Investigating biofilm adhesion and cohesion forces in respect to cleaning applications

Tsiaprazi Stamou, Artemis (2022). Investigating biofilm adhesion and cohesion forces in respect to cleaning applications. University of Birmingham. Ph.D.

Preview

Tsiaprazi-Stamou2022PhD.pdf
Text - Accepted Version
Available under License All rights reserved.
Download (6MB) | Preview

Abstract

Biofilms are complex microbial ecosystems formed by one or more bacteria species immersed in an extracellular matrix of different compositions depending on the environment and the colonizing species. While bacteria are beneficial for several technological bioprocesses, they could be catastrophic for our everyday lives as humans. In schools, healthcare facilities, food processing lines, our homes, we try to keep every surface clean and sterilized from our invisible “friends” in order to prevent ourselves from infectious diseases. Since biofilms are living communities, not traceable with the human eye, reliable methods are needed for the investigation of their attachment, growth and removal on surfaces, three different phenomena all regulated by forces. The goal of this PhD was to understand how the different material surfaces affect the above phenomena.

In the first study the initial biofilm growth as well as the removal of Pseudomonas fluorescens and Pseudomonas putida biofilms from different material surfaces was investigated. After 30 minutes of growth at 25 °C for P. fluorescens and at 30oC for P. putida it was found that P. fluorescens showed higher percentage of surface coverage comparing to P. putida on all surfaces. In terms of different materials, the percentage of the area covered by bacteria was significantly lower on plastic surfaces (PET, PTFE and polypropylene) than on more hydrophilic surfaces like glass, hydroxyapatite and stainless steel. The biofilm residual contamination was investigated using a parallel-plate flow chamber, developed for this thesis, where three different cleaning conditions were tested on stainless steel, polycarbonate and plasma-treated polycarbonate surfaces: 1) Water rinsing under shear stress conditions, 2) NaOH cleaning in static conditions and 3) NaOH cleaning under shear stress conditions. It was found that the procedure that combined NaOH and shear stress was more effective for all material surfaces. In terms of surfaces, it was seen that stainless steel was cleaned more efficiently compared to plastic surfaces. In terms of biofilm residual contamination, a more distinct biofilm removal was observed for P. putida than for P. fluorescens.

In the second study the removal of real mixed-microbial biofilm from common artificial surfaces was investigated using commercial enzymatic detergents and disinfectants used in the food industry. A mixed-microbial sample was sourced from a meat packaging line and biofilm was grown under high shear conditions on stainless steel and PET surfaces and the synergistic effect of enzymes in biofilm cleaning was studied. The cleaning effectiveness was evaluated in response to different formulations containing non-foaming commercial surfactants among with amylase, protease and lipase at neutral pH. The microscopic observation of changes in biofilm structure using SEM and confocal analyses indicated that enzymes were very effective in biofilm removal, especially on stainless steel surfaces. It was observed that the combination of enzymes was more efficient than formulations based in a single enzyme regardless of surfaces. The treatment with formulation combining amylase, protease and lipase, effectively decreased the total biofilm mass, the bacteria viability and the polysaccharide content in the biofilm formulations containing non-foaming commercial surfactants among with amylase, protease and lipase at neutral pH. The microscopic observation of changes in biofilm structure using SEM and confocal analyses indicated that enzymes were very effective in biofilm removal, especially on stainless steel surfaces. It was observed that the combination of enzymes was more efficient than formulations based in a single enzyme regardless of surfaces. The treatment with formulation combining amylase, protease and lipase, effectively decreased the total biofilm mass, the bacteria viability and the polysaccharide content in the biofilm.

The last chapter of this PhD was focused on the biofilm EPS and the role that the forces between EPS and the surrounding interphases play in biofilm cleaning. Regardless of the bacteria species, EPS is generally comprised of soluble, gel-forming polysaccharides, proteins and eDNA, as well as insoluble components such as amyloids, cellulose, fimbriae and pili. Thus, a polysaccharide and specifically alginic acid was chosen as an EPS-related material to be studied. Moreover, surface modification can play a significant role in the prevention of biofilm attachment and growth and consequently the achievement of more effective cleaning. For this reason, the goal of that study was to measure directly the adhesion and cohesion forces, developed between an EPS-related material and surfaces while in air or under simulations of cleaning conditions like water and different pH solutions. Several polymers were studied as material for polycarbonate surface modification under two different pH conditions (3 and 11) and the adhesion and cohesion forces of alginic acid were measured under air, water, NaOH and HCl solutions. Overall, it was seen that during acidic conditions the cohesive strength of alginic acid increases, while in water and in NaOH solution it decreases. Nonetheless, the adhesive strength showed decline during all cleaning conditions which depended highly on the surface. Furthermore, the polymer surface modification of the polycarbonate surfaces had a significant impact on the adhesive strength of the alginic acid in all cases. Of great interest were two polymers, Lupasol and Poly-(2-ethyl-2-oxazoline), as they caused the most important reduction in the adhesive strength of the alginic acid.

Since the results from all studies have been very interesting a future recommendation would be to expand the experiments through the combination of the relevant conditions. Thus, the technique used to measure adhesion and cohesion forces on alginic acid could be adjusted at the micron-mm scale to measure model P. fluorescens and P. putida biofilms under the optimal enzymatic conditions.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):