Detachment of single- and multi-species bacterial biofilms by crude enzymes extracted from wastewater biofilms and bacteria

Abstract

Biofilms are bacterial communities that adhere to biotic and abiotc surfaces, and are embedded in a polymeric matrix composed mainly of polysaccharides and proteins. Not only are biofilms a public health problem, but they are also a hindrance in industrial practices. Due to their intractability by conventional cleaning agents, a number of alternative agents, including enzymes, have been investigated as potential biofilm detachment-promoting agents. Two major types of enzymes, i.e. proteases and polysaccharases, have been used for biofilm removal and their use is aimed at degrading or promoting the collapse of the biofilm matrix. Consequently, the aim of this investigation was primarily to assess the use of enzymes originating from a wastewater biofilm to remove biofilms from three Pseudomonas species, viz. P. aeruginosa PAO1, P. fluorescens and P. putida. To investigate, biofilms were sampled from an aerobic reactor at an industrial wastewater treatment plant. Dissolution of the biofilm, as evidenced by reductions in the soluble chemical oxygen demand (COD) and total suspended solids (TSS), coincided with detectable protease and carbohydrate-degrading enzyme activities. Crude extracellular enzyme extracts prepared from the wastewater biofilm were subsequently shown to remove P. aeruginosa PAO1 biofilms from a glass surface, suggesting that the wastewater biofilms expressed enzymes that may be used towards the removal of detrimental biofilms. Consequently, representative bacteria were isolated from the wastewater biofilm and, based on 16S rRNA gene sequencing and analyses, were found to represent four major phylogenetic divisions of bacteria, i.e. Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. Screening of the bacterial isolates for different enzyme activities indicated that nine isolates produced proteases, while ten isolates produced polysaccharide-degrading enzymes that comprised amylase, xylanase, cellulase, á-glucosidase and â-glucosidase. The ability of these enzymes to degrade proteins and polysaccharides present in purified EPS from P. aeruginosa PAO1, P. putida and P. fluorescens was confirmed by SDS-polyacrylamide gel electrophoresis and an increase in the amount of reducing sugar, respectively, while their efficacy to remove single-and multi-species biofilms cultured in microtiter plates was evaluated using a quantitative spectrophotometric assay. Proteases produced by four of the strains were effective in degrading the EPS proteins of all three Pseudomonas spp., while all bacterial strains that produced polysaccharide-degrading enzymes were capable of degrading the EPS polysaccharides, albeit with different efficiencies. Efficient removal of P. aeruginosa PAO1 biofilms was only achieved when mixtures of enzyme extracts, containing protease and different types of polysaccharase activities, were used. Biofilms of P. putida and P. fluorescens were readily removed with single enzyme extracts prepared from B. subtilis and B. pumilus. Enzyme combinations showing high biofilm removal for all three Pseudomonas species were tested against a mixed species biofilm. These enzyme extracts yielded lower biofilm removal efficiencies than those obtained for mono-species pseudomonad biofilms, possibly due to the heterogenous nature of the EPS. Nevertheless, it may be possible that the enzymes identified in this study could be used in combination with other treatments to increase the biofilm removal effectiveness or in combination with other enzymes to degrade the mixture of proteins and polysaccharides present in the EPS of multi-species biofilms.