Investigating the effects of different physical and chemical stress factors on microbial biofilm

Abstract

Microorganisms that adhere to surfaces in order to protect themselves from many adverse environmental conditions form a layer called biofilm. Biofilms protect bacteria from changing environmental conditions such as starvation, antibiotics, disinfectants, pH and temperature fluctuation, dryness and UV rays. In this study, biofilms were formed on surfaces of glass coupons in a cooling tower model system over a period of 180 days. The biofilms were treated with various stress factors monthly. These stress factors were: exposure to temperatures of 4°C and 60°C, pH of 3, 5, and 11, 3 M aqueous NaCl and distilled water, as well as, monochloramine at 2, 500, and 1 000 mg/L (ppm). Following the treatment with stress factors, both the numbers of actively respiring bacteria and the total bacteria in the biofilms were determined by CTC-DAPI staining. The aerobic heterotrophic plate counts (HPC) in the biofilms were determined by the conventional culture method of spread plating on R2A agar. The aim of this study was to determine the impact of these stressors on the model coolingtower biofilms. Of the stressors tested, those that had the greatest impact were a temperature of 60°C, pH of 3, 3 M NaCl, and monochloramine at both 500 and 1 000 mg/L. However, when using a non-culture-based viability assay (CTC-DAPI staining), an extremely high number of live bacteria were detected even after applying the most effective stress factors (with the exception of pH 3) of 60°C, 3 M NaCl, monochloramine at 500 and 1 000 mg/L. Results showed that biofilm protects the bacteria from extreme physical and chemical stress conditions. Additionally, the conventional culture technique cannot detect the presence of bacteria that have entered the viable but non-culturable (VBNC) phase; the use of different techniques, such as microscopy and cytometry (flow and solid-phase), is therefore important to obtain more accurate results.

Keywords: biofilm, stress factors, monochloramine, CTC-DAPI staining