Liquid Flow in Biofilm Systems

A model biofilm consisting of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Klebsiella pneumoniae was developed to study the relationships between structural heterogeneity and hydrodynamics. Local fluid velocity in the biofilm system was measured by a noninvasive method of particle image velocimetry, using confocal scanning laser microscopy. Velocity profiles were measured in conduit and porous medium reactors in the presence and absence of biofilm. Liquid flow was observed within biofilm channels; simultaneous imaging of the biofilm allowed the liquid velocity to be related to the physical structure of the biofilm.     

Antimicrobial Action of Carvacrol at Different Stages of Dual-Species Biofilm Development by Staphylococcus aureus and Salmonella enterica Serovar Typhimurium

The effects of carvacrol, a natural biocide, on dual-species biofilms formed by Staphylococcus aureus and Salmonella enterica serovar Typhimurium were investigated with a constant-depth film fermentor. Biofilm development reached a quasi-steady state in 12 days at 25°C with S. aureus predominance (≈99%). Cryosectional analysis detected viable S. aureus and S. enterica serovar Typhimurium at depths of 320 and 180 μm from the film surface, respectively. Carvacrol pulses (1.0 mmol/h) inhibited S. aureus by 2.5 log CFU/biofilm during the early stages of film formation, ultimately causing a significant reduction (P < 0.001) of the staphylococcal population at quasi-steady state. Initial carvacrol pulsing elicited a 3 log CFU/biofilm reduction in viable S. enterica serovar Typhimurium, and additional periodic carvacrol pulses instigated significant inhibition of salmonellae (1 to 2 log CFU/biofilm) during biofilm development. Carvacrol pulsing reduced protein levels fivefold (P < 0.001) during initial biofilm development. Comparative studies with a peroxide-based commercial sanitizer (Spor-Klenz RTU) revealed that this commercial sanitizer was more biocidal than carvacrol during early biofilm development. When the biofilm reached quasi-steady state, however, periodic pulses with 1 mmol of carvacrol per h (P = 0.021) elicited a significantly higher inhibition than Spor-Klenz RTU (P = 0.772). Dual-species microcolonies formed under the influence of continuously fed low carvacrol concentrations (1.0 mmol/h) but failed to develop into a mature quasi-steady-state biofilm and did not reach any stage of film formation in the presence of high concentrations (5.0 mmol/h). These data show that carvacrol is an effective natural intervention to control dual-species biofilm formation.     

The Role of the Liquid Phase in the Degradation of Toluene and m‐Cresol within a Biofilm Trickle‐Bed Reactor

The degradation of toluene and m‐cresol in a biofilm trickle‐bed reactor was experimentally and theoretically investigated. Degradation is the result of the cooperation between suspended and immobilized microorganisms in the trickling film and the biofilm. The role of the trickling film is that of a barrier for mass transfer to the biofilm or that of an additional reaction space. This is the result of physical availability of pollutants to the liquid phase as well as co‐substrate degradation of inherent biomass. An instationary reactor balance model is presented. In addition to this the change in wetting behavior of carrier surface due to biofilm formation is discussed. A partial wetting of biofilm surface by rivulets of the trickling film is proposed. The model was verified by experimental data. The different reactor operation modes denoted as biofilm regime versus trickling film regime for the chosen pollutant system were expressed in terms of dimensionless reactions and transfer numbers. It is shown that the volumetric reaction rates for toluene in a trickling film regime reaches values twice as high as that of a biofilm regime due to the presence of the second substrate m‐cresol. The limiting step in both cases is the mass transfer of oxygen to bacteria in the biofilm or trickling film.     

3D finite element model of biofilm detachment using real biofilm structures from CLSM data

In this work, a three‐dimensional model of fluid–structure interactions (FSI) in biofilm systems is developed in order to simulate biofilm detachment as a result of mechanical processes. Therein, fluid flow past the biofilm surface results in a mechanical load on the structure which in turn causes internal stresses in the biofilm matrix. When the strength of the matrix is exceeded parts of the structure are detached. The model is used to investigate the influence of several parameters related to the mechanical strength of the biofilm matrix, Young's modulus, Reynolds number, and biofilm structure on biofilm detachment. Variations in biofilm strength and flow conditions significantly influence the simulation outcome. With respect to structural properties the model is widely independent from a change of Young's modulus. A further result of this work indicates that the change of biofilm structure due to growth or other processes will significantly change the stress distribution in the biofilm and thereby the detachment rate. An increase of the mechanical load by increasing fluid flow results in a flat surface of the remaining biofilm structure. It is concluded that the change of structure during biofilm development is the key determinant in terms of the detachment behavior. Biotechnol. Bioeng. 2009;103: 177–186. © 2008 Wiley Periodicals, Inc.     

Two-Fluid Model of Biofilm Disinfection

We consider a dynamic model of biofilm disinfection in two dimensions. The biofilm is treated as a viscous fluid immersed in a fluid of less viscosity. The bulk fluid moves due to an imposed external parabolic flow. The motion of the fluid is coupled to the biofilm inducing motion of the biofilm. Both the biofilm and the bulk fluid are dominated by viscous forces, hence the Reynolds number is negligible and the appropriate equations are Stokes equations. The governing partial differential equations are recast as boundary integral equations using a version of the Lorenz reciprocal relationship. This allows for robust treatment of the simplified fluid/biofilm motion. The transport of nutrients and antimicrobials, which depends directly on the velocities of the fluid and biofilm, is also included. Disinfection of the bacteria is considered under the assumption that the biofilm growth is overwhelmed by disinfection. Supported by NSF award DMS-0612467.     

Steady state model for evaluation of external and internal mass transfer effects in an immobilized biofilm

A steady model for the evaluation of external liquid film diffusion and internal pore diffusion effects in an immobilized biofilm system under continuous mode of operation was developed. The model takes into account, substrate diffusion through external liquid film and biofilm. Average rate of substrate consumption in the biofilm was considered. The overall efficiency of the biofilm was mathematically represented by considering the combined effects of substrate penetration and substrate utilization in the biofilm. The model was illustrated using a case study of pyridine biodegradation in a rotating biological contactor immobilized with pyridine degrading microbial film. The model is able to effectively predict both internal and external mass transfer effects in an immobilized biofilm system.     

Permeability of a growing biofilm in a porous media fluid flow analyzed by magnetic resonance displacement‐relaxation correlations

Biofilm growth in porous media is difficult to study non‐invasively due to the opaqueness and heterogeneity of the systems. Magnetic resonance is utilized to non‐invasively study water dynamics within porous media. Displacement‐relaxation correlation experiments were performed on fluid flow during biofilm growth in a model porous media of mono‐dispersed polystyrene beads. The spin–spin T₂ magnetic relaxation distinguishes between the biofilm phase and bulk fluid phase due to water–biopolymer interactions present in the biofilm, and the flow dynamics are measured using PGSE NMR experiments. By correlating these two measurements, the effects of biofilm growth on the fluid dynamics can be separated into a detailed analysis of both the biofilm phase and the fluid phase simultaneously within the same experiment. Within the displacement resolution of these experiments, no convective flow was measured through the biomass. An increased amount of longitudinal hydrodynamic dispersion indicates increased hydrodynamic mixing due to fluid channeling caused by biofilm growth. The effect of different biofilm growth conditions was measured by varying the strength of the bacterial growth medium. Biotechnol. Bioeng. 2013; 110: 1366–1375. © 2012 Wiley Periodicals, Inc.     

Reproducible Biofilm Cultivation of Chemostat-Grown Escherichia coli and Investigation of Bacterial Adhesion on Biomaterials Using a Non-Constant-Depth Film Fermenter

Biomaterials-associated infections are primarily initiated by the adhesion of microorganisms on the biomaterial surfaces and subsequent biofilm formation. Understanding the fundamental microbial adhesion mechanisms and biofilm development is crucial for developing strategies to prevent such infections. Suitable in vitro systems for biofilm cultivation and bacterial adhesion at controllable, constant and reproducible conditions are indispensable. This study aimed (i) to modify the previously described constant-depth film fermenter for the reproducible cultivation of biofilms at non-depth-restricted, constant and low shear conditions and (ii) to use this system to elucidate bacterial adhesion kinetics on different biomaterials, focusing on biomaterials surface nanoroughness and hydrophobicity. Chemostat-grown Escherichia coli were used for biofilm cultivation on titanium oxide and investigating bacterial adhesion over time on titanium oxide, poly(styrene), poly(tetrafluoroethylene) and glass. Using chemostat-grown microbial cells (single-species continuous culture) minimized variations between the biofilms cultivated during different experimental runs. Bacterial adhesion on biomaterials comprised an initial lag-phase I followed by a fast adhesion phase II and a phase of saturation III. With increasing biomaterials surface nanoroughness and increasing hydrophobicity, adhesion rates increased during phases I and II. The influence of materials surface hydrophobicity seemed to exceed that of nanoroughness during the lag-phase I, whereas it was vice versa during adhesion phase II. This study introduces the non-constant-depth film fermenter in combination with a chemostat culture to allow for a controlled approach to reproducibly cultivate biofilms and to investigate bacterial adhesion kinetics at constant and low shear conditions. The findings will support developing and adequate testing of biomaterials surface modifications eventually preventing biomaterial-associated infections.     

Architecture of a Nascent Sphingomonas sp. Biofilm under Varied Hydrodynamic Conditions

The architecture of a Sphingomonas biofilm was studied during early phases of its formation, using strain L138, a gfp-tagged derivative of Sphingomonas sp. strain LB126, as a model organism and flow cells and confocal laser scanning microscopy as experimental tools. Spatial and temporal distribution of cells and exopolymer secretions (EPS) within the biofilm, development of microcolonies under flow conditions representing varied Reynolds numbers, and changes in diffusion length with reference to EPS production were studied by sequential sacrificing of biofilms grown in multichannel flow cells and by time-lapse confocal imaging. The area of biofilm in terms of microscopic images required to ensure representative sampling varied by an order of magnitude when area of cell coverage (2 × 10⁵ μm²) or microcolony size (1 × 10⁶ μm²) was the biofilm parameter under investigation. Hence, it is necessary to establish the inherent variability of any biofilm metric one is attempting to quantify. Sphingomonas sp. strain L138 biofilm architecture consisted of microcolonies and extensive water channels. Biomass and EPS distribution were maximal at 8 to 9 μm above the substratum, with a high void fraction near the substratum. Time-lapse confocal imaging and digital image analysis showed that growth of the microcolonies was not uniform: adjacently located colonies registered significant growth or no growth at all. Microcolonies in the biofilm had the ability to move across the attachment surface as a unit, irrespective of fluid flow direction, indicating that movement of microcolonies is an inherent property of the biofilm. Width of water channels decreased as EPS production increased, resulting in increased diffusion distances in the biofilm. Changing hydrodynamic conditions (Reynolds numbers of 0.07, 52, and 87) had no discernible influence on the characteristics of microcolonies (size, shape, or orientation with respect to flow) during the first 24 h of biofilm development. Inherent factors appear to have overriding influence, vis-à-vis environmental factors, on early stages of microcolony development under these laminar flow conditions.     

A comparative study of the effect of probiotics on cariogenic biofilm model for preventing dental caries

Dental caries is induced by oral biofilm containing Streptococcus mutans. Probiotic bacteria were mainly studied for effect on the gastrointestinal tract and have been known to promote human health. However, the information of probiotics for oral health has been lack yet. In this study, we investigated influence of various probiotics on oral bacteria or cariogenic biofilm and evaluated candidate probiotics for dental caries among them. The antimicrobial activity of the spent culture medium of probiotics for oral streptococci was performed. Probiotics were added during the biofilm formation with salivary bacteria including S. mutans. The oral biofilms were stained with a fluorescent dye and observed using the confocal laser scanning microscope. To count bacteria in the biofilm, the bacteria were plated on MSB and BHI agar plates after disrupting the biofilm and cultivated. Glucosyltransferases (gtfs) expression of S. mutans and integration of lactobacilli into the biofilm were evaluated by real-time RT-PCR. Among probiotics, Lactobacillus species strongly inhibited growth of oral streptococci. Moreover, Lactobacillus species strongly inhibited formation of cariogenic biofilm model. The expression of gtfs was significantly reduced by Lactobacillus rhamnosus. The integration of L. rhamnosus into the biofilm model did not exhibit. However, L. acidophilus and L casei integrated into the biofilm model. These results suggest that L. rhamnosus may inhibit oral biofilm formation by decreasing glucan production of S. mutans and antibacterial activity and did not integrate into oral biofilm, which can be a candidate for caries prevention strategy.     

Effect of biofilm growth on steady-state biofilm models

The results of numerical simulations for a growing biological film are presented to justify the use of steady-state biofilm models for approximating the behavior of both unlimited and shear-limited biofilms. For an unlimited biofilm we show that although the total biofilm thickness may continue to increase over time, the active biofilm volume will reach a constant value. We also show that the profile of active microorganisms within the biofilm will become constant with respect to the biofilm/fluid interface and simply move outward as the biofilm thickness increases. For a shear-limited biofilm we similarly show that once a "limiting" thickness has been reached the active biofilm volume, substrate consumption, and profile of active microorganisms within the biofilm will also be independent of the biofilm thickness.     

Stratified mixed-culture biofilm model for anaerobic digestion

Development of a novel two-layer anaerobic biofilm model is based on substrate utilization kinetics and mass transport. The model is applied to steady-state conditions for a fixed-film anaerobic reactor. The microbial film is considered to consist of two distinct biofilm layers, one adjacent to the second, with an acidogenic bacteria biofilm forming the outer layer and a methanogenic film the inner one. The model assumes that sugars are only metabolized by the first layer and converted into volatile fatty acids (VFA), while fatty acids are taken up only by the inner layer. The model is able to predict both substrate flux net uptake and methane production for steady-state conditions. The results of modeling agree with methane production experimental data published elsewhere. Further, the model shows why layered fixed-film reactors can withstand high and inhibitory concentrations of volatile fatty acids as well as severe overloading without failure.     

Computational study of the drag and oscillatory movement of biofilm streamers in fast flows

Hydrodynamic conditions have a significant impact on the biofilm lifecycle. Not well understood is the fact that biofilms, in return, also affect the flow pattern. A decade ago, it was already shown experimentally that under fast flows, biofilm streamers form and oscillate with large amplitudes. This work is a first attempt to answer the questions on the mechanisms behind the oscillatory movement of the streamers, and whether this movement together with the special streamlined form of the streamers, have both a physical and biological benefit for biofilms. In this study, a state of the art two‐dimensional fluid–structure interaction model of biofilm streamers is developed, which implements a transient coupling between the fluid and biofilm mechanics. Hereby, it is clearly shown that formation of a Kármán vortex street behind the streamer body is the main source of the periodic oscillation of the streamers. Additionally it is shown that the formation of streamers reduces the fluid forces which biofilm surface experiences. Biotechnol. Bioeng. 2010; 105: 600–610. © 2009 Wiley Periodicals, Inc.     

Steady-state solution of a two-species biofilm problem

Through a thorough investigation of the boundary conditions for a general two-species biofilm model, a simple and fast method for solving the steady-state case is developed and presented. The methods used may be extended to biofilm models in which more than two species are considered. Four different sets of boundary conditions are possible for the two-species biofilm model. Each set is shown to be asymptotically stable. A biofilm model describing the competition between autotrophic and heterotrophic bacteria and a biofilm model considering only Nitrosomonas and Nitrobacter are used for illustration. A parameter L(crit), critical film thickness for bacterial coexistence, is introduced from which criteria on the bulk concentrations for coexistence are derived. From these criteria it is seen that the thinner the biofilm, the more restrictive the conditions are for steady-state coexistence. For thin biofilms there may, in many cases, be no point in considering more than one species in the biofilm model. Furthermore, the gradients of the bacterial concentrations are in many cases negligible in thin biofilms, and the biofilm may then be assumed to be homogeneous. The criteria on the bulk concentrations together with the four sets of boundary conditions provide the necessary information for a direct solution of the steady-state two-species biofilm model by means of an ordinary differential and algebraic equation solver. (c) 1996 John Wiley & Sons, Inc.     

Understanding photosynthetic biofilm productivity and structure through 2D simulation

We present a spatial model describing the growth of a photosynthetic microalgae biofilm. In this 2D-model we consider photosynthesis, cell carbon accumulation, extracellular matrix excretion, and mortality. The rate of each of these mechanisms is given by kinetic laws regulated by light, nitrate, oxygen and inorganic carbon. The model is based on mixture theory and the behaviour of each component is defined on one hand by mass conservation, which takes into account biological features of the system, and on the other hand by conservation of momentum, which expresses the physical properties of the components. The model simulates the biofilm structural dynamics following an initial colonization phase. It shows that a 75 μm thick active region drives the biofilm development. We then determine the optimal harvesting period and biofilm height which maximize productivity. Finally, different harvesting patterns are tested and their effect on biofilm structure are discussed. The optimal strategy differs whether the objective is to recover the total biofilm or just the algal biomass.     

Identification of Genes Involved in Polysaccharide-Independent Staphylococcus aureus Biofilm Formation

Staphylococcus aureus is a potent biofilm former on host tissue and medical implants, and biofilm growth is a critical virulence determinant for chronic infections. Recent studies suggest that many clinical isolates form polysaccharide-independent biofilms. However, a systematic screen for defective mutants has not been performed to identify factors important for biofilm formation in these strains. We created a library of 14,880 mariner transposon mutants in a S. aureus strain that generates a proteinaceous and extracellular DNA based biofilm matrix. The library was screened for biofilm defects and 31 transposon mutants conferred a reproducible phenotype. In the pool, 16 mutants overproduced extracellular proteases and the protease inhibitor α₂-macroglobulin restored biofilm capacity to 13 of these mutants. The other 15 mutants in the pool displayed normal protease levels and had defects in genes involved in autolysis, osmoregulation, or uncharacterized membrane proteins. Two transposon mutants of interest in the GraRS two-component system and a putative inositol monophosphatase were confirmed in a flow cell biofilm model, genetically complemented, and further verified in a community-associated methicillin-resistant S. aureus (CA-MRSA) isolate. Collectively, our screen for biofilm defective mutants identified novel loci involved in S. aureus biofilm formation and underscored the importance of extracellular protease activity and autolysis in biofilm development.     

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.     

Tissue Plasminogen Activator Coating on Implant Surfaces Reduces Staphylococcus aureus Biofilm Formation

Staphylococcus aureus biofilm infections of indwelling medical devices are a major medical challenge because of their high prevalence and antibiotic resistance. As fibrin plays an important role in S. aureus biofilm formation, we hypothesize that coating of the implant surface with fibrinolytic agents can be used as a new method of antibiofilm prophylaxis. The effect of tissue plasminogen activator (tPA) coating on S. aureus biofilm formation was tested with in vitro microplate biofilm assays and an in vivo mouse model of biofilm infection. tPA coating efficiently inhibited biofilm formation by various S. aureus strains. The effect was dependent on plasminogen activation by tPA, leading to subsequent local fibrin cleavage. A tPA coating on implant surfaces prevented both early adhesion and later biomass accumulation. Furthermore, tPA coating increased the susceptibility of biofilm infections to antibiotics. In vivo, significantly fewer bacteria were detected on the surfaces of implants coated with tPA than on control implants from mice treated with cloxacillin. Fibrinolytic coatings (e.g., with tPA) reduce S. aureus biofilm formation both in vitro and in vivo, suggesting a novel way to prevent bacterial biofilm infections of indwelling medical devices.     

Effect of operational conditions on the rate of citric acid production by rotating disk contactor using Aspergillus niger

The effect of operational conditions such as the specific biofilm area, oxygen concentration, and rotation speed on citric acid production by a rotating disk contactor (RDC) was examined. The overall productivity for a repeated batch culture was also compared with that for a non-repeated batch culture. The citric acid production rate per unit biofilm area (P) was almost constant regardless of the value of the specific film area. The rotation speed of the disks had no effect on P in the range of 5–20 rpm. The apparent saturation constant of dissolved oxygen concentration was 2.05 mg/l. Overall citric acid productivity for a 3-times-repeated batch culture was 1.7 times higher than that for the non-repeated batch culture.