School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, 599489, Singapore, Singapore.
College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, PR China.
PLoS One. 2018 Jun 14;13(6):e0199193. doi: 10.1371/journal.pone.0199193. eCollection 2018.
One of the newly developed methods for Assimilable organic carbon (AOC) determination is leveraged on the cell enumeration by flow cytometry (FC) which could provide a rapid and automated solution for AOC measurement. However, cell samples staining with fluorescence dye is indispensable to reduce background and machine noise. This step would bring additional cost and time consuming for this method. In this study, a green fluorescence protein (GFP) tagged strain derived of AOC testing strain Pseudomonas fluorescens P-17 (GFP-P17) was generated using Tn5 transposon mutagenesis. Continuous culture of this mutant GFP-P17 showed stable expression of eGFP signal detected by flow cytometry without staining step. In addition, this GFP-P17 strain displayed faster growth rate and had a wider range of carbon substrate utilization patterns as compared with P17 wild-type. With this strain, the capability of a new FC method with no dye staining was explored in standard acetate solution, which suggests linear correlation of counts with acetate carbon concentration. Furthermore, this FC method with GFP-P17 strain is applicable in monitoring GAC/BAC efficiency and condition as similar trends of AOC level in water treatment process were measured by both FC method and conventional spread plating count method. Therefore, this fast and easily applicable GFP-P17 based FC method could serve as a tool for routine microbiological drinking water monitoring.
一种新开发的可同化有机碳 (AOC) 测定方法是利用流式细胞术 (FC) 进行细胞计数，这为 AOC 测量提供了快速和自动化的解决方案。然而，细胞样品用荧光染料染色是必不可少的，以减少背景和机器噪音。这一步骤将为该方法带来额外的成本和时间消耗。在本研究中，使用 Tn5 转座子诱变生成了一种源自 AOC 测试菌株荧光假单胞菌 P-17 (GFP-P17) 的绿色荧光蛋白 (GFP) 标记菌株。该突变体 GFP-P17 的连续培养显示出通过流式细胞术检测到的 GFP 信号的稳定表达，而无需染色步骤。此外，与 P17 野生型相比，该 GFP-P17 菌株表现出更快的生长速度和更广泛的碳底物利用模式。使用该菌株，在标准乙酸盐溶液中探索了一种无需染色的新型 FC 方法的能力，这表明计数与乙酸碳浓度呈线性相关。此外，该基于 GFP-P17 菌株的 FC 方法适用于监测 GAC/BAC 效率和条件，因为在水处理过程中通过 FC 方法和常规平板计数法测量到 AOC 水平的相似趋势。因此，这种快速且易于应用的基于 GFP-P17 的 FC 方法可以作为常规微生物饮用水监测的工具。
