Division of Environmental Engineering, Faculty of Engineering, Hokkaido University, North-13, West-8, Sapporo 060-8628, Japan.
Cellspect Co., Ltd., 2-4-23 Kitaiioka, Morioka, Iwate 020-0857, Japan.
Water Res. 2023 Nov 1;246:120689. doi: 10.1016/j.watres.2023.120689. Epub 2023 Oct 2.
As urban rivers are domestic, industrial, and agricultural water resources, fecal pollution poses human health and environmental risks. In this study, we developed a simple and rapid method to detect fecal pollution in urban rivers. Water samples were mixed with liquid medium, including a fluorescent substrate and fluorescence intensity (F.I.) was measured using a microplate reader to determine Escherichia coli (E. coli) β-D-glucuronidase (GUS) activity instead of E. coli concentration. GUS activities measurements in pure E. coli cultures revealed that E. coli incubated with a GUS substrate accumulated GUS enzymes in their cells, whereas those incubated without a GUS substrate did not. The increase in GUS activity corresponded to the proliferation of E. coli and the GUS activity increased linearly even during the lag growth phase of E. coli, indicating the presence of intrinsic GUS (iGUS) in E. coli cells before incubation. iGUS activity persisted at 81 % in the chlorinated samples, even though the E. coli concentration was reduced by a factor of 10. The iGUS activity persisted for approximately three days. Therefore, we assumed that E. coli present in fecal contaminants, in which GUS substrates are present, could be distinguished from those surviving in the natural environment for three days or longer by measuring iGUS activity. River water samples were collected upstream and downstream of the discharge outlets of municipal wastewater treatment plants and a combined sewer outlet. The iGUS activities were <0.24 mMFU/mL for the upstream samples and >0.21 mMFU/mL for the downstream samples. Interestingly, E. coli concentrations were not necessarily associated with fecal pollution. This indicates that by setting a threshold for iGUS activity, our method can be used as a simple and rapid method for detecting fecal pollution in urban rivers. Because the limit of detection for our method is 20 CFU/mL, our method is applicable to detecting high fecal pollution in a small river.
由于城市河流是居民、工业和农业用水的来源，粪便污染会对人类健康和环境造成风险。在本研究中，我们开发了一种简单快速的方法来检测城市河流中的粪便污染。水样与液体培养基混合，其中包括荧光底物，并用微孔板读数器测量荧光强度（FI）来确定大肠杆菌（E. coli）β-D-葡糖醛酸酶（GUS）活性，而不是 E. coli 浓度。在纯大肠杆菌培养物中进行 GUS 活性测量表明，用 GUS 底物孵育的大肠杆菌在其细胞中积累 GUS 酶，而未用 GUS 底物孵育的大肠杆菌则没有。GUS 活性的增加与大肠杆菌的增殖相对应，即使在大肠杆菌的迟滞生长阶段，GUS 活性也呈线性增加，这表明大肠杆菌细胞中存在内在 GUS（iGUS）。即使在 E. coli 浓度降低 10 倍的情况下，氯消毒水样中的 iGUS 活性仍保持在 81%。iGUS 活性持续约三天。因此，我们假设可以通过测量 iGUS 活性来区分存在于粪便污染物中（其中存在 GUS 底物）的大肠杆菌与在自然环境中存活三天或更长时间的大肠杆菌。采集了城市污水处理厂和合流制污水排放口上下游的河水水样。上游样品的 iGUS 活性<0.24 mMFU/mL，下游样品的 iGUS 活性>0.21 mMFU/mL。有趣的是，E. coli 浓度不一定与粪便污染有关。这表明通过设定 iGUS 活性的阈值，我们的方法可以作为检测城市河流中粪便污染的简单快速方法。由于我们的方法的检测限为 20 CFU/mL，因此该方法适用于检测小河中高浓度的粪便污染。
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