Thesis

Biofilm development and regulation in bacillus subtilis under static and flow conditions and effects of Ginkgo biloba leaf extract

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Awarding institution
  • University of Strathclyde
Date of award
  • 2025
Thesis identifier
  • T17502
Person Identifier (Local)
  • 201986457
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Abstract
  • Biofilms are structured communities of microorganisms embedded in an extracellular matrix, capable of adhering to surfaces and withstanding chemical, mechanical, and environmental stresses. They play essential roles in natural ecosystems but also cause major challenges in clinical, industrial, and environmental contexts due to their persistence and resistance to treatment. While Bacillus subtilis (B. subtilis) serves as a well-established model for studying biofilm development, the combined influence of hydrodynamic forces and natural antibiofilm compounds on its structure and regulation remains poorly understood. Existing studies have largely focused on static systems, microfluidic-scale observations, and clinical pathogens, offering limited insight into the macroscale architecture and transcriptional responses of B. subtilis biofilms under flow conditions. Furthermore, although Ginkgo biloba leaf extract (GBLE) has been shown to exhibit antimicrobial or antibiofilm activity against several clinically relevant bacteria, it has not previously been tested against B. subtilis or applied to flow-grown biofilms. Studying these responses under macroscale flow conditions is essential, as such environments are common in natural, clinical and industrial systems, while the use of GBLE reflects the urgent need for environmentally sustainable antibiofilm strategies. This thesis addresses these gaps by investigating the structural and molecular adaptations of B. subtilis biofilms to fluid flow regimes (unidirectional and bidirectional flow) and their responses to GBLE. A combination of confocal laser scanning microscopy, quantitative image analysis, RT-qPCR, and RNA Sequencing was used to examine biofilm morphology, matrix organisation, cellular differentiation, and transcriptional regulation under static and flow conditions, with and without GBLE supplementation. This work shows that GBLE acts primarily as an antibiofilm agent against B. subtilis, with concentration-dependent inhibition of biofilm formation. GBLE greatly influenced agar colony biofilms, promoting motility (except at 400 and 500 µg/mL) and inducing cellular differentiation. Microscopy revealed pronounced morphological changes, including the development of disorganised Van Gogh bundles (single cell chains), intracellular alterations, and increased amyloid production, suggesting differentiation into amyloid-producing cell types as a mechanism to reinforce extracellular matrix and persist under stress. Under unidirectional flow, biofilms were entirely composed of aligned Van Gogh bundles and developed novel higher-order architectures, including spore aggregates, extracellular matrix-rich foundation layers and rope-like twisted bundles of filaments (“Van Gogh ropes”), which are believed to enhance mechanical stability and tensile strength against shear stress. GBLE disrupted these biofilms, reducing biomass and interfering with bundle organisation, thereby compromising the stability of flow-grown biofilms. Exposure to bidirectional flow produced even greater structural heterogeneity, with biofilms exhibiting higher biomass and porosity compared to biofilms under unidirectional flow, and unique raised folds containing nutrient channels. These folds likely facilitate mass transport, spatial differentiation, and resilience under fluctuating hydrodynamic stress. GBLE treatment significantly reduced biomass and disrupted these complex architectures, further underscoring its potential as a natural biofilm-control strategy under dynamic flow conditions. At the molecular level, gene expression analyses in planktonic cultures revealed that GBLE repressed key regulators of matrix production, development, motility, sporulation, and stress tolerance, while selectively inducing pathways linked to oxidative stress resistance and ribosome stabilisation, indicative of a shift toward survival rather than active biofilm development. Comparative transcriptomics across static, unidirectional, and bidirectional flow confirmed that flow regimes strongly activate transcription, with bidirectional flow inducing the broadest response, consistent with the structural complexity observed microscopically. Together, these findings demonstrate that B. subtilis biofilms display remarkable plasticity under flow, adapting through novel structural and genetic strategies to withstand mechanical forces, while GBLE represents a promising environmentally sustainable antibiofilm agent that reduces biomass and disrupts biofilm organisation without strong bactericidal pressure. Together, these findings extend current biofilm development models to incorporate macroscale flow environments and demonstrate the value of integrating microscopic analysis with transcriptomics, applied to industrially relevant environments. The work provides new insight into the physical and regulatory plasticity of B. subtilis biofilms and offers a foundation for developing context-specific, environmentally responsible biofilm control approaches
Advisor / supervisor
  • McConnell, Gail
  • Phoenix, Vernon
Resource Type
DOI

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