Efficacy of a typical clean-in-place protocol against in vitro membrane biofilms

J Dairy Sci. 2022 Nov;105(12):9417-9425. doi: 10.3168/jds.2022-21712. Epub 2022 Oct 12.


This study evaluates the effectiveness of a typical clean-in-place (CIP) protocol against in vitro biofilms on whey reverse osmosis (RO) membranes developed under static condition. Bacterial isolates obtained from RO membrane biofilms were used to develop single and multispecies biofilms under laboratory conditions. A typical commercial CIP protocol was tested against the 24-h-old biofilms, and included 6 sequential treatment steps based on alkali, surfactant, acid, enzyme, a second surfactant, and a sanitizer treatment step. Experiments were conducted in 4 replicates and the data were statistically analyzed. The results revealed a variation in the resistance of mixed-species biofilms against the individual steps in the sequential CIP protocol. The overall 6 steps protocol, although resulted in a greater reduction, also resulted in the detection of survivors even after the final sanitizer step, reflect the ineffectiveness of the CIP protocol for complete removal of biofilms. Posttreatment counts of 0.71 log after the sequential CIP of mixed-species biofilm revealed the resistance of biofilm constitutive microbiota. Mixed-species biofilms, constituting different genera including Bacillus, Staphylococcus, and Streptococcus, were observed to be more resistant than most of the single-species biofilms. However, among the single-species biofilms, significantly different resistance pattern was observed for Bacillus isolates compared with the other bacterial isolates. All 5 isolates of Bacillus were found resistant with survivor counts of more than 1.0 log against the sequential CIP protocol tested. Thus, it can be concluded that the tested CIP protocol had a limited effectiveness to clean membrane biofilms formed on the whey RO membranes.

Keywords: CIP; biofilms; reverse osmosis; whey.

MeSH terms

  • Animals
  • Biofilms*
  • Membranes
  • Microbiota*
  • Surface-Active Agents


  • Surface-Active Agents