Three-Dimensional Free Chlorine and Monochloramine Biofilm Penetration: Correlating Penetration with Biofilm Activity and Viability

Abstract

Disinfectant biofilm penetration and its effect on biofilm aerobic activity and viability are still unclear. In this study, free chlorine and monochloramine were applied until full biofilm penetration occurred, and their effects on biofilm aerobic activity and viability were investigated in three dimensions throughout the entire biofilm depth, extending previous work where viability analysis was limited to the upper biofilm (50 μm depth), free chlorine penetration did not reach completion, and only one-dimensional (depth) profiles were obtained. The free chlorine and monochloramine biofilm concentration profiles were correlated spatially and temporally with aerobic microbial activity and cell-membrane integrity based viability using a combination of (1) microelectrode measurements for disinfectant penetration and (2) LIVE/DEAD BacLight staining, cryo-cross-sectioning, and confocal micrographs analysis for viability measurements throughout the entire biofilm depth. Compared to monochloramine, free chlorine penetration (1) was slower, (2) led to a greater decrease in biofilm thickness from sloughing, and (3) corresponded directly with a viability decrease. In addition, biofilm heterogeneity led to minor differences in either disinfectant's biofilm penetration, and prior biofilm exposure to monochloramine provided little impact to subsequent free chlorine biofilm penetration.

Figure 1

Representative Experiment 1 free chlorine (a) and DO (b) concentration profiles during free chlorine application and Experiment 2 (Phase 1) representative monochloramine (c) and DO (d) concentration profiles during monochloramine application.

Figure 2

Experiment 1 3D free chlorine (a) and DO (b) concentration surfaces during free chlorine application. Times listed are duration of free chlorine exposure. Zero distance represents the slide surface and increasing negative distance values represent distances progressing from the slide surface into the bulk fluid.

Figure 3

Representative CLSM microscopic images of cross–sections of cryoembedded biofilm stained with Live/Dead BacLight. Experiment 1 biofilm viability progression during free chlorine application (a) and Experiment 2 (Phase 1) biofilm viability progression during monochloramine application (b). Green color: SYTO9 stained viable cells, Red color: PI stained nonviable cells (i.e., membrane compromised cells). The images are oriented such that the image bottom represents the slide surface and the image top represents the bulk fluid. The scale bar equals 200 μm.

Figure 4

Experiment 2 (Phase 1) 3D monochloramine (a) and DO (b) concentration surfaces during monochloramine application. Times listed are duration of monochloramine exposure. Zero distance represents the slide surface and increasing negative distance values represent distances progressing from the slide surface into the bulk fluid.

Figure 5

Representative Experiment 2 (Phase 2) free chlorine concentration profiles during free chlorine application to a biofilm previously exposed to monochloramine for 16 days (Experiment 2, Phase 1). Zero distance represents the slide surface and increasing negative distance values represent distances progressing from the slide surface into the bulk fluid.

Figure 6

Experiment 2 (Phase 2) 3D free chlorine concentration surfaces during free chlorine application to a biofilm previously exposed to monochloramine for 16 days (Experiment 2, Phase 1). Times listed are duration of free chlorine exposure. Zero distance represents the slide surface and increasing negative distance values represent distances progressing from the slide surface into the bulk fluid.

Figure 7

Disinfectant concentration inside biofilm (150 μm above the slide surface) as a function of disinfection time for comparison of free chlorine and monochloramine penetration. The initial biofilm thicknesses are shown in Table 1.