Document Type

Thesis - Open Access

Award Date


Degree Name

Master of Science (MS)


Dairy Science

First Advisor

Sanjeev Kumar Anand


Antimicrobial, Bacillus subtilis, Biofilms, Biosanitizer, Competitive Exclusion, Predominance


The microbial attachment and colonization on separation membranes lead to biofilm formation. Some selective isolates within the biofilm constitutive microflora acquire resistance and emerge predominant over prolonged use of the membrane. Thus, proving the cleaning and sanitization protocols to be ineffective in adequately removing resilient biofilms. This subsequently leads to selecting microbial resistance within the constitutive microflora to almost all antimicrobial treatments and hence, creates a need to develop novel alternative strategies to control biofilm formation on membrane surfaces. The first research project under this study was designed to understand the microbial interactions and emergence of predominance within biofilm constitutive microflora of RO membranes. The second study was similar to the first where biofilm microflora from UF membranes was isolated and identified. The third study was conducted to investigate the factors influencing predominance within the biofilm constitutive microflora. This study included the antimicrobial activity of Bacillus subtilis isolate (within the membrane biofilm microflora) as a potential cause of its predominance. The fourth study was conducted as a proof of concept to evaluate the effectiveness of the antimicrobial substance from B. subtilis for cleaning membrane biofilms, under in vitro conditions. In the first study, we isolated and identified six microorganisms from an 18-monthold RO membrane as Bacillus licheniformis, Exiguobacterium aurantiacum, Acinetobacter radioresistens, Bacillus subtilis, and one unidentified species each of Exiguobacterium and Bacillus. The competitive exclusion study helped to establish the emergence of predominance using a co-culture technique. Bacillus subtilis emerged predominant with a mean log count of 7.22 ± 0.22 CFU/ mL. The predominance of B. subtilis was also validated using the process of natural selection in a multi-species growth environment. In the second study, eight microorganisms were identified from two UF membranes, sourced from two different locations. The species identified belonged to genus Exiguobacterium, Enterococcus, Rehnella, Klebsiella, Citrobacter, and Bacillus. It is important to note here that this was only a preliminary study to compare the biofilm microflora of the UF membrane with RO, and further studies are required to draw any conclusions. In the third study, antimicrobial activity as a potential cause of the predominance of B. subtilis was investigated. For which, 12 h freeze-dried cell-free extract (CFE) of B. subtilis was observed to exhibit zones of inhibition when screened against the test strain, Micrococcus luteus. Further, the antimicrobial activity when tested against other constitutive microflora of the membrane biofilm, and some common foodborne pathogens, revealed a broadspectrum of inhibition against gram-positive and gram-negative bacteria. The antimicrobial substance was found to be proteinaceous, having total crude protein 51% (wt/wt), and its amino acid profiling revealed its major constituent to be glutamic acid (11.30% wt/wt). In the final steps of the study, in-vitro biofilms were developed (6.68 ± 0.12 CFU/ inch2) using the isolated B. subtilis. These biofilms were exposed to a commercial cleaning and sanitation protocol. After 4 steps of the process, the residual biofilm counts were reduced to 2.18 ± 0.54 logs CFU/ inch2. These counts got further reduced to 1.20 ± 0.09 logs CFU/ inch2 on using the B. subtilis antimicrobial substance in place of the commercial sanitizer during the cleaning process. The study thus provides a proof of concept for the higher efficacy of the natural antimicrobial substance released by B. subtilis in cleaning resilient biofilms and shows a promise for the future development of a biosanitizer.

Number of Pages



South Dakota State University


In Copyright - Non-Commercial Use Permitted

Available for download on Thursday, June 15, 2023