Institute of Bio- and Geosciences/Plant Sciences (IBG-2), Forschungszentrum Jülich , Wilhelm-Johnen-Straße, D-52428 Jülich, Germany.
Institute of Physics, Faculty of Mathematics and Physics, Charles University , Ke Karlovu 5, CZ-12116 Prague 2, Czech Republic.
Anal Chem. 2017 Nov 21;89(22):12006-12013. doi: 10.1021/acs.analchem.7b02393. Epub 2017 Nov 10.
Polyphosphates have occurred in living cells early in evolution and microalgae contain these important polymers in their cells. Progress in research of polyphosphate metabolism of these ecologically as well as biotechnologically important microorganisms is hampered by the lack of rapid quantification methods. Experiments with the green alga Chlorella vulgaris presented here compared polyphosphate extraction in water, methanol-chloroform, and phenol-chloroform followed by polyphosphate purification by binding to silica columns or ethanol precipitation. The phenol-chloroform extraction of C. vulgaris followed by ethanol precipitation of polyphosphate was shown to be superior to the other tested method variants. Recovery test of added polyphosphate standard to algal biomass showed that the method is accurate. Using this biochemical assay as a validated reference, we show that 2-dimensional, confocal Raman microscopy can serve as a linear proxy for polyphosphate in C. vulgaris with R up to 0.956. With this, polyphosphate quantification can be shortened by use of Raman microscopy from days to hours and, additionally, information about intracellular distribution of polyphosphate and heterogeneity among individual cells in algal culture can be obtained. This offers new insights into the dynamics and role of these polymers crucial for phosphorus uptake and storage. This analytical capability is of particular practical importance because algae aid phosphorus sequestration from wastewater and the thus enriched biomass may serve as organic fertilizer. Both these applications have a strong potential in a future sustainable, circular bioeconomy.
多聚磷酸盐在进化早期就存在于活细胞中，微藻在其细胞中含有这些重要的聚合物。由于缺乏快速定量方法，这些在生态学和生物技术上都很重要的微生物的多聚磷酸盐代谢研究进展受到了阻碍。本文进行的绿藻小球藻实验比较了水、甲醇-氯仿和酚-氯仿中的多聚磷酸盐提取方法，然后通过与硅胶柱结合或乙醇沉淀来纯化多聚磷酸盐。结果表明，小球藻的酚-氯仿提取后再用乙醇沉淀多聚磷酸盐的方法优于其他测试方法。添加多聚磷酸盐标准品到藻生物质的回收测试表明该方法是准确的。使用这种生化测定作为经过验证的参考方法，我们表明二维共聚焦拉曼显微镜可以作为小球藻中多聚磷酸盐的线性替代物，相关系数高达 0.956。通过使用拉曼显微镜，多聚磷酸盐的定量可以从几天缩短到几个小时，并且可以获得多聚磷酸盐在藻类培养物中的细胞内分布和单个细胞之间异质性的信息。这为这些对磷吸收和储存至关重要的聚合物的动态和作用提供了新的见解。这种分析能力具有特殊的实际意义，因为藻类有助于从废水中去除磷，而富磷的生物质可以用作有机肥料。这两种应用都在未来可持续的循环生物经济中具有巨大的潜力。
