Biofilm formation under industrially-relevant conditions.

Allan, Wendy Charlotte (2022). Biofilm formation under industrially-relevant conditions. University of Birmingham. Ph.D.

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Abstract

Biofilms are considered to be one of the most prevalent and successful modes of life on Earth, and the prevailing lifestyle for microorganisms. Enabling bacteria to adapt to an incredibly diverse array of environments and extreme conditions, biofilms are a major contaminant of both medical and industrial settings. Indeed, approximately 80% of microbial infections are associated with biofilm formation, whilst the damage caused by biofilms in industry is estimated at between 2 – 3% of global GDP per annum. In this body of work, the effect of two industrially-relevant shear conditions on biofilm formation by the reference Pseudomonas aeruginosa (Ps. a.) strains PA01 and PA14 was investigated, as well as the effect of growth conditions and growth medium components on curli gene expression in E. coli K12 PHL644.

The CBC biofilm reactor was used to model low and high shear conditions at 75 RPM and 350 RPM respectively, and biofilms grown over a time period of 96 hours. High levels of the intracellular second messenger c-di-GMP are regarded as the determining factor for Ps. a. sessility and progression of the biofilm phenotype, thus the c-di-GMP-responsive cdrA::gfp reporter was used to measure intracellular c-di-GMP levels of PA01 and PA14 under low and high shear conditions. Biofilms were analysed via confocal laser scanning microscopy and staining of extracellular DNA (eDNA) and the exopolysaccharides Psl and Pel, which are all form crucial components of a self-produced and protective extracellular matrix that surrounds and enmeshes Ps. a. within a biofilm. Under high shear at 350 RPM, intracellular c-di-GMP levels of initially adhered (at 24 hours) bacteria were increased, resulting in increased production of exopolysaccharides and formation of early aggregative structures. Shear conditions were shown to impact upon biofilm development and maturation of three-dimensional structures: crucially, mushroom-shaped macrocolonies, which are archetypal of Ps. a. biofilm formation, did not form under high shear. Under low shear at 75 RPM, Psl and Pel were organised into networks of fibre-like structures that penetrated throughout well-established basal biofilms (> 72 hours), which is in agreement with the work of others (with respect to Psl), but is a novel observation of Pel morphology as produced by PA14. The work presented in this thesis therefore provides further insight on the variety of Psl and Pel morphologies that exhibit different structures, spatial and temporal organisation, and function across PA01 and PA14 biofilms in response to either low or high shear conditions. Whilst similarities were observed between the two strains, PA01 and PA14 exhibited distinct responses to the imposed shear regime, in terms of initial surface colonisation, time taken for mature structures to emerge, and exopolysaccharide production.

Biofilms produced by E. coli PHL644 were grown using the Duran bottle model, via a method previously developed by the Overton laboratory. High levels of the transcriptional regulator CsgD are regarded as ‘master switch’ that determines biofilm formation in E. coli, thus the CsgD-responsive csgB::gfp reporter was used to measure curli gene expression in response to growth in LB broth (a rich medium) versus M63+ minimal medium, different concentrations of glucose (at 0 mM, 1 mM, 10 mM and 100 mM), and incubation at different temperatures (at 25 oC, 28 oC, 30 oC and 37 oC), and identify parameters which resulted in maximal expression of curli. Planktonic cell samples were taken from the tops of the Duran bottles, and sedimented cell samples taken from the bottom of the Duran bottles for comparative analysis of growth via spectrophotometry at OD600 and csgB::gfp fluorescence by flow cytometry. Curli gene expression was found to be highest in cultures grown in M63+ minimal medium, with a glucose concentration of 10 mM and at an incubation temperature of 30 oC, which is in agreement with comparable studies in the literature. Of interest was the fact that an inverse relationship between biomass concentration (as defined by OD600 values) and csgB::gfp fluorescence was observed. Curli gene expression in sedimented cell samples was consistently lower than that of planktonic cell cultures across all experimental subsets, suggesting that planktonic cells are physically more capable of surface attachment, and curli expression may be downregulated when in a sediment; exemplifying the importance and function of curli as the initial adhesin of E. coli K12.

Overall, this body of work concludes that different shear conditions can impact upon Ps. a. biofilm development and induce distinct organisation of the ECM, and that the CBC biofilm reactor is a suitable experimental model for assessing the impact of turbulent flow regimes, akin to those experienced in industrial manufacturing plants, on biofilm formation and composition. Additionally, this body of work also demonstrated that the Duran bottle model is also a suitable method for biofilm formation, and for investigating the effect of a wide array of growth conditions on E. coli K12 biofilm formation and curli gene expression. Parameters that resulted in maximal curli gene expression in E. coli K12 PHL644 cultures grown via the Duran bottle method under the conditions tested were identified, and could be further used to optimise production of a physically robust E. coli biofilm for use in biocatalysis or certain industrial settings.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):