A novel biofilm channel for evaluating the adhesion of diatoms to non-biocidal coatings

Laboratory assessment of the adhesion of diatoms to non-toxic fouling-release coatings has tended to focus on single cells rather than the more complex state of a biofilm. A novel culture system based on open channel flow with adjustable bed shear stress values (0–2.4?Pa) has been used to produce biofilms of Navicula incerta. Biofilm development on glass and polydimethylsiloxane elastomer (PDMSe) showed a biphasic relationship with bed shear stress, which was characterised by regions of biofilm stability and instability reflecting cohesion between cells relative to the adhesion to the substratum. On glass, a critical shear stress of 1.3–1.4?Pa prevented biofilm development, whereas on PDMS, biofilms continued to grow at 2.4?Pa. Studies of diatom biofilms cultured on zwitterionic coatings using a bed shear stress of 0.54?Pa showed lower biomass production and adhesion strength on poly(sulfobetaine methacrylate) compared to poly(carboxybetaine methacrylate). The dynamic biofilm approach provides additional information to supplement short duration laboratory evaluations.     

Analysis of the mechanical stability and surface detachment of mature Streptococcus mutans biofilms by applying a range of external shear forces

Well-established biofilms formed by Streptococcus mutans via exopolysaccharide matrix synthesis are firmly attached to tooth surfaces. Enhanced understanding of the physical properties of mature biofilms may lead to improved approaches to detaching or disassembling these highly organized and adhesive structures. Here, the mechanical stability of S. mutans biofilms was investigated by determining their ability to withstand measured applications of shear stress using a custom-built device. The data show that the initial biofilm bulk (~ 50% biomass) was removed after exposure to 0.184 and 0.449 N m^?2 for 67 and 115 h old biofilms. However, removal of the remaining biofilm close to the surface was significantly reduced (vs initial bulk removal) even when shear forces were increased 10-fold. Treatment of biofilms with exopolysaccharide-digesting dextranase substantially compromised their mechanical stability and rigidity, resulting in bulk removal at a shear stress as low as 0.027 N m^?2 and > a two-fold reduction in the storage modulus (G′). The data reveal how incremental increases in shear stress cause distinctive patterns of biofilm detachment, while demonstrating that the exopolysaccharide matrix modulates the resistance of biofilms to mechanical clearance.     

Shear‐induced detachment of biofilms from hollow fiber silicone membranes

A suite of techniques was utilized to evaluate the correlation between biofilm physiology, fluid‐induced shear stress, and detachment in hollow fiber membrane aerated bioreactors. Two monoculture species biofilms were grown on silicone fibers in a hollow fiber membrane aerated bioreactors (HfMBR) to assess detachment under laminar fluid flow conditions. Both physiology (biofilm thickness and roughness) and nutrient mass transport data indicated the presence of a steady state mature biofilm after 3 weeks of development. Surface shear stress proved to be an important parameter for predicting passive detachment for the two biofilms. The average shear stress at the surface of Nitrosomonas europaea biofilms (54.5 ± 3.2 mPa) was approximately 20% higher than for Pseudomonas aeruginosa biofilms (45.8 ± 7.7 mPa), resulting in higher biomass detachment. No significant difference in shear stress was measured between immature and mature biofilms of the same species. There was a significant difference in detached biomass for immature vs. mature biofilms in both species. However, there was no difference in detachment rate between the two species. Biotechnol. Bioeng. 2013; 110: 525–534. © 2012 Wiley Periodicals, Inc.     

The influence of flow cell geometry related shear stresses on the distribution,structure and susceptibility of Pseudomonas aeruginosa 01 biofilms

The effects of non-uniform hydrodynamic conditions resulting from flow cell geometry (square and rectangular cross-section) on Pseudomonas aeruginosa 01 (PAO1) biofilm formation, location, and structure were investigated for nominally similar flow conditions using a combination of confocal scanning laser microscope (CSLM) and computational fluid dynamics (CFD). The thickness and surface coverage of PAO1 biofilms were observed to vary depending on the location in the flow cell and thus also the local wall shear stress. The biofilm structure in a 5:1 (width to height) aspect ratio rectangular flow cell was observed to consist mainly of a layer of bacterial cells with thicker biofilm formation observed in the flow cell corners. For square cross-section (1:1 aspect ratio) flow cells, generally thicker and more uniform surface coverage biofilms were observed. Mushroom shaped structures with hollow centers and wall breaks, indicative of ‘seeding’ dispersal structures, were found exclusively in the square cross-section tubes. Exposure of PAO1 biofilms grown in the flow cells to gentamicin revealed a difference in susceptibility. Biofilms grown in the rectangular flow cell overall exhibited a greater susceptibility to gentamicin compared to those grown in square flow cells. However, even within a given flow cell, differences in susceptibility were observed depending on location. This study demonstrates that the spanwise shear stress distribution within the flow cells has an important impact on the location of colonization and structure of the resultant biofilm. These differences in biofilm structure have a significant impact on the susceptibility of the biofilms grown within flow channels. The impact of flow modification due to flow cell geometry should be considered when designing flow cells for laboratory investigation of bacterial biofilms.     

Roughness effects of diatomaceous slime fouling on turbulent boundary layer hydrodynamics

Abstract

Biofilm fouling significantly impacts ship performance. Here, the impact of biofilm on boundary layer structure at a ship-relevant, low Reynolds number was investigated. Boundary layer measurements were performed over slime-fouled plates using high resolution particle image velocimetry (PIV). The velocity profile over the biofilm showed a downward shift in the log-law region (ΔU⁺), resulting in an effective roughness height (k_s) of 8.8?mm, significantly larger than the physical thickness of the biofilm (1.7?±?0.5?mm) and generating more than three times as much frictional drag as the smooth-wall. The skin-friction coefficient, C_f, of the biofilm was 9.0?×?10^?3 compared with 2.9?×?10^?3 for the smooth wall. The biofilm also enhances turbulent kinetic energy (tke) and Reynolds shear stress, which are more heterogeneous in the streamwise direction than smooth-wall flows. This suggests that biofilms increase drag due to high levels of momentum transport, likely resulting from protruding streamers and surface compliance.     

Impacts of hydrodynamic conditions and microscale surface roughness on the critical shear stress to develop and thickness of early-stage Pseudomonas putida biofilms

Biofilms can increase pathogenic contamination of drinking water, cause biofilm-related diseases, alter the sediment erosion rate, and degrade contaminants in wastewater. Compared with mature biofilms, biofilms in the early-stage have been shown to be more susceptible to antimicrobials and easier to remove. Mechanistic understanding of physical factors controlling early-stage biofilm growth is critical to predict and control biofilm development, yet such understanding is currently incomplete. Here, we reveal the impacts of hydrodynamic conditions and microscale surface roughness on the development of early-stage Pseudomonas putida biofilm through a combination of microfluidic experiments, numerical simulations, and fluid mechanics theories. We demonstrate that early-stage biofilm growth is suppressed under high flow conditions and that the local velocity for early-stage P. putida biofilms (growth time < 14 h) to develop is about 50 μm/s, which is similar to P. putida's swimming speed. We further illustrate that microscale surface roughness promotes the growth of early-stage biofilms by increasing the area of the low-flow region. Furthermore, we show that the critical average shear stress, above which early-stage biofilms cease to form, is 0.9 Pa for rough surfaces, three times as large as the value for flat or smooth surfaces (0.3 Pa). The important control of flow conditions and microscale surface roughness on early-stage biofilm development, characterized in this study, will facilitate future predictions and managements of early-stage P. putida biofilm development on the surfaces of drinking water pipelines, bioreactors, and sediments in aquatic environments.     

Copper affects biofilm inductiveness to larval settlement of the serpulid polychaete Hydroides elegans (Haswell)

Copper (Cu) contamination is a potential threat to the marine environment due to the use of Cu-based antifouling paints. Cu stress on larval settlement of the polychaete Hydroides elegans was investigated, and this was linked to Cu stress on biofilms and on the biofilm development process. The inductiveness of young biofilms was more easily altered by Cu stress than that of old biofilms, indicating the relative vulnerability of young biofilms. This might result from changes in bacterial survival, the bacterial community composition and the chemical profiles of young biofilms. Cu also affected biofilm development and the chemical high performance liquid chromatograph fingerprint profile. The results indicate that Cu affected larval settlement mainly through its effect on the process of biofilm development in the marine environment, and the chemical profile was crucial to biofilm inductiveness. It is strongly recommended that the effects of environmentally toxic substances on biofilms are evaluated in ecotoxicity bioassays using larval settlement of invertebrates as the end point.     

The Role of Exopolymers Produced by Sphingomonas paucimobilis in Biofilm Formation and Composition

Exopolymers have been associated with the initial adhesion of bacteria, which is the primary step for biofilm formation. Moreover, the polymeric matrix of biofilms has a considerable influence on some of the most important physical and physiological properties of biofilms. The role of extracellular polymers in biofilm formation was studied using three mutants of Sphingomonas paucimobilis with increasing capabilities for exopolymer production. The physical, biochemical and physiological properties of three different layers of each biofilm were determined. The layers were detached by submitting the biofilm to increasing shear stress. The results revealed that the presence of exopolymers in the growth medium was essential for biofilm formation. The mutant producing the highest amount of exopolymer formed very thick biofilms, while the biofilms formed by the medium exopolymer producer were on average 8 times thinner. The lowest exopolymer producer did not form biofilm. In both types of biofilms, exopolymer density increased with depth, although this tendency was more significant in thinner biofilms. Cell distribution was also more heterogeneous in thinner biofilms, exhibiting a greater accumulation of cells in the inner layers. The thicker biofilms had very low activity in the inner layer. This was related to a high accumulation of proteins and DNA in this layer due to cell lysis and hydrolytic activity. Activity in the thin biofilm was constant throughout its depth, suggesting that there was no nutrient limitation. The production of exopolymers by each cell was constant throughout the depth of the biofilms, although it was greater in the case of the higher producer.     

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.     

The influence of fluid shear on the structure and material properties of sulphate-reducing bacterial biofilms

Biofilms of sulphate-reducing Desulfovibrio sp. EX265 were grown in square section glass capillary flow cells under a range of fluid flow velocities from 0.01 to 0.4 m/s (wall shear stress, τ_w, from 0.027 to 1.0 N/m²). In situ image analysis and confocal scanning laser microscopy revealed biofilm characteristics similar to those reported for aerobic biofilms. Biofilms in both flow cells were patchy and consisted of cell clusters separated by voids. Length-to-width ratio measurements (l _c:w _c) of biofilm clusters demonstrated the formation of more “streamlined” biofilm clusters (l _c:w _c=3.03) at high-flow velocity (Reynolds number, Re, 1200), whereas at low-flow velocity (Re 120), the l _c:w _c of the clusters was approximately 1 (l _c:w _c of 1 indicates no elongation in the flow direction). Cell clusters grown under high flow were more rigid and had a higher yield point (the point at which the biofilm began to flow like a fluid) than those established at low flow and some biofilm cell aggregates were able to relocate within a cluster, by travelling in the direction of flow, before attaching more firmly downstream. Received 01 February 2002/ Accepted in revised form 16 July 2002     

Effect of Different Concentrations of Ortho-phthalaldehyde on Biofilms Formed by Pseudomonas fluorescens Under Different Flow Conditions

The effectiveness of different concentrations of ortho-phthalaldehyde (OPA) in controlling biofilms of Pseudomonas fluorescens formed on stainless steel slides, using flow cell reactors under laminar and turbulent flow, was investigated by determining the variation in mass and respiratory activity. The physical stability of the biofilm with and without exposure to OPA was studied in a rotating device as variation in the mass of the biofilm on the surface after exposure to different rotation velocities. The activity of OPA against bacterial suspended cultures was evaluated in the presence and absence of bovine serum albumin (BSA) in order to evaluate the interference of proteins on the activity of the biocide. The results showed that biofilms formed under different flow conditions had different properties and reacted differently after biocide application. Biofilms formed under laminar flow were more easily inactivated than those formed under turbulent conditions. However, OPA did not promote the detachment of biofilms from the surface. The exposure of biofilms to different shear stress conditions after OPA treatment enhanced removal from the surface, indicating that OPA may weaken the biofilm matrix. The biocide was more effective on suspended cells than on cells grown in biofilms. This fact may be explained by the reaction of the biocide with proteins of the polymeric matrix of the biofilm as suggested by the significant reduction of biocide action on suspended cells in the presence of BSA.     

The effect of solid surface tension and exposure to elevated hydrodynamic shear on Pseudomonas fluorescens biofilms grown on modified titanium surfaces

Abstract

The solid surface tension of titanium was varied by using organosilane monolayers of various terminations, minimising differences in other material properties. Both the quantity of Pseudomonas fluorescens biofilms grown on the modified surfaces, and the percentage of biofilm remaining after exposure to hydrodynamic shear stress, varied significantly as a function of solid surface tension. The quantity of biofilm was less on chloropropyl-terminated surfaces than on an alkyl-terminated surfaces. However, the percentage of biofilm remaining after exposure to hydrodynamic shear stress (which depends on the adhesion and cohesion strengths of the biofilm) was less for the alkyl-terminated surface than for the chloropropyl-terminated surface, for one of the two sample sets analysed. These results demonstrate the importance of differentiating between the quantity of biofilm on a surface and the adhesion and cohesion strength of the biofilm, and may help explain discrepancies in the existing literature regarding the effect of solid surface tension on the propensity of a surface for microfouling.     

The Effect of Shear on the Desorption of Liposomes Adsorbed to Bacterial Biofilms

Abstract

With the aid of a flow cell assembly the desorption of cationic liposomes prepared from mixtures of dipalmitoylphoshatidylcholine (DDPC), cholesterol, and either dimethyldioctadecylammonium bromide (DDAB) or 3,β[N-(N¹,N-dimethylethylenediamine)-carbamoyl]cholesterol (DC-chol) from immoblized biofilms of Staphylococcus aureus has been studied as a function of shear stress by confocal microscopy. A shear stress theory has been adapted from fluid mechanics of laminar flow between parallel plates and used to determine the critical shear stress for liposome desorption. The critical shear stress for both DDAB and DC-chol liposomes has been determined as a function of cationic lipid content and hence surface charge as reflected in their zeta potentials. The critical shear stress has been used to obtain the potential energy of liposome–biofilm interaction which together with the electrostatic interaction energy has enabled estimates of the London-Hamaker constants to be made. The values of the London-Hamaker constants at small liposome-bacterial cell separation were found to be independent of liposome composition.     

Development and testing of a novel microcantilever technique for measuring the cohesive strength of intact biofilms

Cohesive strength is an important parameter for understanding and modeling the mechanics of biomass detachment from bacterial biofilms. It is challenging to measure the mechanical properties of biofilms, however, because biofilms may desiccate when removed from liquid medium and they are inherently fragile. Poppele and Hozalski (Poppele and Hozalski, 2003, J Microb Methods 55:607–615) presented a microcantilever method for measuring the tensile strength of detached biofilm fragments while submersed in liquid medium. Here we present a modification of the microcantilever method to quantify the strength of intact bacterial biofilms. Initial testing was performed on Pseudomonas aeruginosa biofilms and on Staphylococcus epidermidis biofilms grown in rotating disk reactors. The cohesive strength values were highly variable (i.e., coefficients of variation ranging from 71% to 143%) and ranged from 59 to 18,900 Pa for the P. aeruginosa biofilms and from 61 to 5,840 Pa for the S. epidermidis biofilms. The biofilms also appeared to be isotropic as strength did not vary with angle of testing relative to the direction of applied shear. Strength testing using both the intact and fragment methods was performed on five samples of P. aeruginosa biofilms, and the strength populations were not from the same distribution in three cases. Equivalent diameters for the fragments detached from biofilms during strength testing ranged from 5 to 500 µm, which is within the range of size of biofilm fragments observed in the effluents of lab‐scale and full‐scale bioreactors. The microcantilever is a simple yet powerful tool for measuring the cohesive strength of intact biofilms at a relevant scale. Biotechnol. Bioeng. 2010;105: 924–934. © 2009 Wiley Periodicals, Inc.     

Comparative proteomic analysis of biofilm and planktonic cells of Lactobacillus plantarum DB200

This study investigated the relative abundance of extracellular and cell wall associated proteins (exoproteome), cytoplasmic proteins (proteome), and related phenotypic traits of Lactobacillus plantarum grown under planktonic and biofilm conditions. Lactobacillus plantarum DB200 was preliminarily selected due to its ability to form biofilms and to adhere to Caco2 cells. As shown by fluorescence microscope analysis, biofilm cells became longer and autoaggregated at higher levels than planktonic cells. The molar ratio between glucose consumed and lactate synthesised was markedly decreased under biofilm compared to planktonic conditions. DIGE analysis showed a differential exoproteome (115 protein spots) and proteome (44) between planktonic and biofilm L. plantarum DB200 cells. Proteins up‐ or downregulated by at least twofold (p < 0.05) were found to belong mainly to the following functional categories: cell wall and catabolic process, cell cycle and adhesion, transport, glycolysis and carbohydrate metabolism, exopolysaccharide metabolism, amino acid and protein metabolisms, fatty acid and lipid biosynthesis, purine and nucleotide metabolism, stress response, oxidation/reduction process, and energy metabolism. Many of the above proteins showed moonlighting behavior. In accordance with the high expression levels of stress proteins (e.g., DnaK, GroEL, ClpP, GroES, and catalase), biofilm cells demonstrated enhanced survival under conditions of environmental stress.     

Effect of shear on morphology,viability and metabolic activity of succinic acid-producing Actinobacillus succinogenes biofilms

Two custom-designed bioreactors were used to evaluate the effect of shear on biofilms of a succinic acid producer, Actinobacillus succinogenes. The first bioreactor allowed for in situ removal of small biofilm samples used for microscopic imaging. The second bioreactor allowed for complete removal of all biofilm and was used to analyse biofilm composition and productivity. The smooth, low porosity biofilms obtained under high shear conditions had an average cell viability of 79% compared to 57% at the lowest shear used. The maximum cell-based succinic acid productivity for high shear biofilm was 2.4 g g⁻¹DCW h⁻¹ compared to the 0.8 g g⁻¹DCW h⁻¹ of the low shear biofilm. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays confirmed higher cell metabolic activities for high shear developed biofilm compared to biofilm developed at low shear conditions. Results clearly indicated that high shear biofilm cultivation has beneficial morphological, viability, and cell-based productivity characteristics.

     

Inactivation of Pseudomonas aeruginosa biofilms formed under high shear stress on various hydrophilic and hydrophobic surfaces by a continuous flow of ozonated water

Abstract

The inactivation of Pseudomonas aeruginosa biofilms grown on glass under high shear stress and exposed to a range of dissolved ozone concentrations (2, 5 and 7?ppm) at 10 and 20?min was investigated. The regression equation, log reduction (biofilm)?=?0.64?+?0.59×(C – 2)?+?0.33×(T – 10), described the dependence of biofilm inactivation on the dissolved ozone concentration (C, ppm) and contact time (T, min). The predicted D-values were 11.1, 5.7 and 2.2?min at 2, 5 and 7?ppm, respectively. Inactivation of biofilms grown on various surfaces was tested at a single dissolved ozone concentration of 5?ppm and a single exposure time of 20?min. Biofilms grown on plastic materials showed inactivation results similar to that of biofilms on glass, while biofilms grown on ceramics were statistically significantly more difficult to inactivate, suggesting the importance of utilizing non-porous materials in industrial and clinical settings.     

Effect of heterogeneous structure in mechanically unstressed biofilms on overall growth

In contrast to their name, biofilms are not always flat and homogeneous but instead often exhibit complex structural heterogeneity. It has been suggested that nonhomogeneous geometry is selected in order to increase biofilm growth rate. A previous study (Dockery and Klapper (2002) SIAM J. Appl. Math., 62, 853–869) of a model biofilm system in a static bulk fluid demonstrated that under some circumstances a flat biofilm-bulk fluid interface is linearly unstable to perturbation due to growth induced forces. Computations indicated that subsequent nonlinear evolution results in fingers and mushrooms of biofilm similar to structures observed in actual biofilms. However, the important complementary issue of biological functionality was not considered. Here a weakly nonlinear analysis of the simple growing biofilm layer model in Dockery and Klapper (2002, SIAM J. Appl. Math., 62, 853–869) is presented. It is argued that, at least in the case of biofilms free of external mechanical stress, overall growth is in fact generally inhibited by the presence of growing perturbations in the linear stage. Hence a more complex explanation of function is necessary.     

In situ effective diffusion coefficient profiles in live biofilms using pulsed‐field gradient nuclear magnetic resonance

Diffusive mass transfer in biofilms is characterized by the effective diffusion coefficient. It is well documented that the effective diffusion coefficient can vary by location in a biofilm. The current literature is dominated by effective diffusion coefficient measurements for distinct cell clusters and stratified biofilms showing this spatial variation. Regardless of whether distinct cell clusters or surface‐averaging methods are used, position‐dependent measurements of the effective diffusion coefficient are currently: (1) invasive to the biofilm, (2) performed under unnatural conditions, (3) lethal to cells, and/or (4) spatially restricted to only certain regions of the biofilm. Invasive measurements can lead to inaccurate results and prohibit further (time‐dependent) measurements which are important for the mathematical modeling of biofilms. In this study our goals were to: (1) measure the effective diffusion coefficient for water in live biofilms, (2) monitor how the effective diffusion coefficient changes over time under growth conditions, and (3) correlate the effective diffusion coefficient with depth in the biofilm. We measured in situ two‐dimensional effective diffusion coefficient maps within Shewanella oneidensis MR‐1 biofilms using pulsed‐field gradient nuclear magnetic resonance methods, and used them to calculate surface‐averaged relative effective diffusion coefficient (D_rs) profiles. We found that (1) D_rs decreased from the top of the biofilm to the bottom, (2) D_rs profiles differed for biofilms of different ages, (3) D_rs profiles changed over time and generally decreased with time, (4) all the biofilms showed very similar D_rs profiles near the top of the biofilm, and (5) the D_rs profile near the bottom of the biofilm was different for each biofilm. Practically, our results demonstrate that advanced biofilm models should use a variable effective diffusivity which changes with time and location in the biofilm. Biotechnol. Bioeng. 2010;106: 928–937. © 2010 Wiley Periodicals, Inc.