The Effects of Chemical and Mechanical Stresses on Bacillus cereus and Pseudomonas fluorescens Single- and Dual-Species Biofilm Removal

Abstract

Biofilm control is mainly based on chemical disinfection, without a clear understanding of the role of the biocides and process conditions on biofilm removal. This study aims to understand the effects of a biocide (benzyldimethyldodecyl ammonium chloride-BDMDAC) and mechanical treatment (an increase of shear stress -τw) on single- and dual-species biofilms formed by Bacillus cereus and Pseudomonas fluorescens on high-density polyethene (HDPE). BDMDAC effects were initially assessed on bacterial physicochemical properties and initial adhesion ability. Then, mature biofilms were formed on a rotating cylinder reactor (RCR) for 7 days to assess the effects of chemical and mechanical treatments, and the combination of both on biofilm removal. The results demonstrated that the initial adhesion does not predict the formation of mature biofilms. It was observed that the dual-species biofilms were the most susceptible to BDMDAC exposure. The exposure to increasing τw emphasised the mechanical stability of biofilms, as lower values of τw (1.66 Pa) caused high biofilm erosion and higher τw values (17.7 Pa) seem to compress the remaining biofilm. In general, the combination of BDMDAC and the mechanical treatment was synergic in increasing biofilm removal. However, these were insufficient to cause total biofilm removal (100%; an average standard deviation of 11% for the method accuracy should be considered) from HDPE.

Keywords: BDMDAC; disinfection; high-density polyethene; rotating cylinder reactor; shear stress.

Figure 1

Scheme representative of the rotating cylinder reactor: (a)—inlet of sterile air; (b)—inlet of concentrated nutrient medium; (c)—inlet of diluted nutrient medium; (d)—peristaltic pumps; (e)—chemostat; (f)—rotating cylinder reactor; (g)—testing cylinders (samplers); (h)—synchronising belt that connect the three cylinders; (i)—stirrer that controls rotation speed; (j)—outlet of bacterial suspension.

Figure 2

Number of B. cereus and P. fluorescens in single- and dual-species adhered on HDPE surface, before (□) and after (■) BDMDAC treatment. Mean values ± SDs for at least three replicates are illustrated.

Figure 3

Biofilm removal after submitting the B. cereus, P. fluorescens, and dual-species biofilms to BDMDAC at 300 µg/mL under 0.04 Pa. Mean values ± SDs for at least three replicates are given.

Figure 4

Biofilm removal caused by the increasing series of shear stress (mechanical treatment) on chemically (BDMDAC) untreated (A) and treated (B) biofilms). ■—exposure to 1.66 Pa; ■—exposure to 5.50 Pa; ■—exposure to 10.9 Pa; ■—exposure to 17.7 Pa. The white bar (□) represents the amount of biofilm remaining after the mechanical treatment. Mean values ± SDs for at least three replicates are given.