Browsing by Author "Beyenal, Haluk"
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Item Author's response(2002-02) Lewandowski, Zbigniew; Harkin, Gary; Beyenal, HalukItem Biofilm image reconstruction for assessing structural parameters(2011-06) Renslow, Ryan; Lewandowski, Zbigniew; Beyenal, HalukThe structure of biofilms can be numerically quantified from microscopy images using structural parameters. These parameters are used in biofilm image analysis to compare biofilms, to monitor temporal variation in biofilm structure, to quantify the effects of antibiotics on biofilm structure and to determine the effects of environmental conditions on biofilm structure. It is often hypothesized that biofilms with similar structural parameter values will have similar structures; however, this hypothesis has never been tested.The main goal was to test the hypothesis that the commonly used structural parameters can characterize the differences or similarities between biofilm structures. To achieve this goal (1) biofilm image reconstruction was developed as a new tool for assessing structural parameters, (2) independent reconstructions using the same starting structural parameters were tested to see how they differed from each other, (3) the effect of the original image parameter values on reconstruction success was evaluated, and (4) the effect of the number and type of the parameters on reconstruction success was evaluated. It was found that two biofilms characterized by identical commonly used structural parameter values may look different, that the number and size of clusters in the original biofilm image affect image reconstruction success and that, in general, a small set of arbitrarily selected parameters may not reveal relevant differences between biofilm structures.Item Biofilm monitoring: a perfect solution in search of a problem(2003) Lewandowski, Zbigniew; Beyenal, HalukThe main problem with monitoring biofilms is data interpretation. Biofilm heterogeneity causes monitored parameters to vary from location to location in the same biofilm, and it is difficult to assess to what extent these variations are caused by biofilm heterogeneity and to what extent they reflect other properties of the biofilm. We have used the concept of discretized biofilms, which is an integrated system of biofilm monitoring and data interpretation, to assess the effect of biofilm heterogeneity on biofilm activity. Using this approach we have estimated that a heterogeneous biofilm can be ten times more active, in terms of glucose consumption rate, than a homogeneous biofilm of the same thickness but with uniformly distributed density.Item Biofilms: Their structure, activity, and effect on membrane filtration(2005-03) Lewandowski, Zbigniew; Beyenal, HalukThe goal of this presentation is to identify biofouling mechanisms that cause undesirable effects to the membrane separation processes of flux decline and pressure drop. The underlying assumption of this presentation is that biofouling is unavoidable and that the operator cannot eliminate it entirely. This premise justifies research efforts toward understanding the mechanisms by which biofouling affects the membrane processes, rather than expecting that technology can entirely eliminate membrane biofouling in the near future. An improved understanding of biofouling mechanisms may lead to better membrane design, better membrane modules, and better membrane cleaning procedures.Item Characterizing temporal development of biofilm porosity using artificial neural networks(2008-06) Veluchamy, Raaja R. A.; Lewandowski, Zbigniew; Beyenal, HalukWe used artificial neural networks (ANN) to compute parameters characterising biofilm structure from biofilm images and to interpolate a limited number of experimental data characterising the effects of nutrient concentration and flow velocity on the areal porosity of biofilms. ANN were trained using a set of experimental data characterising structural parameters of biofilms of Pseudomonas aeruginosa (ATCC #700829), Pseudomonas fluorescens (ATCC #700830) and Klebsiella pneumoniae (ATCC #700831) for various flow velocities and glucose concentrations. We used 80% of the data to train ANN and 10% of the data to validate the results, which is routinely carried out as a countermeasure against overtraining. Trained ANN were used to interpolate into the data set and evaluate the missing 10% of the data. To compare ANN accuracy in evaluating the missing data with the accuracies achieved using other interpolation algorithms, we used spline, cubic, linear and nearest neighbour interpolation algorithms to evaluate the missing data. ANN estimates were consistently closer to the experimental data than the estimates made using the other methods.Item Combined effect of substrate concentration and flow velocity on effective diffusivity in biofilms(2000-02) Beyenal, HalukWe have evaluated the influence of glucose concentration and flow velocity at which biofilms were grown on the distribution of effective diffusivity in biofilms consisting of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Klebsiella pneumoniae. From the local diffusivity profiles, measured by a novel microelectrode, the surface averaged relative effective diffusivities ( Ds ) were calculated at different depths in the biofilms. The Ds decreased toward the bottom of the biofilms and the Ds profiles were affected by growth conditions. The Ds was higher in biofilms grown at high glucose concentrations and at low flow velocities to a much lager extent than by the flow velocity.Item Compromised host defense on Pseudomonas aeruginosa biofilms: Characterization of neutrophil and biofilm interactions(2003-10) Jesaitis, A. J.; Franklin, Michael J.; Berglund, Deborah L.; Sasaki, Maiko; Lord, Connie I.; Bleazard, Justin Brock; Duffy, James E.; Beyenal, Haluk; Lewandowski, ZbigniewPseudomonas aeruginosa is an opportunistic pathogen that forms biofilms on tissues and other surfaces. We characterized the interaction of purified human neutrophils with P. aeruginosa, growing in biofilms, with regard to morphology, oxygen consumption, phagocytosis, and degranulation. Scanning electron and confocal laser microscopy indicated that the neutrophils retained a round, unpolarized, unstimulated morphology when exposed to P. aeruginosa PAO1 biofilms. However, transmission electron microscopy demonstrated that neutrophils, although rounded on their dorsal side, were phagocytically active with moderate membrane rearrangement on their bacteria-adjacent surfaces. The settled neutrophils lacked pseudopodia, were impaired in motility, and were enveloped by a cloud of planktonic bacteria released from the biofilms. The oxygen consumption of the biofilm/neutrophil system increased 6- and 8-fold over that of the biofilm alone or unstimulated neutrophils in suspension, respectively. H(2)O(2) accumulation was transient, reaching a maximal measured value of 1 micro M. Following contact, stimulated degranulation was 20-40% (myeloperoxidase, beta-glucuronidase) and 40-80% (lactoferrin) of maximal when compared with formylmethionylleucylphenylalanine plus cytochalasin B stimulation. In summary, after neutrophils settle on P. aeruginosa biofilms, they become phagocytically engorged, partially degranulated, immobilized, and rounded. The settling also causes an increase in oxygen consumption of the system, apparently resulting from a combination of a bacterial respiration and escape response and the neutrophil respiratory burst but with little increase in the soluble concentration of H(2)O(2). Thus, host defense becomes compromised as biofilm bacteria escape while neutrophils remain immobilized with a diminished oxidative potential.Item The double substrate growth kinetics of pseudomonas aeruginosa(2003-01) Beyenal, Haluk; Chen, Suet Nee; Lewandowski, ZbigniewGrowth parameters of Pseudomonas aeruginosa were quantified based on steady-state concentrations, utilization rates of glucose and dissolved oxygen, and microorganism concentration in a chemostat that was operated at 25 °C, pH 7.2, and an agitation rate 350 rpm. The results showed that the microbial growth was limited by the concentration of glucose and the concentration of oxygen. A dual-substrate, Tessier growth kinetics for oxygen and glucose, was in good agreement with the experimental data using the following biokinetic parameters: max=0.29 h-1, Kg=26.9 mg/l, Ko=1.18 mg/l, Yx/g=0.628 g microorganism/g glucose and, Yx/o=0.635 g microorganism/g oxygen. Maintenance factors for glucose and oxygen were: mg=0.0078 g glucose consumed/g microorganism h, and mo=0.014 g oxygen consumed/g microorganism h.Item Dynamics of lead immobilization in sulfate-reducing biofilms(2004-06) Beyenal, Haluk; Lewandowski, ZbigniewWe have evaluated the effects of selected minerals present in subsoil environment on the effciency of lead removal from contaminated groundwaters using biofilms composed of sulfate-reducing microorganisms, and examined the stability of metal deposits after the biofilms had been temporarily exposed to the air. To quantify the studied effects, lead was immobilized in biofilms of Desulfovibrio desulfuricans grown anaerobically in two flat-plate flow reactors, one filled with hematite and the other with quartz. While the biofilms in both reactors were heterogeneous and consisted of voids and channels, the biofilms grown on hematite were denser, thicker, and more porous than those grown on quartz. The average H2S concentrations, measured using microelectrodes, were higher in the biofilms grown on quartz than those measured in the biofilms grown on hematite. During 18 weeks of operation, iron was continuously released from the hematite. Lead was immobilized more efficiently in the biofilms grown on quartz than it was in the biofilms grown on hematite. Lead deposits were partially reoxidized, especially in biofilms grown on hematite, and the biofilms in both reactors responded to the presence of oxygen by lowering their density and increasing the H2S production rate. © 2004 Elsevier Ltd. All rights reserved.Item The effect of detachment on biofilm structure and activity: the oscillating pattern of biofilm accumulation(2007-05) Lewandowski, Zbigniew; Beyenal, Haluk; Stookey, D.In our previous papers we have demonstrated that biofilm structure never reaches a steady state in biofilm reactors; in this paper we link this fact to biofilm detachment and to the oscillating pattern of biofilm accumulation. In one respect reactors supporting suspended microbial growth and reactors supporting attached microbial growth (biofilms) are similar: in both the biomass accumulates in the reactor and is disposed of with the effluent. However, while in reactors with suspended microbial growth biomass accumulation and disposal occur simultaneously, in biofilm reactors these two processes are separated in time. Biomass accumulation in biofilm reactors shows a distinct pattern composed of three phases: (1) growth, (2) detachment, (3) regrowth. Despite this distinct pattern of biofilm accumulation observed at the microscale, biofilm reactors do reach a steady state of substrate removal.Item An electrochemical technique to measure local flow velocity in biofilms(1998-12) Xia, Fan; Beyenal, Haluk; Lewandowski, ZbigniewThe limiting current technique and a mobile microelectrode were used to measure local flow velocity in biofilms. The microelectrode was calibrated using the particle tracking technique in conjunction with confocal scanning laser microscopy (CSLM). A relationship between the limiting current density and the local flow velocity was found using nonlinear regression. This relationship was the same whether biofilm was present or not. Therefore, we used the measurements of limiting current density to evaluate flow velocity distribution around biofilm clusters. The flow velocity distribution was affected by the geometry of interstitial voids and by their orientation with respect to the flow direction in the main conduit. Flow velocity measurements using the limiting current technique were faster than similar measurements using particle tracking with confocal scanning laser microscopy.Item Electrochemically active biofilms: Facts and fiction. A review(2012-09) Babauta, J.; Renslow, Ryan; Lewandowski, Zbigniew; Beyenal, HalukThis review examines the electrochemical techniques used to study extracellular electron transfer in the electrochemically active biofilms that are used in microbial fuel cells and other bioelectrochemical systems. Electrochemically active biofilms are defined as biofilms that exchange electrons with conductive surfaces: electrodes. Following the electrochemical conventions, and recognizing that electrodes can be considered reactants in these bioelectrochemical processes, biofilms that deliver electrons to the biofilm electrode are called anodic, ie electrode-reducing, biofilms, while biofilms that accept electrons from the biofilm electrode are called cathodic, ie electrode-oxidizing, biofilms. How to grow these electrochemically active biofilms in bioelectrochemical systems is discussed and also the critical choices made in the experimental setup that affect the experimental results. The reactor configurations used in bioelectrochemical systems research are also described and the authors demonstrate how to use selected voltammetric techniques to study extracellular electron transfer in bioelectrochemical systems. Finally, some critical concerns with the proposed electron transfer mechanisms in bioelectrochemical systems are addressed together with the prospects of bioelectrochemical systems as energy converting and energy-harvesting devices.Item Evaluation of biofilm image thresholding methods(2001-04) Yang, Xinmin; Beyenal, Haluk; Harkin, Gary; Lewandowski, ZbigniewTo evaluate biomass distribution in heterogeneous biofilms from their microscope images, it is often necessary to perform image thresholding by converting the gray-scale images to binary images consisting of a foreground of biomass material and a background of interstitial space. The selection of gray-scale intensity used for thresholding is arbitrary but under the control of the operator, which may produce unacceptable levels of variability among operators. The quality of numerical information extracted from the images is diminished by such variability, and it is desirable to find a method that improves the reproducibility of thresholding operation. Automatic methods of thresholding provide this reproducibility, but often at the expense of accuracy, as they consistently set thresholds that differ significantly from what human operators would chose. The performance of five automatic image thresholding algorithms was tested in this study; (1) local entropy; (2) joint entropy; (3) relative entropy; (4) Renyi’s entropy; and (5) iterative selection. Only the iterative selection method was satisfactory in that it was consistently setting the threshold level near that set manually. The extraction of feature information from biofilm images benefits from automatic thresholding and can be extended to other fields, such as medical imaging.Item Fiber-optic microsensors to measure backscattered light intensity in biofilms(2000-07) Beyenal, Haluk; Lewandowski, Zbigniew; Yakymyshyn, Chris; Lemley, B.; Wehri, J.We have constructed tapered fiber-optic microsensors with a tip diameter of less than 10 um to measure profiles of backscattered light in biofilms, which are thin layers of micro-organisms firmly attached to surfaces. The observed response agrees well with local effective diffusivity microelectrode measurements, with R2 > 0.85. A strong relation between signal intensity and wavelength has been observed at 670 and 1320 nm. These sensors have the potential to replace local effective diffusivity microelectrodes for true in situ biofilm measurements.Item Growing reproducible biofilms with respect to structure and viable cell counts(2001-10) Jackson, Gary; Beyenal, Haluk; Rees, Wayne M.; Lewandowski, ZbigniewWe have developed a new method of growing 4-day-old biofilms that are reproducible, with respect to viable cell number and biofilm structure. To demonstrate the utility of the method, we grew biofilms composed of Pseudomonas aeruginosa (ATCC#700829), P. fluorescens (ATCC#700830) and Klebsiella pneumoniae (ATCC#700831), 18 times in flat-plate reactors under well-defined conditions of: flow rate, nutrient concentration, temperature, inoculum and growth rate. The resulting 4-day-old biofilms were approximately 2 µm thick and exhibited a high degree of reproducibility. The number of viable cells that accumulated per unit surface area and the biofilm areal porosity were reproduced within 10% error. We have also quantified other parameters characterizing biofilm structure using biofilm-imaging techniques: fractal dimension, textural entropy and diffusion distance as auxiliary parameters characterizing the reproducibility of biofilm accumulation. As a result of analysis, we have introduced a new parameter to better quantify and characterize the number of viable cells in biofilms, "specific number of viable cells" (SNVC). This parameter is the viable cell number normalized with respect to the surface area covered by the biofilm and with respect to the biomass of the biofilm. This new descriptor represents the dynamics of biofilm accumulation better than the traditionally used colony-forming unit (CFU) per surface area covered by the biofilm because it accounts not only for the surface coverage but also for the biofilm thickness.Item An improved Severinghaus-type carbon dioxide microelectrode for use in biofilms(2004-02) Beyenal, Haluk; Davis, Catherine C.; Lewandowski, ZbigniewSeveringhaus-type carbon dioxide microelectrodes with a tip diameter of less than 20 m were constructed using anodically grown iridium oxide film (AIROF) as the internal pH sensor. The AIROF, which was formed at the tip of the iridium wire by cyclic voltammetry in diluted sulfuric acid, showed a super-Nernstian response to pH changes, with a slope between 65 and 80 mV/pH. Therefore, our microelectrodes were more sensitive to changes in carbon dioxide concentration than the previously described microelectrodes, which used liquid ion exchanger (LIX) pH membranes and had a Nernstian response of 59 mV/pH. When calibrated, our microelectrodes showed 65–76 mV per decade of µatm pCO2, and were more sensitive than the microelectrodes using LIX membranes, which showed 57 mV per decade of µatm pCO2. AIROF electrodes were serviceable after 1 month of storage, showing only a modest potential drift of 0.03 ± 0.01 mV/h. Their utility was demonstrated by measuring CO2 concentration profiles in Staphylococcus aureus (MN8) biofilms.Item Internal and external mass transfer in biofilms grown at various flow velocities(2002-02) Beyenal, Haluk; Lewandowski, ZbigniewIt appears that biofilms arrange their internal structure according to the flow velocity at which they are grown, which affects the internal mass transfer rate and microbial activity. In biofilms grown at various flow velocities we determined the vertical profiles of the local relative effective diffusivity (termed Dsa) at several locations within each biofilm. From these profiles we calculated the surface-averaged relative effective diffusivity (termed D1) at various distances from the bottom and plotted it against these distances. The Dsa decreased linearly toward the bottom, forming well-defined profiles that were different for each biofilm. The gradients of these profiles were multiplied by the diffusivity of oxygen, ζ= Dw dDa/dz, and plotted versus the flow velocity at which each each biofilm was grown. The gradients were low at flow velocities below 10 cm/s, reached a maximum at a flow velocity of 10 cm/s, and decreased again at flow velocities exceeding 10 cm/s. The existence of a maximum indicates a possibility that two opposing forces were affecting the slope of the profiles. To explain these observations we hypothesized that biofilms, depending on the flow velocity at which they are grown, arrange their internal architecture to control (1) the nutrient transport rate and (2) the mechanical pliability needed to resist the shear stress of the water flowing past them. It appears that biofilms attempt to satisfy the second goal first, to increase their mechanical strength, and that they do so at the expense of the nutrient transfer rate to deeper layers. This strength increase is associated with an increase in biofilm density, which slows down the internal mass transport rate. Biofilms grown at low flow velocities exhibit low density and high effective diffusivity but cannnot resist higher shear stress, whereas biofilms grown at higher flow velocities are denser and can resist higher shear stress but have a lower effective diffusivity.Item Limiting-current-type microelectrodes for quantifying mass transport dynamics in biofilms(2001) Lewandowski, Zbigniew; Beyenal, HalukItem Mass-transport dynamics, activity, and structure of sulfate-reducing biofilms(2001-07) Beyenal, Haluk; Lewandowski, ZbigniewFactors limiting hydrogen sulfide production were identified in a two-species biofilm containing sulfate-reducing bacteria (Desulfovibrio desulfuricans) and nonsulfate-reducing bacteria (Pseudomonas fluorescens). Profiles ofhydrogen sulfide (H2S) concentration, pH, local mass-transport coefficient, local flow velocity, and local relative effective diffusivity in the biofilm were measured using microelectrodes. Biofilms had a heterogeneous structure consisting of cell clusters separated by voids. Typically, the H2S concentration was lower in the voids than in the adjacent cell clusters, demonstrating that the voids acted as transport channels for removing H2S from cell clusters. The extent of biofilm heterogeneity was directly correlated with the flux of H2S from cell clusters. At flow velocities below 2 cm/s, the flux of H2S from cell clusters depended on the flow velocity. We concluded that at these flow velocities the H2S production rate was limited by the delivery rate of sulfate ions to the biofilm. At flow velocities above 2 cm/s, the H2S production rate was nearly constant and did not depend on the flow velocity. At high flow velocities (<2 cm/s) the H2S production rate was limited by metabolic reactions in the biofilm. Local intrabiofilm flow velocity profiles were influenced strongly by biofilm heterogeneity without significant pH variation within biofilms. Surprisingly, profiles of local relative effective diffusivity indicated that the biofilm was made up of two layers, which could be related to the specimen with a two-species biofilm.Item Measurement of local effective diffusivity in heterogeneous biofilms(1998-11) Beyenal, Haluk; Tanyolac, A.; Lewandowski, ZbigniewWe have developed a novel technique to measure local effective diffusivity distribution in heterogeneous biofilms. Mobile microelectrodes (tip diameter 10 μm) and the limiting current technique were employed to measure the effective diffusivity of electroactive species introduced to natural and artificial biofilms. We calibrated the microelectrodes in artificial biofilms of known effective diffusivity and known density. In mixed population biofilms, local effective diffusivity varied from one location to another and decreased toward the bottom of the biofilm. We related local effective diffusivity to local biofilm density using an empirical equation. Surface-averaged biomass density depended on liquid flow velocity at which the biofilms were grown. The higher the flow velocity, the denser were the biofilms. Our technique permits fast evaluation of local effective diffusivity and biofilm density in heterogeneous biofilms.
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