Browsing by Author "de Beer, Dirk"

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Biofilm structural heterogeneity visualized by three microscopic methods
(1995-08) Stewart, Philip S.; Murga, Ricardo; Srinivasan, Rohini; de Beer, Dirk
The structural heterogeneity of a microbial biofilm was demonstrated by scanning electron microscopy, confocal scanning laser microscopy (CSLM), and cryoembedding followed by sectioning and microscopic examination. Biofilm was composed of a binary population of Pseudomonas aeruginosa and Klebsiella pneumoniae grown in a continuous flow annular reactor. The three microscopic methods provided a consistent picture of the biofilm as a non-uniform structure characterized by variable thickness and variable local cell and polymer densities. Significant changes in these parameters occurred in the biofilm over distances of 10 μm or less. Though the biofilm was several hundred microns thick in places, areas of bare substratum were also observed on the same sample coupon. Cell-free pores and channels in the biofilm interior were evident. Specific staining of cellular nucleic acids with ethidium bromide and extracellular polymeric substances (EPS) with calcofluor showed that cell and EPS distributions did not always overlap. The ethidium bromide-stained region was contained within the larger region of calcofluor staining; thus, some cell-free areas actually were filled with EPS. CSLM and cryoembedding approaches are superior to SEM in their ability to image the biofilm interior and in their potential to provide quantitative information.
Comparison of respiratory activity and culturability during monochloramine disinfection of binary population biofilms
(1994-05) Stewart, Philip S.; Griebe, Thomas; Srinivasan, Rohini; Chen, Cheng-Sao; Yu, Feipeng Philip; de Beer, Dirk; McFeters, Gordon A.
Biofilm bacteria challenged with monochloramine retained significant respiratory activity, even though they could not be cultured on agar plates. Microbial colony counts on agar media declined by approximately 99.9% after 1 h of disinfection, whereas the number of bacteria stained by a fluorescent redox dye experienced a 93% reduction. Integrated measures of biofilm respiratory activity, including net oxygen and glucose utilization rates, showed only a 10 to 15% reduction. In this biofilm system, measures of microbial respiratory activity and culturability yielded widely differing estimates of biocide efficacy.
Direct measurement of chlorine penetration into biofilms during disinfection
(1994-12) de Beer, Dirk; Srinivasan, Rohini; Stewart, Philip S.
Effects of biofilm structures on oxygen distribution and mass transport
(1994-05) de Beer, Dirk; Stoodley, Paul; Roe, Frank L.; Lewandowski, Zbigniew
Aerobic biofilms were found to have a complex structure consisting of microbial cell clusters (discrete aggregates of densely packed cells) and interstitial voids. The oxygen distribution was strongly correlated with these strutures. The voids facilitated oxygen transport from the bulk liquid through the biofilm, supplying approximately 50% of the total oxygen consumed by the cells. The mass transport rate from the bulk liquid is influenced by the biofilm structure; the observed exchange surface of the biofilm is twice that calculated for a simple planar geometry. The oxygen diffusion occurred in the direction normal to the cluster surfaces, the horizontal and vertical components of the oxygen gradients were of equal importance. Consequently, for calculations of mass transfer rates a three-dimensional model is necessary. These findings imply that to accurately describe biofilm activity, the relation between the arrangement of structural components and mass transfer must be undrstood. © 1994 John Wiley & Sons, Inc.
The Guaymas Basin Hiking Guide to Hydrothermal Mounds, Chimneys, and Microbial Mats: Complex Seafloor Expressions of Subsurface Hydrothermal Circulation
(2016-02) Teske, Andreas; de Beer, Dirk; McKay, Luke J.; Tivey, Margaret K.; Biddle, Jennifer F.; Hoer, Daniel; Lloyd, Karen G.; Lever, Mark A.; Roy, Hans; Albert, Daniel B.; Mendlovitz, Howard P.; MacGregor, Barbara J.
The hydrothermal mats, mounds, and chimneys of the southern Guaymas Basin are the surface expression of complex subsurface hydrothermal circulation patterns. In this overview, we document the most frequently visited features of this hydrothermal area with photographs, temperature measurements, and selected geochemical data; many of these distinct habitats await characterization of their microbial communities and activities. Microprofiler deployments on microbial mats and hydrothermal sediments show their steep geochemical and thermal gradients at millimeter-scale vertical resolution. Mapping these hydrothermal features and sampling locations within the southern Guaymas Basin suggest linkages to underlying shallow sills and heat flow gradients. Recognizing the inherent spatial limitations of much current Guaymas Basin sampling calls for comprehensive surveys of the wider spreading region.
Liquid flow and mass transport in heterogeneous biofilms
(1996-11) de Beer, Dirk; Stoodley, Paul; Lewandowski, Zbigniew
Convective mass transport in heterogeneous biofilms, consisting of cell clusters and voids, was investigated using oxygen microelectrodes. Oxygen concentration profiles were measured and contour plots constructed at different (average) flow velocities (Uavg). The profiles were used to determine the thickness of the mass transfer boundary layer (δh) above the voids and the cell clusters. The δh above the biofilm was inversely related to flow, as expected, and decreased exponentially with increasing flow velocity. However, the δh above the voids decreased more rapidly than the δh above the cell clusters resulting in two distinct situations; at low flow velocities the oxygen contours were parallel to the substratum but at high velocities were parallel to the irregular biofilm surface. It was concluded that at low flow velocities the biofilm could be modeled one-dimensionally, with fluxes perpendicular to the substratum and the exchange area being equal to the substratum area, but at higher velocities biofilm voids facilitate mass transport and a more complex, three-dimensional model would be more appropriate. In this latter case fluxes are multidirectional, and the exchange area is equal to that of the convoluted biofilm surface.
Liquid flow in biofilm systems
(1994-08) Stoodley, Paul; de Beer, Dirk; Lewandowski, Zbigniew
A model biofilm consisting of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Kiebsiella 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.
Liquid flow in heterogeneous biofilms
(1994-08) de Beer, Dirk; Stoodley, Paul; Lewandowski, Zbigniew
Liquid flow was studied in aerobic biofilms, consisting of microbial cell clusters (discrete aggregates of densely packed cells) and interstitial voids. Fluorescein microinjection was used as a qualitative technique to determine the presence of flow in cell clusters and voids. Flow velocity profiles were determined by tracking fluorescent latex spheres using confocal microscopy. Liquid was flowing through the voids and was stagnant in the cell clusters. Consequently, in voids both diffusion and convection may contribute to mass transfer, whereas in cell clusters diffusion is the dominant factor. The flow velocity in the biofilm depended on the average flow velocity of the bulk liquid. The velocity profiles in biofilms were linear and the velocity was zero at the substratum surface. The velocity gradients within biofilms were 50% of that near walls without biofilm coverage. The influence of the biofilm roughness on the flow velocity profiles was similar to that caused by rigid roughness elements. © 1994 John Wiley & Sons, Inc.
Measurement of local diffusion coefficients in biofilms by microinjection and confocal microscopy
(1997-01) de Beer, Dirk; Stoodley, Paul; Lewandowski, Zbigniew
A new technique for the determination of local diffusion coefficients in biofilms is described. It is based on the microinjection of fluorescent dyes and quantitative analysis of the subsequent plume formation using confocal laser microscopy. The diffusion coefficients of fluorescein (MW 332), TRITC-IgG (MW 150000) and phycoerythrin (MW 240000) were measured in the cell clusters and interstitial voids of a heterogeneous biofilm. The diffusivities measured in the voids were close to the theoretical values in water. Fluorescein had the same diffusivity in cell clusters, voids, and sterile medium. TRITC-IgG did not diffuse in cell clusters, presumably due to binding to the cell cluster matrix. After treatment of the biofilm with bovine serum albumin, binding capacity decreased and the diffusion coefficient could be measured. The diffusivity of phycoerythrin in cell clusters was impeded by 41%, compared to interstitial voids. From the diffusion data of phycoerythrin it was further calculated that the cell cluster matrix had the characteristics of a gel with 0.6 nm thick fibers and pore diameters of 80 nm. © 1997 John Wiley & Sons, Inc.
Microbial biofilms
(2000) de Beer, Dirk; Stoodley, Paul
Minireview: Biofilms, the customized microniche
(1994-04) Costerton, J. William; Lewandowski, Zbigniew; de Beer, Dirk; Caldwell, D. E.; Korber, D. R.; James, Garth A.
Modular spectral imaging system for discrimination of pigments in cells and microbial communities
(2008-12) Polerecky, Lubos; Bissett, Andrew; Al-Najjar, Mohammad; Faerber, Paul; Osmers, Harald; Suci, Peter A.; Stoodley, Paul; de Beer, Dirk
Here we describe a spectral imaging system for minimally invasive identification, localization, and relative quantification of pigments in cells and microbial communities. The modularity of the system allows pigment detection on spatial scales ranging from the single-cell level to regions whose areas are several tens of square centimeters. For pigment identification in vivo absorption and/or autofluorescence spectra are used as the analytical signals. Along with the hardware, which is easy to transport and simple to assemble and allows rapid measurement, we describe newly developed software that allows highly sensitive and pigment-specific analyses of the hyperspectral data. We also propose and describe a number of applications of the system for microbial ecology, including identification of pigments in living cells and high-spatial-resolution imaging of pigments and the associated phototrophic groups in complex microbial communities, such as photosynthetic endolithic biofilms, microbial mats, and intertidal sediments. This system provides new possibilities for studying the role of spatial organization of microorganisms in the ecological functioning of complex benthic microbial communities or for noninvasively monitoring changes in the spatial organization and/or composition of a microbial community in response to changing environmental factors.
Oxygen and ph microprofiles above corroding mild steel covered with a biofilm
(1995-05) Lee, Whonchee; de Beer, Dirk
O2 and pH microprofiles were measured above corroding mild steel covered with a biofilm. The pH in the anodic areas (tubercles) ranged from 5 to 7 and was always 9.45 at the surface of the cathodic areas. After 1 month of biofilm development, O2 was depleted at the anodic area but could reach the cathodic surface where it was reduced. Consequently, differential O2 concentration cells were the driving force for corrosion. The O2 microprofiles indicated that O2 was consumed in the tubercles, probably by microbial activity, while O2 was reduced electrochemically in the cathodic areas. It was concluded that O2 transfer to the cathodic surface was the rate limiting step for the corrosion process.
Relation between the structure of an aerobic biofilm and transport phenomena
(1995) de Beer, Dirk; Stoodley, Paul
An aerobic biofilm was characterized using confocal scanning laser microscopy (CSLM), O2 micro-electrodes, particle tracking and microinjection of fluorescent dyes. The biofilms were found to consist of microbial clusters of cells and Extra-cellular Polymeric Substance (EPS) separated by interstitial voids. The cell clusters were ca 300 μm and the voids were ca 100 μm wide. The voids were open channels connected with the bulk fluid. Fluorescein micro-injection showed that liquid could flow through the voids, but was always stagnant in the cell clusters. Consequently, in voids both diffusion and convection may contribute to mass transfer, while in cell clusters transport is determined by diffusion only. Particle tracking with CSLM showed that flow velocity inside the biofilm was proportional to the bulk flow velocity. The importance of convective mass transport in biofilms was demonstrated by oxygen distribution measurements. At high flow velocities of the bulk liquid, the mass boundary layer followed the irregular biofilm surface. At lower velocities the mass boundary layer was parallel to the substratum. Mass transfer from voids to cell clusters increased with flow velocity, as result from vonvective mass transport from the bulk to the voids. Convective transport was insignificant at low flow velocities, but at high flow velocities it increased the total mass transport by 200-250%. The local diffusion coefficients in biofilms were measured using microinjection of fluorescent dyes and quantitative analysis of the subsequent plume formation using CSLM. The diffusion coefficient of small, non-binding molecules in cell clusters is close to that in water. Very large molecules were impeded in their diffusion through the biofilm matrix. It was calculated that the cell cluster matrix had the characteristics of a gel network with pore diameters of 80 nm.