Quantification of Enzymatic Biofilm Removal using the Sauerbrey Equation. Application to the case of Pseudomonas protegens.

LEVY, IVANA K.; DÉBORA SALUSTRO; FERNANDO BATTAGLINI; LIZARRAGA, LEONARDO; MURGIDA, DANIEL; AGUSTI, ROSALIA; D´ACCORSO, N.; RAVENTOS SEGURA, DOROTEA; GONZALEZ PALMEN, LORENA; NEGRI RICARDO MARTÍN

ACS Omega

American Chemical Society

Año: 2024

A methodology for the quantitative analysis of enzymatic removal of biofilms was developed, based on Quartz Crystal Microbalance (QCM) under stationary conditions. This was applied to the case of Pseudomonas protegens (PP) biofilms, through a series of five enzymes, whose removal activity was screened using the presented methodology. The procedure is based in the following: when biofilms can be modeled as rigid-materials, QCM can be used as a balance under stationary conditions for determining the biofilms mass reduction by enzymatic removal. For considering a biofilm as a rigid model, energy dissipation effects, associated to viscoelastic properties of the biofilm, must be negligible. Hence, a QCM system with detection of dissipation (referred as QCM-D) was used for evaluating the energy losses which, in fact, resulted negligible in the case of dehydrated PP biofilms, validating the application of the Sauerbrey equation for the change of mass calculations. The stationary methodology reduces operating times and simplifies data analysis, in comparison to dynamic approaches based on flow setups, which requires the incorporation of dissipation effects due to the liquid media. By QCM, glycosidase type enzymes showed biofilm removal higher than 80% at enzyme concentration 50 ppm, reaching removal over 90% in the cases of amylase and cellulase/xylanase enzymes. The highest removal percentage produced a reduction from about 15 µg to 1 µg in the biofilm mass. Amylase enzyme was tested from below 50 ppm to 1 ppm, reaching around 60% of removal at 1 ppm.The obtained results were supported by other instrumental techniques as Raman Spectroscopy (RS), Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR), Atomic Force Microscopy (AFM), High Performance Anion Exchange Chromatography (HPAEC), Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC). The removal quantifications obtained with QCM were compared with those obtained by well-established screening techniques (UV-Vis spectrophotometry using crystal violet (CV) and agar diffusion test). The proposed methodology expands the possibility of using quartz micro balance to perform enzymatic activity screening.