分光光度法;用三维荧光光谱分析藻细胞胞外聚合物 (extracellular polymeric substances,EPS) 组分和含量。
    结果 1) 自养与兼养来源的蛋白核小球藻在低氮胁迫处理下均能生长,自养来源藻细胞更能迅速感应低氮胁迫的条件变化,转接后第1天即开始快速生长。四个胁迫处理中,以硝酸钠6 mmol/L组藻细胞干重和油脂含量最高。该低氮胁迫培养4天后,所有藻细胞样品生长到达稳定期,此时自养与兼养来源的藻细胞干重分别为2.56 g/L和4.62 g/L,藻细胞油脂含量分别为15.5%和39.3%,与正常对照组 (硝酸钠 18 mmol/L) 相比均显著增加。2) 低氮胁迫处理能提高蛋白核小球藻的油脂产率。其中6 mmol/L胁迫处理4天后,兼养藻细胞油脂产率最高,达到129.56 mg/(L·d),同比是自养来源藻细胞的7.95倍。3) 兼养组藻细胞胞内外蛋白、淀粉 (多糖) 初始含量显著高于自养组。藻细胞油脂、蛋白、淀粉含量在低氮胁迫处理培养2天或3天内均显著下降,之后低氮胁迫组藻细胞胞内外蛋白含量持续下降,细胞油脂与胞内淀粉含量开始回升,在第4天出现明显的拐点。4) 6 mmol/L低氮胁迫处理4天后,自养来源藻细胞中,对藻细胞絮凝有促进作用的蛋白类色氨酸物质含量比对照组增加40.3%,兼养来源藻细胞蛋白类色氨酸物质和对藻细胞絮凝有抑制作用的胡敏酸和富里酸类腐殖酸物质的含量分别为对照组的83.6%、74.8%和54.8%。兼养和自养组藻细胞自絮凝率分别为78.5%和80.3%,均比对照组显著提高。
    结论 自养与兼养来源的蛋白核小球藻对低氮胁迫处理的响应存在差异,藻细胞生化组分含量变化受培养基供氮水平和培养时间的影响。低氮胁迫通过影响藻细胞促进和抑制藻细胞自絮凝的生化组分的比例,显著提高其收获期的自絮凝率。针对供试藻种,以硝酸钠6 mmol/L低氮胁迫处理4天对蛋白核小球藻的油脂产率和絮凝性能的提升效果最佳。

     

    Abstract:
    Objectives Chlorella pyrenoidosa are high in protein contents and rich in all kinds of essential amino acids and various nutritional health care factors, which have been listed as a new resource of food in China in 2012. The paper studied the specific nitrogen stress levels and days for culture of Chlorella pyrenoidosa.
    Methods The Chlorella pyrenoidosa was inoculated to the basic BG11 medium for autotrophic cultivation and BG11 medium supplemented with 10 g/L glucose for mixotrophic cultivation, then obtained seed algae cells. The conventional nitrogen supply of 18 mmol/L sodium nitrite in the BG11 basic medium was used as control, mediums with 3, 6 and 9 mmol/L sodium nitrite were prepared as low nitrogen stress treatments. The algae cell biomass was measured by dry weight and the total lipid was quantified by staining method. The protein and starch contents of algae cells were determined by ultraviolet spectrophotometry, and the three-dimensional fluorescence spectra was used to analyze the components and contents of algae extracellular polymeric substances (EPS).
    Results 1) The Chlorella pyrenoidosa cells from both autotrophic and mixotrophic cultivation could grow under low nitrogen stress conditions, algae cells from autotrophic cultivation responded more rapidly to the change of low nitrogen stress and started to grow rapidly on the first day after transfer. The highest cell dry weight and lipid content of the algae were harvested in the NaNO3 6 mmol/L treatment groups. After culturing for 4 days, the algae growth reached stable phase, the cells dry weight from autotrophic and mixotrophic cultivation sources was 2.56 and 4.62 g/L, respectively, and the corresponding lipid contents reached to 15.5% and 39.3%, which were all notably higher than their normal N controls. 2) Low N stress could improve the lipid productivity of Chlorella pyrenoidosa. Both the highest lipid productivities of algae cells derived from mixotrophic cultivation were in NaNO3 6 mmol/L treatment, that for the mixotrophic algae cells was 129.56 mg/(L·d) on the 4th day, which was 7.95 times of that for the autotrophic algae cells. 3) The initial contents of intra- and extra-cellular protein and starch (polysaccharide) in cells from mixotrophic group were notably higher than those from autotrophic group. The lipid, protein, starch contents were decreased significantly in the 2nd or 3rd day of culture, then the intra- and extra-cellular protein contents continued to decline, while the lipid and intracellular starch contents began to increase with an apparent inflection points. The change trends of the lipid and intracellular starch content in algal cells were basically synchronous. 4) The EPS components in the Chlorella pyrenoidosa contained the tryptophan-like proteins (TLP) that advantage to algae cells flocculating and some humic substances including humilic acid (HA) and fulvic acid (FA) that disadvantage to flocculation. After culturing for 4 days in NaNO3 6 mmol/L medium, the content of TLP in algea cells from autotrophic cultivation was increased by 40.3% than the control, while the content of TLP, HA and FA in algae cells from mixotrophic cultivation were 83.6%, 74.8% and 54.8% of that in control groups, respectively. The corresponding self-flocculation rate of algae cells from autotrophic and mixotrophic cultivation source were 78.5% and 80.3% respectively, which were notably improved than their corresponding controls.
    Conclusions The response to low-nitrogen stress varied between the autotrophic and mixotrophic Chlorella pyrenoidosa cells. The biochemical component contents of algal cells are influenced by the culture medium nitrogen supply levels and culturing days. The low nitrogen stress influences the self-flocculation rate of algae cells remarkably by changing the EPS compositions of Chlorella pyrenoidosa. For the tested Chlorella pyrenoidosa, the suitable nitrogen stress level is NaNO3 6 mmol/L and the cultural time is 4 days for the highest lipid productivity and flocculation performance.

     

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