Functionalized microparticles for online biofilm control

Biofilms are ubiquitous in nature and its formation is a strategy that bacteria use in order to survive in hostile environments. Although biofilms play crucial roles in many processes they often are unwanted and cause serious problems in various areas like the food industry, medical industry, industrial heat exchangers and cooling water systems, which can lead to increase in the costs of production and maintenance, as well as to public health and environmental concerns and impacts. Although various methods of biofilm control exist, these present limitations and often fail to remove completely the biofilms from surfaces, contributing to the dissemination of resistance. The control of undesirable biofilms often include the use of chemical products with antimicrobial properties such as biocides and surfactants. However, these substances can be harmful to the environment and public health, therefore they should be used in as small quantities as possible. In the past few decades there has been a dilemma between effective disinfection and formation of harmful disinfection by-products (DBPs). Advances in nanotechnology science led to its environmental application. The focus of this work was the development of microparticles with functionalized surfaces that carry the antimicrobial agent. This strategy significantly reduces the amount of antimicrobial agent needed, prevents undesired reactions with other components that can lead sometimes to the formation of organochlorinated compounds (potentially carcinogenic), and finally reduces the costs of wastewater treatment due to the presence of residual concentrations of biocides in the effluent. In the beginning of this study, the action of the quaternary ammonium compound benzyldimethyldodecylammonium chloride (BDMDAC) was assessed against the Gram-negative bacterium Pseudomonas fluorescens. The targets of antimicrobial action were studied using different bacterial physiological indices. The minimum inhibitory concentration (MIC) was 20 mg/L and the minimum bactericidal concentration (MBC) value was 10 mg/L. BDMDAC led to a significant change in cell surface hydrophobicity and induced propidium iodide uptake. Such results suggest cytoplasmic membrane damage, corroborated by the release of intracellular potassium. The results obtained from the zeta potential assay indicated the existence of interactions between the cationic antimicrobial agent and the cell surface. Scanning electron microscopy (SEM) allowed to detect that cells treated with 20 mg/L were less bulky and their membrane seems to be rougher, wrinkled, deformed comparatively to untreated cells. The microparticles of polystyrene (PS) were functionalized with BDMDAC using the layer-by-layer (LbL) self-assembly technique. Zeta potential measurements indicated significant changes in the surface charge compatible with the BDMDAC/particle interaction. The efficacy of microparticles carrying BDMDAC was assessed against P. fluorescens in planktonic and biofilm states. This biocidal carrier structure had a significant stability verified by the release of only 15% of BDMDAC when immersed in water for 18 months. The evaluation of biocidal carrier activity was carried out through the determination of the survival ratio of P. fluorescens planktonic and biofilm cells after different exposure periods to BDMDAC coated particles. Tests with biofilm cells were also performed with free BDMDAC. MBC of BDMDAC coated polystyrene (PS) microparticles was 9.2 mg/L and 6.5 mg/L for exposure periods of 30 and 60 minutes, respectively. Biofilms were developed in a chemostat reactor and polyvinylchloride was used as adhesion surface. Seven days old biofilms were exposed to BDMDAC coated PS microparticles (0.87 mg/L) resulting in viability decreases of 60.5% and 66.5% of the total biofilm population for a 30 and 60 min exposure time, respectively. The exposure during 60 min to 6.33 mg/L and 11.75 mg/L of BDMDAC in PS coated microparticles promoted inactivation of 80.6% and 87.2% of the total population, respectively. The antimicrobial effects obtained with the application of free BDMDAC were similar to those promoted by the application of BDMDAC coated particles. Due to the high cost of PS microparticles, calcium carbonate (CaCO3) microparticles were selected for testing BDMDAC immobilization with the LbL assembly technique. These microparticles have the advantage of being cheaper and porous (and the disadvantage of being susceptible to low pH values). P. fluorescens biofilms were exposed to 6.33 mg/L and 11.75 mg/L of BDMDAC-coated CaCO3 microparticles for 60 min. This strategy promoted inactivation of 81.9% and 93.3% of the total population. The effects of flowing conditions on biofilm susceptibility to BDMDAC-coated CaCO3 microparticles was tested using a flow cell reactor, mimicking industrial pipes, for biofilm formation and antimicrobial testing. SEM images of the microparticles show that they are spherical and have a spongy surface. These microparticles in suspension have an average size diameter of 5 μm and a zeta potential of -20 ± 3 mV. The exposure of biofilms to 50 mg/L of BDMDAC-coated CaCO3 microparticles promoted inactivation of 100% of the biofilm population after 90 minutes of exposure. The overall results indicate that this novel strategy has a strong potential to control flow-generated biofilms and has many advantages over traditional biofilm control strategies, particularly the possibility for the reuse of the biocidal particles and consequent reduction of discharges of antimicrobials into the environment.