Antimicrobial activity as a potential factor influencing the predominance of Bacillus subtilis within the constitutive microflora of a whey reverse osmosis membrane biofilm

Abstract

Current cleaning and sanitation protocols may not be adequately effective in cleaning separation membranes and can result in the formation of resilient multispecies biofilms. The matured biofilms may result in a bacterial predominance with resilient strains on membranes with a prolonged use. In our previous study, we isolated organisms such as Bacillus subtilis, Bacillus licheniformis, Exiguobacterium aurantiacum, and Acinetobacter radioresistens from an 18-mo-old reverse osmosis membrane. The competitive exclusion studies revealed the predominance of B. subtilis within the membrane biofilm microflora. This study investigated the antimicrobial activity of the B. subtilis isolate as a potential cause of its predominance. The culture isolate was propagated in tryptic soy broth at 37°C, and microfiltered to prepare cell-free extracts (CFE) at 8-, 10-, 12-, 14-, 16-, and 18-h intervals. The CFE were freeze-dried and suspended in minimum quantities of HPLC-grade water to prepare concentrated solutions. The antimicrobial activities of CFE were tested using the agar-well assay against the biofilm constitutive microflora. The experiments were conducted in triplicates and means were compared for significant differences using a general linear mixed model procedure. The results indicated the highest antimicrobial activity of 12-h CFE of B. subtilis against other constitutive microflora such as Exiguobacterium sp., E. auranticum, and A. radioresistens, with average inhibition zone sizes of 16.5 ± 0.00, 16.25 ± 0.66, and 20.6 ± 0.00 mm, respectively. Upon treatment with proteinase K, the CFE completely lost its antimicrobial activity, establishing it to be a proteinaceous compound. The AA profiling revealed the total crude protein in CFE to be 51% (wt/wt), with its major constituent as glutamic acid (11.30% wt/wt). The freeze-dried CFE was thermally stable on exposure to the common temperature used for sanitizer applications (23.8°C for 5 and 10 min) and over a pH range of 3.0 to 6.3. The study helped us understand the role of the antimicrobial compound produced by B. subtilis as a potential cause of its predominance within the biofilm constitutive microflora.