Characterization of two quaternary ammonium chloride-resistant bacteria isolated from papermaking processing water and the biocidal effect on their biofilm formation

Abstract

Biofilm formation and growth on equipment surfaces is detrimental to papermaking processes. However, a fundamental understanding leading to an optimal control strategy is yet to be found. Quaternary ammonium compounds (QAC) are being increasingly applied in the papermaking processes. Among them, the most frequently applied, N-alkyl-benzyl-dimethyl ammonium chloride, was employed in this study. To foster fundamental understanding of QAC efficacy towards biofilm control, two of the highest QAC-resistant strains of bacteria were isolated from the papermaking processing water and employed as model organisms. By the 16S rRNA gene sequencing technique, two Gram-negative rods with QAC resistance were identified as Morganella morganii (HB22) and the biofilm-forming Pseudomonas putida (HB45). The minimal inhibition concentration (MIC) values were 8 mg L −1 for HB22 and 16 mg L −1 for HB45, respectively, against QAC in basal medium (BM). However, both strains could grow under more than 150 mg L −1 QAC in basal medium at neutral pH. As observed by crystal violet assay and fluorescent confocal microscopy, HB45 formed biofilm more slowly on stainless steel coupon which is the prime material of papermachine than on the surface of polystyrene, the most common material for food packaging and semi-finished/finished products. HB45 formed biofilm more slowly on stainless steel coupons than on polystyrene Petri dish surfaces, as observed by crystal violet assay and fluorescent confocal microscopy. For HB45, there was a marginal increase of inhibition of biofilm formation by increasing QAC concentration from 50 to 75 mg L −1. By comparison of inhibition concentration in liquid state and in biofilm formation, the results implicate that the current practice in papermaking processes of adding biocide to qualitatively control planktonic bacterial communities does not ensure control of biofilm formation.