Quantification of Enzymatic Biofilm Removal Using the Sauerbrey Equation: Application to the Case of Pseudomonas protegens

LEVY, I.K.; SALUSTRO, D.; BATTAGLINI, F.; LIZARRAGA, L.; MURGIDA, D.H.; AGUSTI, R.; D'ACCORSO, N.; RAVENTOS SEGURA, D.; GONZALEZ PALMEN, L.; NEGRI, R.M.

ACS Omega

American Chemical Society

Año: 2024 vol. 9 p. 10445 - 10458

A methodologyfor the quantitativeanalysisofenzymaticremovalof biofilms(BF)was developed,basedon aquartzcrystalmicrobalance(QCM)understationaryconditions.Thiswas appliedto the case ofPseudomonasprotegens(PP)BFs,througha seriesof five enzymes,whoseremovalactivitywasscreenedusingthe presentedmethodology.The procedureis basedon the following:whenBFs can be modeledas rigidmaterials,QCMcan be usedas a balanceunderstationaryconditionsfordeterminingthe BFs massreduction by enzymaticremoval.Forconsideringa BF as a rigidmodel,energydissipationeffects,associatedwith viscoelasticpropertiesof the BF, mustbe negligible.Hence,a QCMsystemwith detectionof dissipation(referredto asQCMwithdissipation)was usedfor evaluatingthe energylosses,which,in fact, resultedin negligibleenergylossesin the case of dehydratedPP BFs,validatingthe applicationof the Sauerbreyequationfor the changeof masscalculations.The stationarymethodologyreducesoperatingtimesand simplifiesdata analysisincomparisonto dynamicapproachesbasedon flow setups,whichrequiresthe incorporationof dissipationeffectsdue to the liquidmedia.By carryingout QCM,glycosidase-typeenzymesshowedBF removalhigherthan80%at enzymeconcentration50 ppm,reachingremovalover90%in the casesof amylaseand cellulase/xylanaseenzymes.The highestremovalpercentageproducedareductionfromabout15 to 1μg in the BF mass.Amylaseenzymewas testedfrombelow50 to 1 ppm,reachingaround60%ofremovalat 1 ppm.The obtainedresultsweresupportedby otherinstrumentaltechniquessuchas Ramanspectroscopy,attenuatedtotalreflectionFouriertransforminfraredspectroscopy,atomicforcemicroscopy,highperformanceanionexchangechromatography,thermogravimetricanalysis,and differentialscanningcalorimetry.TheremovalquantificationsobtainedwithQCMwerecomparedwith thoseobtainedby well-establishedscreeningtechniques(UV−visspectrophotometryusingcrystalvioletand agar diffusiontest).The proposedmethodologyexpandsthe possibilityof usinga quartzmicrobalanceto performenzymaticactivityscreening