A microbiology-based assay for quantification of bacterial early stage biofilm formation on reverse-osmosis and nanofiltration membranes

Membrane-based water treatment and purification technologies are crucial globally, and are especially essential in areas that suffer from a shortage of water. In the source water bacteria are ubiquitous and microbial attachment and growth on membranes is a major problem associated with these technologies. Here we describe an assay to quantify early stage biofilm growth on reverse-osmosis (RO) and nanofiltration (NF) polyamide membranes. The adherence and growth of Klebsiella oxytoca on RO and NF membranes was monitored over time under static conditions, with repeated removal of planktonic bacteria, and this assay was effective in showing especially the early stages of bacterial attachment and growth. Comparison of bacterial growth on NF270, SW30, TM820-400, and LE-400 membranes showed only slight differences, and in all cases the bacterial growth rate accelerated between 5 and 10 h of incubation time. The average surface growth rate was 1.3 × 10⁶ CFU/(cm² × h) during 5-10 h of incubation, and was measured by counting colony-forming units (CFU) after detachment of bacteria from the membranes. Fluorescence microscopy confirmed early stage biofilm growth between 5 and 10 h of incubation time. This assay will be useful to assess the susceptibility of membranes to bacterial attachment and subsequent biofilm growth and will be especially useful in screening novel antimicrobial and anti-biofilm membrane coatings and modifications and gaining insight into the mode of action of surface-tethered antimicrobial agents. Since initial irreversible adhesion is a critical step in the development of biofilms and hence biofouling on membranes, a deeper understanding of the process may lead to novel strategies for biofilm prevention.