A model of fluid-biofilm interaction using a Burger material law

A two-dimensional finite element model of the biofilm response to flow was developed. The numerical code sequentially coupled the fluid dynamics of turbulent, incompressible flow with the mechanical response of a single hemispherical biofilm cluster (approximately 100 microm) attached to the flow boundary. A non-linear Burger material law was used to represent the viscoelastic response of a representative microbial biofilm. This constitutive law was incorporated into the numerical model as a Prony series representation of the biofilm's relaxation modulus. Model simulations illuminated interesting details of this fluid-structure interaction. Simulations revealed that softer biofilms (characterized by lower elastic moduli) were highly susceptible to lift forces and consequently were subject to even greater drag forces found higher in the velocity field. A bimodal deformation path due to the two Burger relaxation times was also observed in several simulations. This suggested that interfacial biofilm may be most susceptible to hydrodynamically induced detachment during the initial relaxation time. This result may prove useful in developing removal strategies. Additionally, plots of lift versus drag suggested that the deformation paths taken by viscoelastic biofilms are largely insensitive to specific material coefficients. Softer biofilms merely seem to follow the same path (as a stiffer biofilm) at a faster rate. These relationships may be useful in estimating the hydrodynamic forces acting on an attached biofilm based on changes in scale and cataloged material properties.     

Predicting biofilm deformation with a viscoelastic phase-field model: Modeling and experimental studies

Biofilms commonly develop in flowing aqueous environments, where the flow causes the biofilm to deform. Because biofilm deformation affects the flow regime, and because biofilms behave as complex heterogeneous viscoelastic materials, few models are able to predict biofilm deformation. In this study, a phase-field (PF) continuum model coupled with the Oldroyd-B constitutive equation was developed and used to simulate biofilm deformation. The accuracy of the model was evaluated using two types of biofilms: a synthetic biofilm, made from alginate mixed with bacterial cells, and a Pseudomonas aeruginosa biofilm. Shear rheometry was used to experimentally determine the mechanical parameters for each biofilm, used as inputs for the model. Biofilm deformation under fluid flow was monitored experimentally using optical coherence tomography. The comparison between the experimental and modeling geometries, for selected horizontal cross sections, after fluid-driven deformation was good. The relative errors ranged from 3.2 to 21.1% for the synthetic biofilm and from 9.1 to 11.1% for the P. aeruginosa biofilm. This is the first demonstration of the effectiveness of a viscoelastic PF biofilm model. This model provides an important tool for predicting biofilm viscoelastic deformation. It also can benefit the design and control of biofilms in engineering systems.     

Viscoelastic properties of a mixed culture biofilm from rheometer creep analysis

The mechanical properties of mixed culture biofilms were determined by creep analysis using an AR1000 rotating disk rheometer. The biofilms were grown directly on the rheometer disks which were rotated in a chemostat for 12 d. The resulting biofilms were heterogeneous and ranged from 35 microns to 50 microns in thickness. The creep curves were all viscoelastic in nature. The close agreement between stress and strain ratio of a sample tested at 0.1 and 0.5 Pa suggested that the biofilms were tested in the linear viscoelastic range and supported the use of linear viscoelastic theory in the development of a constitutive law. The experimental data was fit to a 4-element Burger spring and dashpot model. The shear modulus (G) ranged from 0.2 to 24 Pa and the viscous coefficient (eta) from 10 to 3000 Pa. These values were in the same range as those previously estimated from fluid shear deformation of biofilms in flow cells. A viscoelastic biofilm model will help to predict shear related biofilm phenomena such as elevated pressure drop, detachment, and the flow of biofilms over solid surfaces.     

Viscoelastic properties of Staphylococcus aureus and Staphylococcus epidermidis mono‐microbial biofilms

The viscoelastic properties of mono‐microbial biofilms produced by ocular and reference staphylococcal strains were investigated. The microorganisms were characterized for their haemolytic activity and agr typing and the biofilms, grown on stainless steel surface under static conditions, were analysed by Confocal Laser Scanning Microscopy. Static and dynamic rheometric tests were carried out to determine the steady‐flow viscosity and the elastic and viscous moduli. The analysed biofilms showed the typical time‐dependent behaviour of viscoelastic materials with considerable elasticity and mechanical stability except for Staphylococcus aureus ATCC 29213 biofilm which showed a very fragile structure. In particular, S. aureus 6ME biofilm was more compact than other staphylococcal biofilms studied with a yield stress ranging between 2 and 3 Pa. The data obtained in this work could represent a starting point for developing new therapeutic strategies against biofilm‐associated infections, such as improving the drug effect by associating an antimicrobial agent with a biofilm viscoelasticity modifier.     

Applying the digital image correlation method to estimate the mechanical properties of bacterial biofilms subjected to a wall shear stress

A digital image correlation (DIC) method was applied to characterize the mechanical behavior of Pseudomonas aeruginosa biofilms in response to wall shear stress using digital video micrographs taken from biofilm flow cells. The appearance of the biofilm in the transmitted light photomicrographs presented a natural texture which was highly conducive to random encoding for DIC. The displacement fields were calculated for two biofilm specimens. The DIC method concurred with previous analysis showing that biofilms exhibit viscoelastic behavior, but had the advantage over simple length measurements of longitudinal strain that it could precisely measure local strains in length (x) and width (y) within biofilm clusters with a 2 μm resolution as a function of time and wall shear stress. It was concluded that DIC was more accurate at measuring elastic moduli than simple length measurements, but that time-lapse 3D images would enable even more accurate estimates to be performed.     

Viscoelastic Properties of a Mixed Culture Biofilm from Rheometer Creep Analysis

The mechanical properties of mixed culture biofilms were determined by creep analysis using an AR1000 rotating disk rheometer. The biofilms were grown directly on the rheometer disks which were rotated in a chemostat for 12 d. The resulting biofilms were heterogeneous and ranged from 35?μm to 50?μm in thickness. The creep curves were all viscoelastic in nature. The close agreement between stress and strain ratio of a sample tested at 0.1 and 0.5 Pa suggested that the biofilms were tested in the linear viscoelastic range and supported the use of linear viscoelastic theory in the development of a constitutive law. The experimental data was fit to a 4-element Burger spring and dashpot model. The shear modulus (G) ranged from 0.2 to 24 Pa and the viscous coefficient (η) from 10 to 3000 Pa. These values were in the same range as those previously estimated from fluid shear deformation of biofilms in flow cells. A viscoelastic biofilm model will help to predict shear related biofilm phenomena such as elevated pressure drop, detachment, and the flow of biofilms over solid surfaces.     

Mini-review: convection around biofilms

Water that flows around a biofilm influences the transport of solutes into and out of the biofilm and applies forces to the biofilm that can cause it to deform and detach. Engineering approaches to quantifying and understanding these phenomena are reviewed in the context of biofilm systems. The slow-moving fluid adjacent to the biofilm acts as an insulator for diffusive exchange. External mass transfer resistance is important because it can exacerbate oxygen or nutrient limitation in biofilms, worsen product inhibition, affect quorum sensing, and contribute to the development of tall, fingerlike biofilm clusters. Measurements of fluid motion around biofilms by particle velocimetry and magnetic resonance imaging indicate that water flows around, but not through biofilm cell clusters. Moving fluid applies forces to biofilms resulting in diverse outcomes including viscoelastic deformation, rolling, development of streamers, oscillatory movement, and material failure or detachment. The primary force applied to the biofilm is a shear force in the main direction of fluid flow, but complex hydrodynamics including eddies, vortex streets, turbulent wakes, and turbulent bursts result in additional force components.     

Long-term monitoring of biofilm growth and disinfection using a quartz crystal microbalance and reflectance measurements

A biofilm reactor was constructed to monitor the long-term growth and removal of biofilms as monitored by the use of a quartz crystal microbalance (QCM) and a novel optical method. The optical method measures the reflectance of white light off the surface of the quartz crystal microbalance electrode (gold) for determination of the biofilm thickness. Biofilm growth of Pseudomonas aeruginosa (PA) on the surface was used as a model system. Bioreactors were monitored for over 6 days. Expressing the QCM data as the ratio of changes in resistance to changes in frequency (DeltaR/Deltaf) facilitated the comparison of individual biofilm reactor runs. The various stages of biofilm growth and adaptation to low nutrients showed consistent characteristic changes in the DeltaR/Deltaf ratio, a parameter that reflects changes in the viscoelastic properties of the biofilm. The utility of white light reflectance for thickness measurements was shown for those stages of biofilm growth when the solution was not turbid due to high numbers of unattached cells. The thickness of the biofilms after 6 days ranged from 48 mum to 68 mum. Removal of the biofilm by a disinfectant (chlorine) was also measured in real time. The combination of QCM and reflectance allowed us to monitor in real time changes in the viscoelastic properties and thickness of biofilms over long periods of time.     

Adaptive responses to antimicrobial agents in biofilms

Bacterial biofilms demonstrate adaptive resistance in response to antimicrobial stress more effectively than corresponding planktonic populations. We propose here that, in biofilms, reaction-diffusion limited penetration may result in only low levels of antimicrobial exposure to deeper regions of the biofilm. Sheltered cells are then able to enter an adapted resistant state if the local time scale for adaptation is faster than that for disinfection. This mechanism is not available to a planktonic population. A mathematical model is presented to illustrate. Results indicate that, for a sufficiently thick biofilm, cells in the biofilm implement adaptive responses more effectively than do freely suspended cells. Effective disinfection requires applied biocide concentration that increases quadratically or exponentially with biofilm thickness.     

Uniaxial compression measurement device for investigation of the mechanical stability of biofilms.

The mechanical stability of biofilms is important for biotechnology, as sloughing of the biomass due to mechanical failure of the biofilm matrix can lead to severe interferences with biofilm processes. In cases of biofouling, biofilms have to be removed, in which case their mechanical stability must be overcome. The apparent modulus of elasticity and the yield strength as obtained from uniaxial compression experiments can be taken as parameters indicative for the mechanical stability of a biofilm. A film rheometer is presented which allows for the determination of these quantities, using model biofilms of Pseudomonas aeruginosa grown on membrane filters. The compressive stress-strain behaviour up to the point of failure is recorded at a compression speed of 1 microm s(-1). In accordance with the stress-strain curve, the investigated biofilm can be described as viscoelastic material, which demonstrates plastic flow properties. The extracellular polymeric substances (EPS), which keep biofilms together, form a temporary network of fluctuating junction points. Above the yield point, the gel structure fails and the system behaves as a highly viscous fluid. The apparent modulus of elasticity and the yield point are considered to be useful parameters for characterizing the mechanical properties of biofilms.     

Structural deformation of bacterial biofilms caused by short-term fluctuations in fluid shear: an in situ investigation of biofilm rheology.

The physical properties (rheology) of biofilms will determine the shape and mechanical stability of the biofilm structure and consequently affect both mass transfer and detachment processes. Biofilm viscoelasticity is also thought to increase fluid energy losses in pipelines. Yet there is very little information on the rheology of intact biofilms. This is due in part to the difficulty in using conventional testing techniques. The size and nature of biofilms makes them difficult to handle, while removal from a surface destroys the integrity of the sample. We have developed a method which allowed us to conduct simple stress-strain and creep experiments on mixed and pure culture biofilms in situ by observing the structural deformations caused by changes in hydrodynamic shear stress (tau(w)). The biofilms were grown under turbulent pipe flow (flow velocity (u) = 1 m/s, Reynolds number (Re) = 3600, tau(w) = 5. 09 N/m(2)) for between 12 and 23 days. The resulting biofilms were heterogeneous and consisted of filamentous streamers that were readily deformed by changes in tau(w). At tau(w) of 10.11 N/m(2) the streamers were flattened so that the thickness was reduced by 25%. We estimated that the shear modulus (G) of the mixed culture biofilm was 27 N/m(2) and the apparent elastic modulus (E(app)) of both biofilms was in the range of 17 to 40 N/m(2). The biofilms behaved like elastic and viscoelastic solids below the tau(w) at which they were grown but behaved like viscoelastic fluids at elevated tau(w). The implications of these results for fluid energy losses and the processes of mass transfer and detachment are discussed.     

Effect of biofilm on the rheological properties of cohesive sediment

Biofilm, a product of metabolic activity, has an important effect on the physico-chemical properties of cohesive sediment. However, little effort has been made to determine the substantial effects of biofilm growth on specific sediment properties, for example rheological properties. Understanding the changes associated with biofilm growth and quantifying the time scales over which these changes occur are important for understanding how biofilms mediate sediment properties and processes and the development of sediment transport mechanics. The effect of biofilm on the rheological properties of cohesive sediment was investigated experimentally. The rheological properties of sediment slurries with and without biofilm at different growth phases were measured and compared. Measurement showed biofilm growth has a significant effect on the rheological properties of cohesive sediment. Rheological equations for biofilm sediment and expressions for rheological properties which change over time are proposed. These equations, and information on biofilm sediment, are important for inclusion of biosedimentological processes in models of sediment dynamics.     

Membrane-attached biofilms for VOC wastewater treatment I: Novel in situ biofilm thickness measurement technique

This article reports a novel nondisruptive technique for measuring the thicknesses of membrane-attached biofilms in situ, using a single tube extractive membrane bioreactor (STEMB). The biodegradation of a toxic volatile organic compound (VOC) (1,2-dichloroethane [DCE]) by Xanthobacter autotrophicus GJ10 has been used as a model system to develop the technique. The results give information on the biomass-silicone rubber attachment phenomena, and on the development over time of biofilms growing on the silicone membrane, without disrupting operation. Experimental results are presented showing the evolution over time of biofilm thickness, and also the density of biofilms for four experimental runs. The hydrodynamic conditions on the biomedium side of the membrane were found to influence the initial attachment phenomena and subsequent biofilm growth. (c) 1995 John Wiley & Sons, Inc.     

Absolute Quantitation of Bacterial Biofilm Adhesion and Viscoelasticity by Microbead Force Spectroscopy

Bacterial biofilms are the most prevalent mode of bacterial growth in nature. Adhesive and viscoelastic properties of bacteria play important roles at different stages of biofilm development. Following irreversible attachment of bacterial cells onto a surface, a biofilm can grow in which its matrix viscoelasticity helps to maintain structural integrity, determine stress resistance, and control ease of dispersion. In this study, a novel application of force spectroscopy was developed to characterize the surface adhesion and viscoelasticity of bacterial cells in biofilms. By performing microbead force spectroscopy with a closed-loop atomic force microscope, we accurately quantified these properties over a defined contact area. Using the model gram-negative bacterium Pseudomonas aeruginosa, we observed that the adhesive and viscoelastic properties of an isogenic lipopolysaccharide mutant wapR biofilm were significantly different from those measured for the wild-type strain PAO1 biofilm. Moreover, biofilm maturation in either strain also led to prominent changes in adhesion and viscoelasticity. To minimize variability in force measurements resulting from experimental parameter changes, we developed standardized conditions for microbead force spectroscopy to enable meaningful comparison of data obtained in different experiments. Force plots measured under standard conditions showed that the adhesive pressures of PAO1 and wapR early biofilms were 34 ± 15 Pa and 332 ± 47 Pa, respectively, whereas those of PAO1 and wapR mature biofilms were 19 ± 7 Pa and 80 ± 22 Pa, respectively. Fitting of creep data to a Voigt Standard Linear Solid viscoelasticity model revealed that the instantaneous and delayed elastic moduli in P. aeruginosa were drastically reduced by lipopolysaccharide deficiency and biofilm maturation, whereas viscosity was decreased only for biofilm maturation. In conclusion, we have introduced a direct biophysical method for simultaneously quantifying adhesion and viscoelasticity in bacterial biofilms under native conditions. This method could prove valuable for elucidating the contribution of genetic backgrounds, growth conditions, and environmental stresses to microbial community physiology.     

Hydroelectricity and fish: a synopsis of comprehensive studies of upstream and downstream passage of anadromous wild Atlantic salmon, <Emphasis Type="Italic">Salmo salar</Emphasis>, on the Exploits River,Canada

Microalgal biofilms are associated with considerable variability in the properties of natural sediments, yet little effort has been made to isolate micro-scale spatial and temporal changes in sediment properties caused by the growth of a biofilm. Understanding the changes associated with biofilm growth and quantifying the time scales over which these changes occur is important for developing suitable experimental designs and for understanding how biofilms mediate sediment properties and processes. The development of a microphytobenthic biofilm and associated changes in the sediment was investigated over 45 days in the laboratory. The biogeochemical properties of the sediment: bulk density, water content, chlorophyll a concentration and colloidal carbohydrate concentration were measured on a sub-millimetre scale in the top 2 mm. The erosion threshold was measured with a Cohesive Strength Meter (CSM). Biofilm development was rapid, with changes in the properties occurring after 1 day and a visible film forming after just 3 days. The largest changes in sediment properties tended to occur in the surface 200 μm through time, with some variables also showing a differing response with depth. There were significant changes in water content, chlorophyll a concentration, colloidal carbohydrate concentration and erosion threshold in the surface 2 mm, with a general trend to increase with time. Bulk density was highly variable and did not show a consistent pattern of change with time. Erosion threshold was positively correlated with water content, chlorophyll a and colloidal carbohydrate in the surface 200 μm and these were also positively correlated with each other. Low Temperature Scanning Electron Microscopy (LTSEM) images revealed changes in the surface sediment structure and the formation of a thick multi-layer biofilm. The rapidity of biofilm growth and development and the associated changes to the sediment should be considered when designing experiments that investigate biofilms and properties of sediments and/or that involve biocide treatments or disturbance to the sediment.     

Effects of biofilm heterogeneity on the apparent mechanical properties obtained by shear rheometry

Rheometry is an experimental technique widely used to determine the mechanical properties of biofilms. However, it characterizes the bulk mechanical behavior of the whole biofilm. The effects of biofilm mechanical heterogeneity on rheometry measurements are not known. We used laboratory experiments and computer modeling to explore the effects of biofilm mechanical heterogeneity on the results obtained by rheometry. A synthetic biofilm with layered mechanical properties was studied, and a viscoelastic biofilm theory was employed using the Kelvin–Voigt model. Agar gels with different concentrations were used to prepare the layered, heterogeneous biofilm, which was characterized for mechanical properties in shear mode with a rheometer. Both experiments and simulations indicated that the biofilm properties from rheometry were strongly biased by the weakest portion of the biofilm. The simulation results using linearly stratified mechanical properties from a previous study also showed that the weaker portions of the biofilm dominated the mechanical properties in creep tests. We note that the model can be used as a predictive tool to explore the mechanical behavior of complex biofilm structures beyond those accessible to experiments. Since most biofilms display some degree of mechanical heterogeneity, our results suggest caution should be used in the interpretation of rheometry data. It does not necessarily provide the “average” mechanical properties of the entire biofilm if the mechanical properties are stratified.     

A porous elastic model for bacterial biofilms: application to the simulation of deformation of bacterial biofilms under microfluidic jet impingement

The presence of bacterial biofilms is detrimental in a wide range of healthcare situations especially wound healing. Physical debridement of biofilms is a method widely used to remove them. This study evaluates the use of microfluidic jet impingement to debride biofilms. In this case, a biofilm is treated as a saturated porous medium also having linear elastic properties. A numerical modeling approach is used to calculate the von Mises stress distribution within a porous medium under fluid-structure interaction (FSI) loading to determine the initial rupture of the biofilm structure. The segregated model first simulates the flow field to obtain the FSI interface loading along the fluid-solid interface and body force loading within the porous medium. A stress-strain model is consequently used to calculate the von Mises stress distribution to obtain the biofilm deformation. Under a vertical jet, 60% of the deformation of the porous medium can be accounted for by treating the medium as if it was an impermeable solid. However, the maximum deformation in the porous medium corresponds to the point of maximum shear stress which is a different position in the porous medium than that of the maximum normal stress in an impermeable solid. The study shows that a jet nozzle of 500 μm internal diameter (ID) with flow of Reynolds number (Re) of 200 can remove the majority of biofilm species.     

Effects of carbon and oxygen limitations and calcium concentrations on biofilm removal processes

Bacterial biofilm removal processes due to shear and catastrophic sloughing have been investigated in a turbulent flow system under conditions of carbon versus oxygen substrate limitations and varying aqueous phase calcium concentrations. Biofilm cellular and extracellular polymer carbon, total biofilm carbon and mass, and biofilm calcium concentrations are measured for pure culture biofilms of the facultative aerobe, Pseudomonas putida ATCC 11172. Results indicate oxygen-limited biofilms reach a higher steady-state biofilm organic carbon level than carbon-limited biofilms. Oxygen-limited biofilms also exhibit (1) a higher extracellular polymer-carbon: cell-carbon ratio throughout biofilm development and (2) a higher biofilm calcium content than carbon-limited biofilms. Increasing aqueous phase calcium concentrations increase the amount of biofilm calcium in both cases; the rate of calcium accumulation in oxygen-limited biofilms increases with increasing liquid phase calcium concentrations over the entire range studied while the rates of calcium accumulation in carbon-limited biofilms appear independent of aqueous phase calcium concentrations above 11.0 mg/L. Oxygen-limited biofilms with their higher extracellular polymer and calcium content exhibit shear removal rates that are 20-40% of those observed for carbon-limited biofilms. However, it is the oxygen-limited biofilms that experience catastrophic sloughing events. The carbon-limited biofilms studied here never sloughed even if subjected to intentional long-term deprivation of all nutrients. Reduced shear removal and the susceptibility to sloughing of the oxygen-limited biofilms are attributed to their more cohesive structure bought about by their relatively greater extracellular polymer production.     

Mechanical properties and failure of Streptococcus mutans biofilms, studied using a microindentation device

Knowledge of mechanical properties and failure mechanisms of biofilms is needed to determine how biofilms react on mechanical stress. Methods currently available cannot be used to determine mechanical properties of biofilms on a small scale with high accuracy. A novel microindentation apparatus in combination with a confocal microscope was used to determine the viscoelastic properties of Streptococcus mutans biofilms. The apparatus comprises a small glass indenter and a highly sensitive force transducer. It was shown that the present biofilm, grown under still conditions, behaves as a viscoelastic solid with a storage modulus of 1-8 kPa and a loss modulus of 5-10 kPa at a strain of 10%. Biofilm failure was investigated visually through a confocal microscope by dragging the indenter through the biofilm. It was shown that the tensile strength of the biofilm is predominantly determined by the tensile strength of the extracellular polysaccharide matrix. The combination of microindentation and confocal microscopy is a promising technique to determine and characterize the mechanical properties of soft materials in various fields of microbiology.