Resistance of the constitutive microflora of biofilms formed on whey reverse-osmosis membranes to individual cleaning steps of a typical clean-in-place protocol

This experiment evaluates the effectiveness of individual steps of a clean-in-place protocol against the biofilm constitutive microflora isolated from the biofilms developed on whey reverse-osmosis membranes, aged 2 to 14 mo, under industrial processing conditions. The isolates used for the in vitro resistance studies included species of Bacillus, Enterococcus, Streptococcus, Staphylococcus, Micrococcus, Aeromonas, Corynebacterium, Pseudomonas, Klebsiella, and Escherichia. The 6 cleaning steps (alkali, surfactant, acid, enzyme, a second surfactant, and sanitizer treatment) revealed resistance of isolates in both planktonic and biofilm-embedded cell states. The most effective step was the acid treatment, which resulted in 4.54 to 7.90 and 2.09 to 5.02 log reductions of the planktonic and biofilm-embedded cells, respectively. Although the sanitizer step causing a reduction of 4.91 to 8.33 log in the case of planktonic cells, it was less effective against the biofilm-embedded cells, resulting in a reduction of 0.59 to 1.64 log. Bacillus spp. showed the highest resistance in both planktonic, as well as embedded cell states.