Effects of chlorination on the survival of sewage bacteria in seawater microcosms

Abstract

Chlorination is a commonly used disinfection method in sewage treatment process. However, resistant bacteria may survive chlorination and enter the receiving aquatic environment upon effluent discharge. There has been limited research on the effects of chlorination on bacterial survival in seawater. To address this knowledge gap, microcosm experiments were conducted to simulate the discharge of chlorinated effluents into coastal seawater. The results revealed that bacterial communities in seawater-based effluents survived better in seawater than those in freshwater-based effluents. High chlorine dosages could significantly reduce the viable bacterial populations and their chance of regrowth in seawater. Additionally, faecal indicator bacteria (FIB) that entered the viable but non-culturable (VBNC) state under chlorination tended to persist in the VBNC state without resuscitation during seawater incubation. Because of the prevalence of VBNC indicator bacteria, qPCR quantification of FIB was more effective than conventional culture-based methods in tracing viable pathogenic chlorine-resistant bacteria, although the correlation strength varied depending on the type of effluent. This study sheds light on how chlorine dosages and the intrinsic properties of effluents affect bacterial survival in seawater and highlights the potential and limitations of using FIB in monitoring the health risks associated with the discharge of chlorinated effluents.

FIGURE 1

Total, viable and culturable concentrations of Escherichia coli and Enterococcus in the effluents of two sewage treatment works under different chlorine dosages. The total and viable 23S rRNA gene copy numbers detected in qPCR assays were converted to numbers of cell equivalent (log₁₀ cell equivalent 100 mL⁻¹) by dividing the median gene copies per cell, for comparison with the culturable plate count (log₁₀ CFU 100 mL⁻¹). Data are shown as mean ± 1 S.D. log₁₀ copies per 100 mL of effluent derived from three replicates. Pre and post indicate samples before and after chlorination, respectively. T0 indicates samples upon dilution with seawater, T1–T3 indicate samples taken from the microcosms at 24, 48 and 72 h. Shaded area illustrates the difference between total, viable and culturable proportions.

FIGURE 2

Constrained correspondence analysis (CCA) plot showing the correlations between bacterial community structures and physicochemical parameters. The Chi‐squared distance matrices of sequence variant compositions are explained by the physicochemical parameters. All measured physicochemical parameters are significant constraining variables (p < 0.05, PERMANOVA). The direction of arrow indicates the increase in amount of the variable, and the length of arrow is relative to the strength of correlation with the variable. The percentage of variation explained by each constrained axis is indicated. Pre and post indicate samples before and after chlorination, respectively. T0 indicates samples upon dilution with seawater, T1–T3 indicate samples taken from the microcosms at 24, 48 and 72 h. HD, high chlorine dosage; LD, low chlorine dosage; N, no chlorine; S, seawater control.

FIGURE 3

Proportions of effluent and seawater communities in the effluent‐seawater mixtures during incubation. SourceTracker analysis was utilised to evaluate the total and viable proportions of effluents communities after disinfection treatments (post) and seawater controls communities (Seawater) in the effluent‐seawater mixtures of SC and ST for each dosage condition. The genus counts in each samples were used for the analysis. Solid lines and symbols indicate total populations, while dotted lines and hollow symbols indicate viable populations. T0 indicates samples upon dilution with seawater, T1–T3 indicate samples taken from the microcosms at 24, 48 and 72 h. Data shown are mean ± 1 S.D. percentages.

FIGURE 4

Viable concentrations of the genera from effluent communities during incubation in the microcosms. Genera originated from the source of disinfected effluent communities were classified by SourceTracker. Those with relative abundance over 0.01% and common in both SC and ST samples were shown in the heatmaps. The concentrations were averaged over viable triplicates. Colour scale represented the Z‐scores of the concentrations in rows. T0 indicates samples upon dilution with seawater, T1–T3 indicate samples taken from the microcosms at 24, 48 and 72 h. HD: high chlorine dosage; LD: low chlorine dosage; N: no chlorine.

FIGURE 5

Alterations of the relative abundances and concentrations of viable chlorine‐resistant bacteria (CRB) under different chlorine dosages and during seawater incubation. Viable chlorine‐resistant genera and species identified in the amplicon sequences of SC and ST samples are shown. Estimated concentrations of the CRB are represented by the size of bubbles. The bubble sizes of the largest value, geometric mean of the smallest and largest value, and the smallest value in the data set are shown in the legend. Relative abundances of the CRB are represented by the colour scale. Pre and post indicate samples before and after chlorination, respectively. T0 indicates samples upon dilution with seawater, T1–T3 indicate samples taken from the microcosms at 24, 48 and 72 h.

FIGURE 6

Spearman's rank correlations between the concentrations of CRB and indicator bacteria. The correlations between the viable concentrations of CRB and the total, viable and culturable concentrations of Escherichia coli (EC) and Enterococcus (ENT) in SC and ST samples are shown. The range of correlation is depicted by the size and colour of bubbles, while the numbers indicate the values of correlation coefficient.