The effect of hydrodynamic conditions on the phenotype of Pseudomonas fluorescens biofilms

Abstract

This study investigated the phenotypic characteristics of monoculture P. fluorescens biofilms grown under turbulent and laminar flow, using flow cells reactors with stainless steel substrata. The cellular physiology and the overall biofilm activity, structure and composition were characterized, and compared, within hydrodynamically distinct conditions. The results indicate that turbulent flow-generated biofilm cells were significantly less extensive, with decreased metabolic activity and a lower protein and polysaccharides composition per cell than those from laminar flow-generated biofilms. The effect of flow regime did not cause significantly different outer membrane protein expression. From the analysis of biofilm activity, structure and composition, turbulent flow-generated biofilms were metabolically more active, had twice more mass per cm², and higher cellular density and protein content (mainly cellular) than laminar flow-generated biofilms. Conversely, laminar flow-generated biofilms presented higher total and matrix polysaccharide contents. Direct visualisation and scanning electron microscopy analysis showed that these different flows generate structurally different biofilms, corroborating the quantitative results. The combination of applied methods provided useful information regarding a broad spectrum of biofilm parameters, which can contribute to control and model biofilm processes.     

Sodium dodecyl sulfate allows the persistence and recovery of biofilms of Pseudomonas fluorescens formed under different hydrodynamic conditions

The effect of the anionic surfactant sodium dodecyl sulfate (SDS) on Pseudomonas fluorescens biofilms was investigated using flow cell reactors with stainless steel substrata, under turbulent (Re = 5200) and laminar (Re = 2000) flow. Steady-state biofilms were exposed to SDS in single doses (0.5, 1, 3 and 7 mM) and biofilm respiratory activity and mass measured at 0, 3, 7 and 12 h after the SDS application. The effect of SDS on biofilm mechanical stability was assessed using a rotating bioreactor. Whilst high concentrations (7 mM) of SDS promoted significant biofilm inactivation, it did not significantly reduce biofouling. Turbulent and laminar flow-generated biofilms had comparable susceptibility to SDS application. Following SDS exposure, biofilms rapidly recovered over the following 12 h, achieving higher respiratory activity values than before treatment. This phenomenon of post-treatment recovery was more pronounced for turbulent flow-generated biofilms, with an increase in SDS concentration. The mechanical stability of the biofilms increased with surfactant application, except for SDS concentrations near the critical micellar concentration, as measured by biofilm removal due to an increase in external shear stress forces. The data suggest that although SDS exerts antimicrobial action against P. fluorescens biofilms, even if only partial and reversible, it had only limited antifouling efficacy, increasing biofilm mechanical stability at low concentrations and allowing significant and rapid recovery of turbulent flow-generated biofilms.     

Effect of flow regime on the architecture of a Pseudomonas fluorescens biofilm

A comparison of the effects of laminar versus turbulent flow regime on the characteristics of a single-species biofilm is presented. The study was carried out by growing Pseudomonas fluorescens biofilms in a flow cell and studying the different layers of the biological matrix with a confocal laser-scanning microscope. The following conclusions were obtained: i) a higher concentration of cells was found in the upper layers of the microbial films than in their inner layers, regardless of the flow regime; ii) the fraction of cells in the overall biofilm mass decreased with time as the film grew; and iii) under laminar flow the total number of cells was higher than in biofilms formed under turbulent flow, but the latter had a higher number of cells per unit volume. Such conclusions, together with the fact that the biofilms were more dense and stable when formed in contact with turbulent flows, favor the design of more compact and efficient biofilm reactors operating in turbulent conditions.     

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.     

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.     

Influence of hydrodynamics and cell signaling on the structure and behavior of Pseudomonas aeruginosa biofilms

Biofilms were grown from wild-type (WT) Pseudomonas aeruginosa PAO1 and the cell signaling lasI mutant PAO1-JP1 under laminar and turbulent flows to investigate the relative contributions of hydrodynamics and cell signaling for biofilm formation. Various biofilm morphological parameters were quantified using Image Structure Analyzer software. Multivariate analysis demonstrated that both cell signaling and hydrodynamics significantly (P < 0.000) influenced biofilm structure. In turbulent flow, both biofilms formed streamlined patches, which in some cases developed ripple-like wave structures which flowed downstream along the surface of the flow cell. In laminar flow, both biofilms formed monolayers interspersed with small circular microcolonies. Ripple-like structures also formed in four out of six WT biofilms, although their velocity was approximately 10 times less than that of those that formed in the turbulent flow cells. The movement of biofilm cell clusters over solid surfaces may have important clinical implications for the dissemination of biofilm subject to fluid shear, such as that found in catheters. The ability of the cell signaling mutant to form biofilms in high shear flow demonstrates that signaling mechanisms are not required for the formation of strongly adhered biofilms. Similarity between biofilm morphologies in WT and mutant biofilms suggests that the dilution of signal molecules by mass transfer effects in faster flowing systems mollifies the dramatic influence of signal molecules on biofilm structure reported in previous studies.     

Influence of Hydrodynamics and Cell Signaling on the Structure and Behavior of Pseudomonas aeruginosa Biofilms

Biofilms were grown from wild-type (WT) Pseudomonas aeruginosa PAO1 and the cell signaling lasI mutant PAO1-JP1 under laminar and turbulent flows to investigate the relative contributions of hydrodynamics and cell signaling for biofilm formation. Various biofilm morphological parameters were quantified using Image Structure Analyzer software. Multivariate analysis demonstrated that both cell signaling and hydrodynamics significantly (P < 0.000) influenced biofilm structure. In turbulent flow, both biofilms formed streamlined patches, which in some cases developed ripple-like wave structures which flowed downstream along the surface of the flow cell. In laminar flow, both biofilms formed monolayers interspersed with small circular microcolonies. Ripple-like structures also formed in four out of six WT biofilms, although their velocity was approximately 10 times less than that of those that formed in the turbulent flow cells. The movement of biofilm cell clusters over solid surfaces may have important clinical implications for the dissemination of biofilm subject to fluid shear, such as that found in catheters. The ability of the cell signaling mutant to form biofilms in high shear flow demonstrates that signaling mechanisms are not required for the formation of strongly adhered biofilms. Similarity between biofilm morphologies in WT and mutant biofilms suggests that the dilution of signal molecules by mass transfer effects in faster flowing systems mollifies the dramatic influence of signal molecules on biofilm structure reported in previous studies.     

Influence of hydrodynamics and nutrients on biofilm structure

Hydrodynamic conditions control two interlinked parameters; mass transfer and drag, and will, therefore, significantly influence many of the processes involved in biofilm development. The goal of this research was to determine the effect of flow velocity and nutrients on biofilm structure. Biofilms were grown in square glass capillary flow cells under laminar and turbulent flows. Biofilms were observed microscopically under flow conditions using image analysis. Mixed species bacterial biofilms were grown with glucose (40 mg/l) as the limiting nutrient. Biofilms grown under laminar conditions were patchy and consisted of roughly circular cell clusters separated by interstitial voids. Biofilms in the turbulent flow cell were also patchy but these biofilms consisted of patches of ripples and elongated 'streamers' which oscillated in the flow. To assess the influence of changing nutrient conditions on biofilm structure the glucose concentration was increased from 40 to 400 mg/l on an established 21 day old biofilm growing in turbulent flow. The cell clusters grew rapidly and the thickness of the biofilm increased from 30 μ to 130 μ within 17 h. The ripples disappeared after 10 hours. After 5 d the glucose concentration was reduced back to 40 mg/l. There was a loss of biomass and patches of ripples were re-established within a further 2 d.     

Flowing biofilms as a transport mechanism for biomass through porous media under laminar and turbulent conditions in a laboratory reactor system

Fluid flow has been shown to be important in influencing biofilm morphology and causing biofilms to flow over surfaces in flow cell experiments. However, it is not known whether similar effects may occur in porous media. Generally, it is assumed that the primary transport mechanism for biomass in porous media is through convection, as suspended particulates (cells and flocs) carried by fluid flowing through the interstices. However, the flow of biofilms over the surfaces of soils and sediment particles, may represent an important flux of biomass, and subsequently affect both biological activity and permeability. Mixed species bacterial biofilms were grown in glass flow cells packed with 1 mm diameter glass beads, under laminar or turbulent flow (porous media Reynolds number = 20 and 200 respectively). The morphology and dynamic behavior reflected those of biofilms grown in the open flow cells. The laminar biofilm was relatively uniform and after 23 d had inundated the majority of the pore spaces. Under turbulent flow the biofilm accumulated primarily in protected regions at contact points between the beads and formed streamers that trailed from the leeward face. Both biofilms caused a 2 to 3-fold increase in friction factor and in both cases there were sudden reductions in friction factor followed by rapid recovery, suggesting periodic sloughing and regrowth events. Time-lapse microscopy revealed that under both laminar and turbulent conditions biofilms flowed over the surface of the porous media. In some instances ripple structures formed. The velocity of biofilm flow was on the order of 10 mum h(-1) in the turbulent flow cell and 1.0 mum h(-1) in the laminar flow cell.     

Flowing biofilms as a transport mechanism for biomass through porous media under laminar and turbulent conditions in a laboratory reactor system

Abstract

Fluid flow has been shown to be important in influencing biofilm morphology and causing biofilms to flow over surfaces in flow cell experiments. However, it is not known whether similar effects may occur in porous media. Generally, it is assumed that the primary transport mechanism for biomass in porous media is through convection, as suspended particulates (cells and flocs) carried by fluid flowing through the interstices. However, the flow of biofilms over the surfaces of soils and sediment particles, may represent an important flux of biomass, and subsequently affect both biological activity and permeability. Mixed species bacterial biofilms were grown in glass flow cells packed with 1 mm diameter glass beads, under laminar or turbulent flow (porous media Reynolds number = 20 and 200 respectively). The morphology and dynamic behavior reflected those of biofilms grown in the open flow cells. The laminar biofilm was relatively uniform and after 23 d had inundated the majority of the pore spaces. Under turbulent flow the biofilm accumulated primarily in protected regions at contact points between the beads and formed streamers that trailed from the leeward face. Both biofilms caused a 2 to 3-fold increase in friction factor and in both cases there were sudden reductions in friction factor followed by rapid recovery, suggesting periodic sloughing and regrowth events. Time-lapse microscopy revealed that under both laminar and turbulent conditions biofilms flowed over the surface of the porous media. In some instances ripple structures formed. The velocity of biofilm flow was on the order of 10 μm h^?1 in the turbulent flow cell and 1.0 μm h^?1 in the laminar flow cell.     

Biophysical Controls on Community Succession in Stream Biofilms

Biofilm formation is controlled by an array of coupled physical, chemical, and biotic processes. Despite the ecological relevance of microbial biofilms, their community formation and succession remain poorly understood. We investigated the effect of flow velocity, as the major physical force in stream ecosystems, on biofilm community succession (as continuous shifts in community composition) in microcosms under laminar, intermediate, and turbulent flow. Flow clearly shaped the development of biofilm architecture and community composition, as revealed by microscopic investigation, denaturing gradient gel electrophoresis (DGGE) analysis, and sequencing. While biofilm growth patterns were undirected under laminar flow, they were clearly directed into ridges and conspicuous streamers under turbulent flow. A total of 51 biofilm DGGE bands were detected; the average number ranged from 13 to 16. Successional trajectories diverged from an initial community that was common in all flow treatments and increasingly converged as biofilms matured. We suggest that this developmental pattern was primarily driven by algae, which, as “ecosystem engineers,” modulate their microenvironment to create similar architectures and flow conditions in all treatments and thereby reduce the physical effect of flow on biofilms. Our results thus suggest a shift from a predominantly physical control to coupled biophysical controls on bacterial community succession in stream biofilms.     

Biophysical controls on community succession in stream biofilms

Biofilm formation is controlled by an array of coupled physical, chemical, and biotic processes. Despite the ecological relevance of microbial biofilms, their community formation and succession remain poorly understood. We investigated the effect of flow velocity, as the major physical force in stream ecosystems, on biofilm community succession (as continuous shifts in community composition) in microcosms under laminar, intermediate, and turbulent flow. Flow clearly shaped the development of biofilm architecture and community composition, as revealed by microscopic investigation, denaturing gradient gel electrophoresis (DGGE) analysis, and sequencing. While biofilm growth patterns were undirected under laminar flow, they were clearly directed into ridges and conspicuous streamers under turbulent flow. A total of 51 biofilm DGGE bands were detected; the average number ranged from 13 to 16. Successional trajectories diverged from an initial community that was common in all flow treatments and increasingly converged as biofilms matured. We suggest that this developmental pattern was primarily driven by algae, which, as "ecosystem engineers," modulate their microenvironment to create similar architectures and flow conditions in all treatments and thereby reduce the physical effect of flow on biofilms. Our results thus suggest a shift from a predominantly physical control to coupled biophysical controls on bacterial community succession in stream biofilms.     

Investigation of the mesoscale structure and volumetric features of biofilms using optical coherence tomography

Optical coherence tomography (OCT) was successfully applied to visualize the mesoscale structure of three different heterotrophic biofilms. For this purpose, biofilm volumes of 4 × 4 × 1.6 mm³ were scanned with spatial resolutions lower than 20 µm within an acquisition time of 2 min. A heterogeneous structure was detected for biofilms cultivated in laminar as well as transient flow conditions. The structure was found to be more homogeneous for the biofilm grown in turbulent flow. This biofilm structure was characterized by a volumetric porosity of 0.36, whereas the porosity calculated for biofilms grown in laminar and transient conditions was 0.65. These results were directly generated from the distribution of porosity calculated from the OCT images acquired and can be linked to structural properties. Up to now, the mesoscale biofilm structure was only observable with time‐consuming and expensive studies, for example, magnetic resonance microscopy. OCT will most certainly be helpful for improved understanding and prediction of biofilm physics with respect to macroscale processes, for example, mass transfer and detachment as the information about mesoscale is easily accessible using this method. In the context of this study, we show that CLSM images do not necessarily provide an accurate representation of the biofilm structure at the mesoscale. Additionally, the typical characteristic parameters obtained from CLSM image stacks differ largely from those calculated from OCT images. Nevertheless, to determine the local distribution of biofilm constituents, microscopic methods such as confocal laser scanning microscopy are required. Biotechnol. Bioeng. 2010;107: 844–853. © 2010 Wiley Periodicals, Inc.     

The formation of migratory ripples in a mixed species bacterial biofilm growing in turbulent flow

Mixed-species biofilms, consisting of Klebsiella pneumoniae , Pseudomonas aeruginosa , Pseudomonas fluorescens and Stenotrophomonas maltophilia , were grown in glass flow cells under either laminar or turbulent flow. The biofilms grown in laminar flow consisted of roughly circular-shaped microcolonies separated by water channels. In contrast, biofilm microcolonies grown in turbulent flow were elongated in the downstream direction, forming filamentous 'streamers'. Moreover, biofilms growing in turbulent flow developed extensive patches of ripple-like structures between 9 and 13 days of growth. Using time-lapse microscopic imaging, we discovered that the biofilm ripples migrated downstream. The morphology and the migration velocity of the ripples varied with short-term changes in the bulk liquid flow velocity. The ripples had a maximum migration velocity of 800 μm h⁻¹ (2.2 × 10⁻⁷ m s⁻¹) when the liquid flow velocity was 0.5 m s⁻¹ (Reynolds number = 1800). This work challenges the commonly held assumption that biofilm structures remain at the same location on a surface until they eventually detach.     

Biofilm ecology: On-line methods bring new insights into mic and microbial biofouling

Microbial biofilms were formed on coupons with defined coatings in once-through laminar flow fields of controlled bulk-phase composition and shear. Dilute media were utilized to select for biofilm growth. The formation, succession, and stability of the biofilms were monitored with non-destructive on-line methods (fluorescence, bioluminescence, attenuated total reflectance Fourier transform infrared spectrometry [ATR-FTIR] and electrochemical impedance spectroscopy) and by high resolution destructive analysts (viable and direct counts and phospholipid fatty acid signature methods) at the termination of the experiments. Biofilms of reproducible composition can be formed and the order of inoculation of multi-component biofilms affects their composition at harvest. The corrosion rates of mild steel depended on the biofilm composition but not the attached biomass. Examination of biofilms with the scanning vibrating electrode in a microscope field showed effects of heterogeneity in biofilm structure which promoted localized anodic activity. Pseudomonas stains were engineered to contain the lux gene cassette as a "reporter"; and the formation of the exopolymer alginate was shown not to promote attachment of the strain or secondary colonization by Vibrio. Examination of mutants forming different alginate structures showed differential attachment and biofilm structure. Studies of mutants of lipopolysaccharide structure showed differential attachment to substrata. Specific antifouling and fouling-release coatings showed a wide range of attachment and release properties as well as sublethal toxicity.     

Enhanced high copy number plasmid maintenance and heterologous protein production in an Escherichia coli biofilm

Escherichia coli has been widely used for heterologous protein production (HPP). To determine whether a biofilm environment could benefit E. coli HPP using high copy number plasmids, we compared plasmid maintenance and HPP by E. coli ATCC 33456 containing plasmid pEGFP (a pUC family vector) cultivated in biofilms and in suspended culture. Cells were grown with or without antibiotic selective pressure in flow cells or chemostats for up to 6 days. In biofilms, antibiotic selective pressure increased the plasmid copy number (PCN), but by 144 h, biofilms grown in antibiotic-free media had comparable plasmid concentrations. In the chemostat, the PCN declined steadily, although 100 ppm ampicillin in the medium slowed the rate of plasmid loss. Production of green fluorescent protein (GFP), a representative heterologous protein, was quantified by flow cytometry. In biofilms, at ampicillin concentrations >or=33 ppm, strongly fluorescent cells comprised more than half of the population by 48 h. In the chemostat, more than 50% of the population was non-fluorescent by 48 h in media containing 100 ppm ampicillin, and strongly fluorescent cells were <10% of the population. Biofilm structure was determined by confocal microscopy. Maximum biofilm thickness ranged from 30 to 45 microns, with no significant changes in biofilm structure after 48 h. Plasmid multimer percentages were similar to inocula for cells cultivated in either biofilms or the chemostat. The results indicate that the biofilm environment enhanced both plasmid maintenance and cellular GFP concentrations, and that low levels of antibiotic increased the beneficial effect.     

Drinking water biofilm assessment of total and culturable bacteria under different operating conditions

Monitoring of biofilms subjected to different operating conditions was performed using a flow cell system. The system was fed by chlorine-free tap water, with and without added nutrients (0.5 mg l(-1) carbon, 0.1 mg l(-1) nitrogen and 0.01 mg l(-1) phosphorus), and biofilms were grown on polyvinyl chloride (PVC) and stainless steel (SS) coupons, both in laminar and turbulent flow. The parameters analysed were culturable cells, using R2A, and total bacteria, which was assessed using the 4,6-diamino-2-phenylindole (DAPI) staining method. The impact of the different operating conditions in the studied parameters was established using Multivariate Analysis of Variance (MANOVA). From the most relevant to the least relevant factor, the total and culturable bacteria in biofilms increased due to the addition of nutrients to water (F = 20.005; p < 0.001); the use of turbulent (Re = 11000) instead of laminar (Re = 2000) hydrodynamic flows (F = 9.173; p < 0.001); and the use of PVC instead of SS as the support material (F = 2.848; p = 0.060). Interactions between these conditions, namely between surface and flow (F = 8.235; p < 0.001) and also flow and nutrients (F = 5.498; p < 0.05) have also proved to significantly influence biofilm formation. This work highlights the need for a deeper understanding of how the large spectrum of conditions interact and affect biofilm formation potential and accumulation with the final purpose of predicting the total and culturable bacteria attached to real drinking water distribution pipes based on the system characteristics.     

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.     

Influence of hydrodynamic conditions on biofilm behavior in a methanogenic inverse turbulent bed reactor

This paper presents a study about the influence of gas velocity on a methanogenic biofilm in an inverse turbulent bed reactor. Experimental results indicate a dynamic response of the growing attached biomass to the changes of hydrodynamic conditions, mainly attrition constraints. Short but intensive increases of gas velocity (U(g)) are shown to induce more detachment than a high but constant gas flow rate. Hydrodynamic conditions control the composition of the growing biofilm in terms of cells and exocellular polymeric substances (EPS). The cell fraction within the biofilm (R(cell)) was found to be inversely proportional to the gas velocity. The specific activity expressed in methane production rate or COD removal rate is higher in biofilms formed under high hydrodynamic constraints. The control of the hydrodynamic conditions in a biofilm reactor should make it possible to obtain a resistant and active biofilm.     

Effect of flow regimes on the presence of Legionella within the biofilm of a model plumbing system

AIMS: Stagnation is widely believed to predispose water systems to colonization by Legionella. A model plumbing system was constructed to determine the effect of flow regimes on the presence of Legionella within microbial biofilms. METHODS AND RESULTS: The plumbing model contained three parallel pipes where turbulent, laminar and stagnant flow regimes were established. Four sets of experiments were carried out with Reynolds number from 10,000 to 40,000 and from 355 to 2,000 in turbulent and laminar pipes, respectively. Legionella counts recovered from biofilm and planktonic water samples of the three sampling pipes were compared with to determine the effect of flow regime on the presence of Legionella. Significantly higher colony counts of Legionella were recovered from the biofilm of the pipe with turbulent flow compared with the pipe with laminar flow. The lowest counts were in the pipe with stagnant flow. CONCLUSIONS: We were unable to demonstrate that stagnant conditions promoted Legionella colonization. SIGNIFICANCE AND IMPACT OF THE STUDY: Plumbing modifications to remove areas of stagnation including deadlegs are widely recommended, but these modifications are tedious and expensive to perform. Controlled studies in large buildings are needed to validate this unproved hypothesis.