Phenotypic variation in growth and biofilm formation of Leuconostoc spp. from sugar beet factories.

Authors: JOSHI, SANJAY - Orise Fellow; Bruni, Gillian; Zimmerman, Tia; Terrell, Evan; Salter, Johnathan; KASHEM, NAYEEM HASAN - Orise Fellow; Nam, Sunghyun

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/17/2025
Publication Date: 1/15/2026
Citation: Joshi, S., Bruni, G.O., Zimmerman, T.E., Terrell, E.C., Salter, J.L., Kashem, N., Nam, S. 2026. Phenotypic variation in growth and biofilm formation of Leuconostoc spp. from sugar beet factories.. Frontiers in Microbiology. 16: Article 1745936. https://doi.org/10.3389/fmicb.2025.1745936.
DOI: https://doi.org/10.3389/fmicb.2025.1745936

Interpretive Summary: In sugar processing factories, certain bacteria like Leuconostoc spp. produce sticky substances known as exopolysaccharides (EPS) and form slimy layers called biofilms. During manufacturing of raw sugar, bacterial EPS and biofilm often lead to clogged filters, reduced product quality, and increased operational costs. While these bacteria are common, different strains can behave in surprisingly different ways, and the reasons for this have not been well understood. Here, we examined nine Leuconostoc strains collected from sugar beet factories. The strains were cultivated in laboratory systems simulating factory conditions, with a combination of batch adherence and continuous flow biofilm bioreactor assays. Growth rate, EPS production, and viscosity were also measured. Advanced scanning electron microscopy was used to examine the biofilm structures showing silo-like aggregates and patchy surface colonization. Some strains produced large amounts of EPS and sugar based polymers like dextran, while others, such as strain BSDF2 3, made very strong biofilms. These findings show that Leuconostoc strains exhibit varying characteristics impacting biofilm formation. Understanding these differences can help the sugar industry develop targeted cleaning and prevention strategies to reduce sucrose losses and keep production running efficiently.

Technical Abstract: Leuconostoc bacteria are common colonizers of sugar crop processing environments resulting in sucrose losses and formation of exopolysaccharides (EPS) and biofilms that can lead to reduced product quality and higher operational costs. Although Leuconostoc species are present in abundance, strain-specific differences in biofilm formation, EPS production, and matrix structure are not well understood. In this study, nine sugar beet factory-derived Leuconostoc isolates were grown and evaluated using a combination of batch adherence and continuous flow biofilm bioreactor assays, cryo scanning electron microscopy (SEM), EPS quantification, viscosity testing, and growth rate analysis to determine which phenotypes correlate with biofilm formation. The results from the adherence batch phase biofilms indicated significant phenotypic variation among isolates with the highest bacterial proliferation by L. suionicum BSDF25-7 and BSDF48-3, exceeding 5 x10^8 colony-forming units/cm2 on stainless steel coupons. In contrast, the highest biofilm biomass accumulated was BSDF2-3 and BSDF25-7, indicating differences in cell proliferation and biofilm matrix structure. Indeed CryoSEM imaging revealed diverse biofilm structures, including silo-like aggregates and patchy surface colonization, indicating strain-specific extracellular matrix assembly strategies. Flow-through biofilm bioreactor assays further identified BSDF2-3 and BSDF5-1 as predominant biofilm formers with the highest CFU/cm2 present at 4 x10^8 and 1 x10^9, respectively, while BSDF2-3 accumulated twice the biofilm biomass as BSDF5-1. Leuconostoc strains BSDF25-7 and BSDF48-3 produced high levels of dextran and EPS, while BSDF2-3 consistently formed dense, shear-resistant biofilms despite slow growth and low EPS levels, suggesting alternative matrix composition or structural adaptations. Individual Leuconostoc strains adapt uniquely, adding to the functional diversity of biofilms that impact formation, matrix complexity, and resistance to environmental stressors. This work furthers our understanding of EPS and growth phenotypes involved in biofilm formation while providing a working model enabling the development of future antimicrobial mitigation strategies.