The bacterial planktonic growth and the removal of bacterial cells grown on polypropylene surface coated with covalently immobilized proteases (subtilisin Carlsberg or α-chymotrypsin) was investigated for Enterococcus hirae, Staphyloccocus epidermidis and Eschericha coli. Immobilization of both proteases on plasma-treated polypropylene was carried out using as cross-linking agent i) glutaraldehyde or ii) N'-diisopropylcarbodiimide and N-hydroxysuccinimide. In the presence of immobilized proteases a higher bacterial planktonic growth (up to 40 %) was observed. Instead, a different effect was observed on cell removal, and it varied according to the bacteria strain, the immobilized protease and the immobilization procedure. In particular, the presence of subtilisin in the polypropylene coating increased the cell removal of E. hirae by simple washing of the polypropylene surface and both subtilisin and α-chymotrypsin immobilized by N'-diisopropylcarbodiimide and N-hydroxysuccinimide favored the removal of S. epidermidis after sonication. No significant differences compared to the control where observed in all the other cases. In conclusion this study indicates that proteases can be an enhancer of microbial biomass (a phenomena that could be exploited for industrial fermentation) and can affect the strength of cell adhesion for some bacteria.
Flow device analyses and micromanipulation were used to assess the adhesive and cohesive integrity of the immobilised bacterial populations (biomass) of Pseudomonas fluorescens, which were harvested at different growth times and applied to a substrate made of stainless steel subsequently accommodated in a specially designed flow chamber. After the biomass was exposed to a fluidic environment for a period of time, the biomass samples were removed from the flow chamber and the apparent adhesion and cohesion of the remaining biomass was measured using a micromanipulation technique. The surface area of the substrate covered by the biomass exposed to the fluid flow was monitored by a digital camera and then quantified by image analysis. The results indicate a strong correlation between micromanipulation measurements and flow chamber experiments. The micromanipulation data show that the apparent adhesive strength of the biomass increased with the growth time. Moreover, the apparent adhesive strength was found to be stronger than the bacterial cohesive strength. The data was used to interpret the removal behaviour of the biomass from the flow chamber. Using these techniques, specific mechanisms of biomass detachment from a surface and optimised cleaning strategies can be postulated.
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