The Mechanism of Chlorine Damage Using Enhanced Green Fluorescent Protein-Expressing Escherichia coli

Mizozoe Mizozoe,Aikawa Aikawa,Otaki Otaki

doi:10.3390/w11102156

Abstract

This study investigated how chlorine inactivates and damages Escherichia coli cells. E. coli that had transformed to express enhanced green fluorescent protein (EGFP) at the cytoplasm was treated with chlorine. Damage to the cell membrane and cell wall was analyzed by measuring the fluorescence intensity of the leaked EGFP, then accounting for the fluorescence deterioration. At pH 7, E. coli was lethally damaged after treatment with chlorine, but significant leakage of EGFP was not observed. In contrast, significant leakage of EGFP was observed at pH 9, even though E. coli was not as inactivated as it was at pH 7. Flow cytometry was used to confirm the fluorescence intensity of the remaining EGFP inside the cells. No significant fluorescence loss was observed in the cells at pH 7. However, at pH 9, the fluorescence intensity in the cells decreased, indicating leakage of EGFP. These results suggest that hypochlorous acid inactivates E. coli without damaging its cell membrane and cell wall, whereas the hypochlorite ion inactivates E. coli by damaging its cell membrane and cell wall. It was possible to confirm the chlorine damage mechanism on E. coli by measuring the fluorescence intensity of the leaked EGFP.

Highlights

During water treatment, disinfectants are used to inactivate or sufficiently suppress the proliferation of pathogenic microorganisms
The fluorescence intensity reduction curve, calculated from the first-order decay constant obtained by Equation (5) and shown in Figure 1, allowed the fluorescence intensity retention efficiency to be calculated based on the CT value during leaked enhanced green fluorescent protein (EGFP) quantitation in disinfection experiments
We investigated whether the degree of damage to E. coli by chlorine can be measured by expressing EGFP in the cytoplasm calculating the leakage in response to chlorine treatment

Summary

Introduction

Disinfectants are used to inactivate or sufficiently suppress the proliferation of pathogenic microorganisms. Chlorination, one of the most widely used disinfection methods, employs the powerful oxidizing capacity of chlorine to kill pathogenic microorganisms This technique is relatively inexpensive; a small amount of chlorine can disinfect a large amount of water. The redox potential of sodium hypochlorite, a substance widely used for chlorination, is a straight line with an inflection point at a specific pH corresponding to the pKa of hypochlorous acid (7.537). In this case, hypochlorous acid is the main species at pH ≤ 7.5, and the hypochlorite ion is the main species at pH ≥ 7.5 [1]

Methods

Results

Conclusion