Stability of beta-D-glucuronidase (GUS) in sewage wastewater treatment processes and enumeration of the number of Escherichia coli by measuring GUS activity as a surrogate of E. coli

The conventional determination methods of Escherichia coli in sewage wastewater are complicated and time-consuming. Herein, we propose a novel simple and rapid enumeration method of E. coli in wastewater based on beta-D-glucuronidase (GUS) activity with fluorogenic enzyme substrate using a microplate reader. Determination of the copy numbers of uidA gene encoding GUS and its mRNA showed insignificant growth of E. coli and production of GUS during the initial phase of GUS activity assay, indicating presence of intrinsic GUS activity of the wastewater. The GUS activity was not affected by background matrix of the wastewater. The number of E. coli in primary and secondary clarifiers could be estimated by determining the GUS activity of the wastewater as a surrogate of E. coli within 2 h without any pre- and post-treatment. Seasonal variation of E. coli in secondary clarifiers of two wastewater treatment plants could be also estimated over ten months. Specific GUS activities of E. coli were different among three wastewater treatment plants, which was associated with difference in community of E. coli species. The simple, rapid and low cost (0.02 USD per sample) method enables us to simultaneously (up to 96 samples) enumerate the number of E. coli in wastewater.