On-line quantification of thickness and strength of single and mixed species biofilm grown under controlled laminar flow conditions

https://doi.org/10.1016/j.fbp.2018.08.009Get rights and content

Highlights

  • In-situ measurement of strength and thickness of E. coli and B. cepacia biofilms.

  • Biofilm strength is strongly correlated to its protein content.

  • Biofilm desiccation significantly increased its attachment strength.

  • Biofilm removal on all substrates occurred in two stages.

  • Less energy required to remove biofilm grown less than 5 days and more than 21 days.

Abstract

This study describes cleaning investigations of biofilms comprised of Escherichia coli and Burkholderia cepacia grown on polyethylene, stainless steel and glass substrates. Their adherence behaviour was determined under controlled hydrodynamic conditions using the non-contact technique of fluid dynamic gauging (FDG). FDG utilises flow data to estimate (i) the adhesive (between biofilm and substrate)/cohesive (between cells and extracellular polymeric substances) strengths, and (ii) the thicknesses of biofilms. The thickness of single and mixed species biofilms increased linearly with time and plateaued at 14 days with no significant reduction thereafter. The asymptotic thickness of mixed species biofilm were thinner than E. coli biofilms. The adhesive strength, on the other hand, peaked at approximately 14 days with a significant reduction thereafter. The results showed that the development of biofilm thickness and attachment strength are not affected by the range of surface roughness and surface energy employed. However, the increase in strength is strongly correlated to the protein and glucose content of the biofilms. Confocal laser scanning microscopy results confirmed an increase in the percentage of dead cells after 21 days, contributing to the weakening of the biofilms. Interrupting the flow of media during biofilm development had a negligible impact upon the thickness, but was found to significantly increase the biofilm strength.

Introduction

Biofouling is ubiquitous in a number of fields including food and pharmaceutical production, shipping, steel manufacturing, petrochemicals, water desalination, and drinking water treatment and distribution. Biofilms can grow on all surfaces that are exposed to local bacteria inhabitation such as pipe bends, conveyor belts, floors and rubber seals. Biofilms consist primarily of viable and nonviable microorganisms embedded in polyanionic extracellular polymeric substances anchored to a surface (Carpentier and Cerf, 1993). The initial microorganism attachment is reversible, and may be the rate limiting step of the entire growth process. The bond with the surface is consolidated when irreversible attachment begins to take place, which is initiated by the production of extracellular polymeric substances (EPS) (Whitehead and Verran, 2015, Garnett and Matthews, 2012, Yebra et al., 2006, Momba et al., 2000). EPS are produced and excreted by the microorganisms of interest, with a chemical structure dependent upon both the species involved and the environmental conditions. They may contain polysaccharides, proteins, phospholipids, teichoic and nucleic acids, and other polymeric substances hydrated to 85–95% water. The EPS is responsible for most of the characteristics of the biofilm, ranging from structural benefits, such as instigating the adherence of biofilms to surfaces and the formation of a gel-like network keeping the bacteria together, to the protection of bacteria against potentially damaging influences from the environment. Arguably the most important function of EPS is their role as fundamental structural elements determining the mechanical stability of biofilms (Wingender et al., 1999).

The disinfection of biofilms is a problematic task due to the range of defence mechanisms they possess. The threat of biofouling cannot be entirely eliminated as antifouling measures are only temporary or time-dependent restrictions of growth, and regular disinfection is therefore required in order to prevent their continuous development (Flemming et al., 2011). Current pre-treatment technologies focus on the reduction in microorganisms in the feed source, which may not provide effective biofouling control since biofilm development relies heavily on the surface chemistry of substrates and availability of nutrients (Chen et al., 2013, Jamaly et al., 2014). Chemical agents are often employed to kill microorganisms, but the biofilm structure must be removed to prevent re-growth and maintain sterility. The required concentration of antibacterial agents is also considerably higher, and must be increased by between 10 and 100 times in comparison with the respective equivalent planktonic cultures (Blanchard et al., 1998). To avoid the use of chemical agents that can pose health and environmental risks, the usual methods of biofilm deactivation involve pumping large volumes of cleaning solutions through pipelines to achieve the additional benefits of mechanical cleaning.

There are numerous studies and review articles related to biofilm formation and characterisation of their properties, and mitigation of biofouling (e.g. Bucs et al., 2018, Wang and Lan, 2018, Gule et al., 2016, Srey et al., 2013). In general, these studies can be grouped into three main areas: biofilm surface characteristics, biofilm structure and thickness, and biofilm adhesion to a surface. A variety of lab-based on-line methods for estimating the thickness and development of biofouling have been explored and reported. These methods include microscopic (e.g. confocal laser scanning microscopy) (Mukherjee et al., 2016), spectroscopic (e.g. infrared, nuclear magnetic resonance and Raman spectroscopy) (Kögler et al., 2016) and ultrasonic time-domain reflectometry (Sim et al., 2013). Atomic force microscopy (AFM) is probably the only technique that allows the measurement of the physical adhesive forces of foulants to surfaces in situ, which may include bacterial and biofilm adhesion to surfaces (Powell et al., 2017). However, it is especially challenging to obtain reliable measurements in flow systems commonly employed in industry.

Previous studies from the authors have focused on single species biofouling and cleaning experiments by using static culture (Peck et al., 2015) and turbulent duct-flow (Suwarno et al., 2017) systems. The main aim of this study was to seek more sustainable methods of effective biofilm deactivation and removal whilst reducing chemical, water and energy consumption. This work presents experiments of single and mixed species cultures of Escherichia coli Nissle1917 and Burkholderia cepacia biofilms grown on polyethylene, glass and stainless steel 304 under controlled laminar flow conditions in a modified drip flow reactor. M9 minimal medium containing glucose was used to provide an artificially designed source of minimum nutrients for growth. The technique of fluid dynamic gauging (FDG) was utilised to quantify both the thickness and the strength of the biofilms incubated for up to 28 days in situ. This study also explored the effect of biofilm content and the impact of desiccation which could occur due to flow disturbances or during cleaning (i.e. transition from feed to cleaning formulations) on growth and removal.

Section snippets

Bacteria strains, culture media and substrates

E. coli Nissle1917 and B. cepacia DSM-7288 were used to grow single species and mixed biofilms under controlled laminar flow conditions. E. coli is recognised to form biofilms on many different surfaces, which is essential for the studying and comparison of fouling mechanisms with an industrial focus. Protocols for E. coli biofilm growth are well-established and it has been extensively characterised. B. cepacia was selected as an additional species for mixed species biofilm development.

Surface roughness and energy

Table 1 shows the roughness of polyethylene, stainless and glass substrates determined by using AFM in tapping mode. All surfaces appear to be relatively smooth and exhibit similar roughness profiles. These irregularities are significantly lower in comparison to the size of the cells (both species are typically approximately 2 μm in length) and would offer nothing in the way of shelter or enhance surface area for colonisation. Therefore, it seems unlikely that surface roughness will play an

Conclusions

The aim of this work was to investigate the development and removal of E. coli and B. cepacia biofilms from a range of substrates by using fluid dynamic gauging (FDG). FDG has been used successfully to determine the yield strength of biofilm adhesion and the strength of intercellular cohesion within biofilms, as a function of incubation time. The results indicate a relationship between maturity and biofilm strength, with a peak after a growth period of 14 days, with weakened structures evident

Acknowledgements

O.P.W. Peck would like to thank the University of Bath for the University Research Studentship. The authors would like to thank Kate Meredith (Department of Pharmacy and Pharmacology) for technical support in biofilm growth. E. coli and B. cepacia were kindly provided by Dr. Harold Tjalsma of the Radboud University Nijmegen Medical Centre. We would also like to thank Dr. Alistair Muir and Mr. Fernando Acosta (Department of Chemical Engineering) for their assistance with the optical microscope

References (39)

  • K.A. Whitehead et al.

    Formation, architecture and functionality of microbial biofilms in the food industry

    Curr. Opin. Food Sci.

    (2015)
  • D.M. Yebra et al.

    Effects of marine microbial biofilms on the biocide release rate from antifouling paints—a model-based analysis

    Prog. Org. Coat.

    (2006)
  • D. Alsteens et al.

    Direct measurement of hydrophobic forces on cell surfaces using AFM

    Langmuir: ACS J. Surf. Colloids

    (2007)
  • A.P. Blanchard et al.

    Peroxygen disinfection of Pseudomonas aeruginosa biofilms on stainless steel discs

    Biofouling

    (1998)
  • Z.F. Burton et al.

    Experiments in Molecular Biology: Biochemical Applications

    (1997)
  • B. Carpentier et al.

    Biofilm and their consequences with particular reference to hygiene in the food industry

    J. Appl. Bacteriol.

    (1993)
  • X. Chen et al.

    Dynamics of biofilm formation under different nutrient levels and the effect on biofouling of a reverse osmosis membrane system

    Biofouling

    (2013)
  • Y.M.J. Chew et al.

    Fluid dynamic gauging for measuring the strength of soft deposits

    J. Food Eng.

    (2004)
  • T.R. De Kievit et al.

    Quorum-sensing genes in pseudomonas aeruginosa biofilms: their role and expression patterns

    Appl. Environ. Microbiol.

    (2001)
  • Cited by (5)

    • Lactobacillus plantarum strains show diversity in biofilm formation under flow conditions

      2022, Heliyon
      Citation Excerpt :

      A number of approaches have been used to pursue biofilm formation in flowing conditions including the use of microfluidics, drip flow, and rotating disk reactors (Schwartz et al., 2010; Oder et al., 2018; Lee et al. 2008; Pereira et al., 2002; Rusconi et al., 2011). In most of studies, selected species of gram-negative bacteria including Escherichia coli, Legionella pneumophila, Pseudomonas spp., and Burkholderia cepacia have been studied under flow conditions (Emge et al., 2016; Mampel et al., 2006; Peck et al. 2019; Teodósio et al., 2011). Gram-positive bacteria also play a crucial role in many environmental, food processing, and medical settings, and only a limited number of studies have assessed the influence of flow on the development and growth of biofilms.

    View full text